U.S. patent application number 11/603625 was filed with the patent office on 2007-04-05 for human g protein-coupled receptors and modulators thereof for the treatment of metabolic-related disorders.
This patent application is currently assigned to Arena Pharmaceuticals, Inc.. Invention is credited to Dominic Behan, Derek Chalmers, Ruoping Chen, Bryan Choi, Daniel Connolly, Huong T. Dang, Yaron Hakak, James Leonard, Michael Lerner, Chen Liaw, Kevin P. Lowitz, Jeremy Richman, David J. Unett.
Application Number | 20070079392 11/603625 |
Document ID | / |
Family ID | 32719612 |
Filed Date | 2007-04-05 |
United States Patent
Application |
20070079392 |
Kind Code |
A1 |
Liaw; Chen ; et al. |
April 5, 2007 |
Human G protein-coupled receptors and modulators thereof for the
treatment of metabolic-related disorders
Abstract
The present invention relates to methods of identifying whether
a candidate compound is a modulator of a G protein-coupled receptor
(GPCR). In preferred embodiments, the GPCR is human. In other
preferred embodiments, the GPCR is coupled to Gi and lowers the
level of intracellular cAMP. In other preferred embodiments, the
GPCR is expressed endogenously by adipocytes. In further preferred
embodiments, the GPCR inhibits intracellular lipolysis. In other
further preferred embodiments, the GPCR is a nicotinic acid
receptor. The present invention also relates to methods of using a
modulator of said GPCR. Preferred modulator is agonist. Agonists of
the invention are useful as therapeutic agents for the prevention
or treatment of metabolic-related disorders, including
dyslipidemia, atherosclerosis, coronary heart disease, stroke,
insulin resistance, and type 2 diabetes.
Inventors: |
Liaw; Chen; (San Diego,
CA) ; Behan; Dominic; (San Diego, CA) ;
Chalmers; Derek; (Cardiff, CA) ; Lerner; Michael;
(Rancho Santa Fe, CA) ; Dang; Huong T.; (San
Diego, CA) ; Choi; Bryan; (Bonita, CA) ;
Leonard; James; (San Diego, CA) ; Hakak; Yaron;
(San Diego, CA) ; Lowitz; Kevin P.; (Sunnyvale,
CA) ; Unett; David J.; (San Diego, CA) ; Chen;
Ruoping; (San Diego, CA) ; Richman; Jeremy;
(San Diego, CA) ; Connolly; Daniel; (Carlsbad,
CA) |
Correspondence
Address: |
COZEN O'CONNOR, P.C.
1900 MARKET STREET
PHILADELPHIA
PA
19103-3508
US
|
Assignee: |
Arena Pharmaceuticals, Inc.
San Diego
CA
|
Family ID: |
32719612 |
Appl. No.: |
11/603625 |
Filed: |
November 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10897815 |
Jul 23, 2004 |
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11603625 |
Nov 22, 2006 |
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10314048 |
Dec 6, 2002 |
6902902 |
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10897815 |
Jul 23, 2004 |
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10096511 |
Mar 12, 2002 |
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10314048 |
Dec 6, 2002 |
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09995543 |
Nov 27, 2001 |
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10096511 |
Mar 12, 2002 |
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60399917 |
Jul 29, 2002 |
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60404761 |
Aug 19, 2002 |
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60410747 |
Sep 13, 2002 |
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Current U.S.
Class: |
800/14 ;
435/320.1; 435/325; 435/69.1; 435/7.1; 514/341; 546/268.1;
800/18 |
Current CPC
Class: |
A01K 2217/00 20130101;
G01N 2333/726 20130101; A01K 2267/03 20130101; G01N 33/566
20130101; A61K 31/4439 20130101; A01K 2227/105 20130101; G01N
2500/00 20130101; C07K 14/705 20130101; C07D 403/02 20130101 |
Class at
Publication: |
800/014 ;
435/007.1; 800/018; 435/069.1; 435/320.1; 435/325; 546/268.1;
514/341 |
International
Class: |
A01K 67/027 20060101
A01K067/027; G01N 33/53 20060101 G01N033/53; C12P 21/06 20060101
C12P021/06; C07D 403/02 20060101 C07D403/02; C07K 14/705 20060101
C07K014/705 |
Claims
1. A method of identifying whether a candidate compound is a
modulator of a nicotinic acid GPCR, said receptor comprising an
amino acid sequence selected from the group consisting of: (a) SEQ.
ID. NO.:36 (hRUP25); (b) SEQ. ID. NO.:137 (mRUP25); and (c) SEQ.
ID. NO.:139 (rRUP25); or an allelic variant, a biologically active
mutant, or a biologically active fragment of said amino acid
sequence; comprising the steps of: (a') contacting the candidate
compound with the receptor; and (b') determining whether the
receptor functionality is modulated; wherein a change in receptor
functionality is indicative of the candidate compound being a
modulator of a nicotinic acid GPCR.
2. A modulator of a nicotinic acid GPCR (RUP25) identified
according to the method of claim 1, provided that the modulator is
not identical to a compound selected from the group consisting of:
##STR100## wherein: R.sub.1 is selected from the group consisting
of halogen, hydroxyl, acetylamino, amino, alkoxy, carboalkoxy,
alkylthio, monoalkylamino, dialkylamino, N-alkylcarbamyl,
N,N-dialkylcarbamyl, alkylsulfonyl, said alkyl groups containing
from 1 to 4 carbons, trifluoromethyl, trifluoromethoxy,
trifluoromethylthio, methoxymethyl, carboxy, carbamyl, alkanoyloxy
containing up to 4 carbon atoms, phenyl, p-chlorophenyl,
p-methylphenyl and p-aminophenyl; R.sub.2 is selected from the
group consisting of halogen, alkannoyloxy containing from 1-4
carbon atoms, carboalkoxy containing from 2 to 5 carbon atoms,
carbamyl, N-alkyl carbamyl and N,N-dialkylcarbamyl wherein said
alkyl groups contain from 1-4 carbon atoms and trifluoromethyl; n
is a whole number from 0 to 4; and N-oxides thereof; ##STR101##
R.sub.3 and R.sub.4 are hydrogen, alkyl containing from 1 to 4
carbon atoms or cycloalkyl containing from 3 to 7 carbon atoms; n
is a whole number from 0 to 4; and N-oxides thereof. ##STR102##
wherein: R.sub.5 and R.sub.6 are each selected from the group
consisting of H, halogen, hydroxyl, amino, alkyloxy, alkylthio,
monoalkylamino, dialkylamino, N-alkylcarbamyl, N,N-dialkylcarbamyl,
alkylsulfoxy, alkylsulfony, said alkyl groups containing from 1 to
4 carbons, trifluoromethyl, trifluoromethoxy, trifluoromethylthio,
carboxy, carbamyl, alkanoyloxy containing up to 4 carbon atoms,
phenyl, p-chlorophenyl, p-methylphenyl and p-aminophenyl; n is a
whole number from 0 to 4; and N-oxides thereof; ##STR103## wherein:
at least one of R.sub.7, R.sub.8 and R.sub.9 is C.sub.1-6 alkyl and
the others are hydrogen atoms; R.sub.10 is hydroxy or C.sub.1-6
alkoxy, or a salt of the compounds wherein R.sub.4 is hydoxy with a
pharmaceutically acceptable base; ##STR104## wherein: at least one
of R.sub.7, R.sub.8 and R.sub.9 is C.sub.1-6 alkyl and the others
are hydrogen atoms; each of R.sub.11 and R.sub.12, which may be the
same or different, is hydrogen or C.sub.1-6 alkyl; ##STR105##
wherein: at least one of R.sub.13 represents an alkyl group of 7-11
carbon atoms and R.sub.14 represents H or a lower alkyl group of up
to two carbon atoms, and a pharmaceutically acceptable carrier; g)
pyrazine-2-carboxylic acid amide, 5-chloro-pyrazine-2-carboxylic
acid amide, 5-amino-pyrazine-2-carboxylic acid amide,
5-benzyl-pyrazine-2-carboxylic acid amide,
6-chloro-pyrazine-2-carboxylic acid amide,
6-methoxy-pyrazine-2-carboxylic acid amide,
3-chloro-pyrazine-2-carboxylic acid amide, 3-methoxy
-pyrazine-2-carboxylic acid amide, pyrazine-2-carboxylic acid
ethylamide, morpholin-4-yl-pyrazine-2-ylmethanone,
5-methyl-pyrazine-2-carboxylic acid (6-methyl-pyrazin-2-yl)-amide,
5-methyl-pyrazine-2-carboxylic acid (5-methyl-pyrazin-2-yl)-amide,
5-methyl-pyrazine-2-carboxylic acid (3-methyl-pyrazin-2-yl)-amide,
(5-methyl-pyrazin-2-yl)-morpholin-4-yl-methanone,
5-methyl-pyrazine-2-carboxylic acid hydroxyamide,
pyrazine-2-carboxylic acid, 5-amino-pyrazine-2-carboxylic acid,
5-benzyl-pyrazine-2-carboxylic acid, 6-chloro-pyrazine-2-carboxylic
acid, 6-methoxy-pyrazine-2-carboxylic acid,
3-hydroxy-pyrazine-2-carboxylic acid,
5-methyl-pyrazine-2-carboxylic acid 2-hydroxy-ethyl ester,
5-methyl-pyrazine-2-carboxylic acid allyl ester,
5-methyl-pyrazine-2-carboxylic acid phenyl ester,
5-methyl-pyrazine-2-carboxylic acid ethoxycarbonylmethyl ester,
pyrazine-2-carboxylic acid methyl ester or
2-methyl-5-(1H-tetrazol-5-yl)-pyrazine; and 4-N-oxides thereof; h)
5-(3-(5-Methyl)isoxazolyl)tetrazole; i)
5-(5-(3-Methyl)isoxazolyl)tetrazole; j) 5-(3-Quinolyl)tetrazole; k)
Nicotinic acid; l) Pyridazine-4-carboxylic acid; m) 3-pyridine
acetic acid; n) 5-Methylnicotinic acid; o) 6-Methylnicotinic acid;
p) Nicotinic acid-1-oxide; q) 2-Hydroxynicotinic acid; r)
Furane-3-carboxylic acid; s) 5-Methylpyrazole-3-carboxylic acid;
and t) 3-Methylisoxazole-5-carboxylic acid.
3. A method of modulating the activity of a nicotinic acid GPCR,
said receptor comprising an amino acid sequence selected from the
group consisting of: (a) SEQ. ID. NO.:36 (hRUP25); (b) SEQ. ID.
NO.:137 (mRUP25); and (c) SEQ. ID. NO.:139 (rRUP25); or an allelic
variant, a biologically active mutant, or a biologically active
fragment of said amino acid sequence; comprising the step of
contacting the receptor with the modulator of claim 2.
4. A method of preventing or treating a disorder of lipid
metabolism in an individual comprising contacting a therapeutically
effective amount of the modulator of claim 2 with a nicotinic acid
GPCR, said receptor comprising an amino acid sequence selected from
the group consisting of: (a) SEQ. ID. NO.:36 (hRUP25); (b) SEQ. ID.
NO.:137 (mRUP25); and (c) SEQ. ID. NO.:139 (rRUP25); or an allelic
variant or biologically active fragment of said amino acid
sequence.
5. A method of preventing or treating a metabolic-related disorder
in an individual comprising contacting a therapeutically effective
amount of the modulator of claim 2 with a nicotinic acid GPCR, said
receptor comprising an amino acid sequence selected from the group
consisting of: (a) SEQ. ID. NO.:36 (hRUP25); (b) SEQ. ID. NO.:137
(mRUP25); and (c) SEQ. ID. NO.:139 (rRUP25); or an allelic variant
or biologically active fragment of said amino acid sequence.
6. A method of preparing a composition which comprises identifying
a modulator of a nicotinic acid GPCR and then admixing a carrier
and the modulator, wherein the modulator is identifiable by the
method of claim 1 and provided that the modulator is not identical
to a compound selected from the group consisting of: ##STR106##
wherein: R.sub.1 is selected from the group consisting of halogen,
hydroxyl, acetylamino, amino, alkoxy, carboalkoxy, alkylthio,
monoalkylamino, dialkylamino, N-alkylcarbamyl, N,N-dialkylcarbamyl,
alkylsulfonyl, said alkyl groups containing from 1 to 4 carbons,
trifluoromethyl, trifluoromethoxy, trifluoromethylthio,
methoxymethyl, carboxy, carbamyl, alkanoyloxy containing up to 4
carbon atoms, phenyl, p-chlorophenyl, p-methylphenyl and
p-aminophenyl; R.sub.2 is selected from the group consisting of
halogen, alkannoyloxy containing from 1-4 carbon atoms, carboalkoxy
containing from 2 to 5 carbon atoms, carbamyl, N-alkyl carbamyl and
N,N-dialkylcarbamyl wherein said alkyl groups contain from 1-4
carbon atoms and trifluoromethyl; n is a whole number from 0 to 4;
and N-oxides thereof; ##STR107## R.sub.3 and R.sub.4 are hydrogen,
alkyl containing from 1 to 4 carbon atoms or cycloalkyl containing
from 3 to 7 carbon atoms; n is a whole number from 0 to 4; and
N-oxides thereof ##STR108## wherein: R.sub.5 and R.sub.6 are each
selected from the group consisting of H, halogen, hydroxyl, amino,
alkyloxy, alkylthio, monoalkylamino, dialkylamino, N-alkylcarbamyl,
N,N-dialkylcarbamyl, alkylsulfoxy, alkylsulfony, said alkyl groups
containing from 1 to 4 carbons, trifluoromethyl, trifluoromethoxy,
trifluoromethylthio, carboxy, carbamyl, alkanoyloxy containing up
to 4 carbon atoms, phenyl, p-chlorophenyl, p-methylphenyl and
p-aminophenyl; n is a whole number from 0 to 4; and N-oxides
thereof; ##STR109## wherein: at least one of R.sub.7, R.sub.8 and
R.sub.9 is C.sub.1-6 alkyl and the others are hydrogen atoms;
R.sub.10 is hydroxy or C.sub.1-6 alkoxy, or a salt of the compounds
wherein R.sub.4 is hydoxy with a pharmaceutically acceptable base;
##STR110## wherein: at least one of R.sub.7, R.sub.8 and R.sub.9 is
C.sub.1-6 alkyl and the others are hydrogen atoms; each of R.sub.11
and R.sub.12, which may be the same or different, is hydrogen or
C.sub.1-6 alkyl; ##STR111## wherein: at least one of R.sub.13
represents an alkyl group of 7-11 carbon atoms and R.sub.14
represents H or a lower alkyl group of up to two carbon atoms, and
a pharmaceutically acceptable carrier; g) pyrazine-2-carboxylic
acid amide, 5-chloro-pyrazine-2-carboxylic acid amide,
5-amino-pyrazine-2-carboxylic acid amide,
5-benzyl-pyrazine-2-carboxylic acid amide,
6-chloro-pyrazine-2-carboxylic acid amide,
6-methoxy-pyrazine-2-carboxylic acid amide,
3-chloro-pyrazine-2-carboxylic acid amide,
3-methoxy-pyrazine-2-carboxylic acid amide, pyrazine-2-carboxylic
acid ethylamide, morpholin-4-yl-pyrazine-2-ylmethanone,
5-methyl-pyrazine-2-carboxylic acid (6-methyl-pyrazin-2-yl)-amide,
5-methyl-pyrazine-2-carboxylic acid (5-methyl-pyrazin-2-yl)-amide,
5-methyl-pyrazine-2-carboxylic acid (3-methyl-pyrazin-2-yl)-amide,
(5-methyl-pyrazin-2-yl)-morpholin-4-yl-methanone,
5-methyl-pyrazine-2-carboxylic acid hydroxyamide,
pyrazine-2-carboxylic acid, 5-amino-pyrazine-2-carboxylic acid,
5-benzyl-pyrazine-2-carboxylic acid, 6-chloro-pyrazine-2-carboxylic
acid, 6-methoxy-pyrazine-2-carboxylic acid,
3-hydroxy-pyrazine-2-carboxylic acid,
5-methyl-pyrazine-2-carboxylic acid 2-hydroxy-ethyl ester,
5-methyl-pyrazine-2-carboxylic acid allyl ester,
5-methyl-pyrazine-2-carboxylic acid phenyl ester,
5-methyl-pyrazine-2-carboxylic acid ethoxycarbonylmethyl ester,
pyrazine-2-carboxylic acid methyl ester or
2-methyl-5-(1H-tetrazol-5-yl)-pyrazine; and 4-N-oxides thereof; h)
5-(3-(5-Methyl)isoxazolyl)tetrazole; i)
5-(5-(3-Methyl)isoxazolyl)tetrazole; j) 5-(3-Quinolyl)tetrazole; k)
Nicotinic acid; l) Pyridazine-4-carboxylic acid; m) 3-pyridine
acetic acid; n) 5-Methylnicotinic acid; o) 6-Methylnicotinic acid;
p) Nicotinic acid-1-oxide; q) 2-Hydroxynicotinic acid; r)
Furane-3-carboxylic acid; s) 5-Methylpyrazole-3-carboxylic acid;
and t) 3-Methylisoxazole-5-carboxylic acid.
7. A pharmaceutical or physiologically acceptable composition
comprising, consisting essentially of, or consisting of the
modulator of claim 2.
8. A method of changing lipid metabolism comprising providing or
administering to an individual in need of said change said
pharmaceutical or physiologically acceptable composition of claim
7.
9. A method of preventing or treating a metabolic-related disorder
comprising providing or administering to an individual in need of
said prevention or treatment said pharmaceutical or physiologically
acceptable composition of claim 7.
10. A method of using the modulator of claim 2 for the preparation
of a medicament for the treatment of a disorder in lipid metabolism
in an individual.
11. A method of using the modulator of claim 2 for the preparation
of a medicament for the treatment of a metabolic-related disorder
in an individual.
12. A method of identifying whether a candidate compound binds to a
nicotinic acid GPCR, said receptor comprising an amino acid
sequence selected from the group consisting of: (a) SEQ. ID. NO.:36
(hRUP25); (b) SEQ. ID. NO.:137 (mRUP25); and (c) SEQ. ID. NO.:139
(rRUP25); or an allelic variant or a biologically active fragment
of said amino acid sequence; comprising the steps of: (a')
contacting the receptor with a labeled reference compound known to
bind to the GPCR in the presence or absence of the candidate
compound; and (b') determining whether the binding of said labeled
reference compound to the receptor is inhibited in the presence of
the candidate compound; wherein said inhibition is indicative of
the candidate compound binding to a nicotinic acid GPCR.
13. A method of making a mouse genetically predisposed to a
metabolic-related disorder or a disorder of lipid metabolism
comprising the step of knocking out the gene encoding the
polypeptide of SEQ. ID. NO.:137 (mRUP25) or the polypeptide of SEQ.
ID. NO.:151 (mRUP19).
14. A knockout mouse according to the method of claim 13.
15. A method of using the knockout mouse of claim 14 to identify
whether a candidate compound has therapeutic efficacy for the
prevention or treatment of said metabolic-related disorder or said
disorder of lipid metabolism.
16. A method of making a rat genetically predisposed to a
metabolic-related disorder or a disorder of lipid metabolism
comprising the step of knocking out the gene encoding the
polypeptide of SEQ. ID. NO.:139 (rRUP25) or the polypeptide of SEQ.
ID. NO.:157 (rRUP19).
17. A knockout rat according to the method of claim 16.
18. A method of using the knockout rat of claim 17 to identify
whether a candidate compound has therapeutic efficacy for the
prevention or treatment of said metabolic-related disorder or said
disorder of lipid metabolism.
19. An isolated EFA-hRUP25 polynucleotide selected from the group
consisting of: (a) a polynucleotide comprising the nucleotide
sequence of SEQ. ID. NO.:158; (b) a polynucleotide having the
nucleotide sequence of SEQ. ID. NO.:158; (c) a polynucleotide
comprising a polynucleotide encoding the polypeptide having the
amino acid sequence of SEQ. ID. NO.:159 or a biologically active
fragment of said polypeptide; and (d) a polynucleotide encoding the
polypeptide having the amino acid sequence of SEQ. ID. NO.:159 or a
biologically active fragment of said polypeptide.
20. An isolated EFA-hRUP25 polypeptide selected from the group
consisting of: (a) a polypeptide comprising the amino acid sequence
of SEQ. ID. NO.:159, or a biologically active fragment of said
polypeptide; and (b) a polypeptide having the amino acid sequence
of SEQ. ID. NO.:159, or a biologically active fragment of said
polypeptide.
21. A recombinant vector comprising the polynucleotide of claim
19.
22. A host cell comprising the recombinant vector of claim 21.
Description
[0001] The present application is a Continuation of U.S. Utility
patent application Ser. No. 10/897,815, filed Jul. 23, 2004, which
is a Continuation of U.S. Utility patent application Ser. No.
10/314,048, filed Dec. 6, 2002, which is a Continuation-In-Part of
U.S. Utility patent application Ser. No. 10/096,511, filed Mar. 12,
2002, (now abandoned), which is a Continuation of U.S. Utility
patent application Ser. No. 09/995,543, filed Nov. 27, 2001 (now
abandoned) and claims benefit of priority to U.S. Provisional
Patent Applications: Ser. No. 60/399,917, filed Jul. 29, 2002, Ser.
No. 60/404,761, filed Aug. 19, 2002 and Ser. No. 60/410,747, filed
Sep. 13, 2002, the disclosure of each of which is hereby
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to methods of identifying
whether a candidate compound is a modulator of a G protein-coupled
receptor (GPCR). In preferred embodiments, the GPCR is human. In
other preferred embodiments, the GPCR is coupled to Gi and lowers
the level of intracellular cAMP. In other preferred embodiments,
the GPCR is expressed endogenously by adipocytes. In further
preferred embodiments, the GPCR inhibits intracellular lipolysis.
In other further preferred embodiments, the GPCR is a nicotinic
acid receptor. The present invention also relates to methods of
using a modulator of said GPCR. Preferred modulator is agonist.
Agonists of the invention are useful as therapeutic agents for the
prevention or treatment of metabolic-related disorders, including
dyslipidemia, atherosclerosis, coronary heart disease, stroke,
insulin resistance and type 2 diabetes.
BACKGROUND OF THE INVENTION
[0003] A. Nicotinic Acid as an Antilipolytic Agent
[0004] Atherosclerosis and stroke are the numbers one and number
three leading causes of death of both men and women in the United
States. [See, e.g., Nature Medicine, Special Focus on
Atherosclerosis, (2002) 8:1209-1262; the disclosure of which is
hereby incorporated by reference in its entirely.] Type 2 diabetes
is a public health problem that is serious, widespread and
increasing [Brownlee M, Nature (2001) 414:813-20 and references
therein; Zimmet P et al., Nature (2001) 414:782-7 and references
therein; Saltiel A R et al., Nature (2001) 414:799-806 and
references therein; the disclosure of each of which is hereby
incorporated by reference in its entirety]. Elevated levels of low
density lipoprotein (LDL) cholesterol or low levels of high density
lipoprotein (HDL) cholesterol are, independently, risk factors for
atherosclerosis and associated cardiovascular pathologies. In
addition, high levels of plasma free fatty acids are associated
with insulin resistance and type 2 diabetes. One strategy for
decreasing LDL-cholesterol, increasing HDL-cholesterol, and
decreasing plasma free fatty acids is to inhibit lipolysis in
adipose tissue. This approach involves regulation of hormone
sensitive lipase, which is the rate-limiting enzyme in lipolysis.
Lipolytic agents increase cellular levels of cAMP, which leads to
activation of hormone sensitive lipase within adipocytes. Agents
that lower intracellular cAMP levels, by contrast, would be
antilipolytic.
[0005] It is also worth noting in passing that an increase in
cellular levels of cAMP down-regulates the secretion of adiponectin
from adipocytes [Delporte, M L et al. Biochem J (2002) 367:677-85;
the disclosure of which is incorporated by reference in its
entirety]. Reduced levels of plasma adiponectin have been
associated with metabolic-related disorders, including
atherosclerosis, coronary heart disease, stroke, insulin resistance
and type 2 diabetes [Matsuda, M et al. J Biol Chem (2002)
277:37487-91 and reviewed therein; the disclosure of which is
hereby incorporated by reference in its entirety]. [Also see:
Yamauchi T et al., Nat Med (2002) 8:1288-95; and Tomas E et al.,
Proc Natl Acad Sci USA (2002) Nov. 27; the disclosure of each of
which is hereby incorporated by reference in its entirety.]
Globular adiponectin protected ob/ob mice from diabetes and apoE
deficient mice from atherosclerosis [Yamauchi, T et al. J Biol Chem
(2002) November; the disclosure of which is hereby incorporated by
reference in its entirety]. [Also see Okamoto, Y et al. Circulation
(2002) 26:2767-70; the disclosure of which is hereby incorporated
by reference in its entirety.] There is evidence that the
regulation of human serum adiponectin levels through modulation of
adipocyte intracellular cAMP level is independent of adipocyte
lipolysis [Staiger H et al., Horm Metab Res (2002) 34:601-3; the
disclosure of which is hereby incorporated by reference in its
entirety].
[0006] Nicotinic acid (niacin, pyridine-3-carboxylic acid) is a
water-soluble vitamin required by the human body for health, growth
and reproduction; a part of the Vitamin B complex. Nicotinic acid
is also one of the oldest used drugs for the treatment of
dyslipidemia. It is a valuable drug in that it favorably affects
virtually all of the lipid parameters listed above [Goodman and
Gilman's Pharmacological Basis of Therapeutics, editors Harmon J G
and Limbird L E, Chapter 36, Mahley R W and Bersot T P (2001) pages
971-1002]. The benefits of nicotinic acid in the treatment or
prevention of atherosclerotic cardiovascular disease have been
documented in six major clinical trials [Guyton J R (1998) Am J
Cardiol 82:18U-23U]. Structure and synthesis of analogs or
derivatives of nicotinic acid are discussed throughout the Merck
Index, An Encyclopedia of Chemicals, Drugs, and Biologicals, Tenth
Edition (1983), which is incorporated herein by reference in its
entirety.
[0007] Nicotinic acid and currently existing analogs thereof
inhibit the production and release of free fatty acids from adipose
tissue, likely via an inhibition of adenylyl cyclase, a decrease in
intracellular cAMP levels, and a concomitant decrease in hormone
sensitive lipase activity. Agonists that down-regulate hormone
sensitive lipase activity leading to a decrease in plasma free
fatty acid levels are likely to have therapeutic value. The
consequence of decreasing plasma free fatty acids is two-fold.
First, it will ultimately lower LDL-cholesterol and raise
HDL-cholesterol levels, independent risk factors, thereby reducing
the risk of mortality due to cardiovascular incidence subsequent to
atheroma formation. Second, it will provide an increase in insulin
sensitivity in individuals with insulin resistance or type 2
diabetes. Unfortunately, the use of nicotinic acid as a therapeutic
is partially limited by a number of associated, adverse
side-effects. These include flushing, free fatty acid rebound, and
liver toxicity.
[0008] Agonists of antilipolytic GPCRs having limited tissue
distribution beyond adipose may be especially valuable in view of
the diminished opportunity for potentially undesirable
side-effects.
[0009] The rational development of novel, nicotinic acid receptor
agonists that have fewer side-effects is an area of active
investigation, but to date it has been hindered by the inability to
molecularly identify the nicotinic acid receptor. Recent work
suggests that nicotinic acid may act through a specific GPCR
[Lorenzen A, et al. (2001) Molecular Pharmacology 59:349-357 and
reviewed therein; the disclosure of which is hereby incorporated by
reference in its entirety]. Furthermore, it is important to
consider that other receptors of the same class may exist on the
surface of adipocytes and similarly decrease hormone sensitive
lipase activity through a reduction in the level of intracellular
cAMP but without the elicitation of adverse effects such as
flushing, thereby representing promising novel therapeutic
targets.
[0010] B. G Protein-Coupled Receptors
[0011] Although a number of receptor classes exist in humans, by
far the most abundant and therapeutically relevant is represented
by the G protein-coupled receptor (GPCR) class. It is estimated
that there are some 30,000-40,000 genes within the human genome,
and of these, approximately 2% are estimated to code for GPCRs.
Receptors, including GPCRs, for which the endogenous ligand has
been identified, are referred to as "known" receptors, while
receptors for which the endogenous ligand has not been identified
are referred to as "orphan" receptors.
[0012] GPCRs represent an important area for the development of
pharmaceutical products: from approximately 20 of the 100 known
GPCRs, approximately 60% of all prescription pharmaceuticals have
been developed. For example, in 1999, of the top 100 brand name
prescription drugs, the following drugs interact with GPCRs (the
primary diseases and/or disorders treated related to the drug is
indicated in parentheses): TABLE-US-00001 Claritin .RTM.
(allergies) Prozac .RTM. (depression) Vasotec .RTM. (hypertension)
Paxil .RTM. (depression) Zoloft .RTM. (depression) Zyprexa
.RTM.(psychotic disorder) Cozaar .RTM. (hypertension) Imitrex .RTM.
(migraine) Zantac .RTM. (reflux) Propulsid .RTM. (reflux disease)
Risperdal .RTM. (schizophrenia) Serevent .RTM. (asthma) Pepcid
.RTM. (reflux) Gaster .RTM. (ulcers) Atrovent .RTM. (bronchospasm)
Effexor .RTM. (depression) Depakote .RTM. (epilepsy) Cardura
.RTM.(prostatic ypertrophy) Allegra .RTM. (allergies) Lupron .RTM.
(prostate cancer) Zoladex .RTM. (prostate cancer) Diprivan .RTM.
(anesthesia) BuSpar .RTM. (anxiety) Ventolin .RTM. (bronchospasm)
Hytrin .RTM. (hypertension) Wellbutrin .RTM. (depression) Zyrtec
.RTM. (rhinitis) Plavix .RTM. (MI/stroke) Toprol-XL .RTM.
(hypertension) Tenormin .RTM. (angina) Xalatan .RTM. (glaucoma)
Singulair .RTM. (asthma) Diovan .RTM. (hypertension) Harnal .RTM.
(prostatic hyperplasia) (Med Ad News 1999 Data).
[0013] GPCRs share a common structural motif, having seven
sequences of between 22 to 24 hydrophobic amino acids that form
seven alpha helices, each of which spans the membrane (each span is
identified by number, i.e., transmembrane-1 (TM-1), transmembrane-2
(TM-2), etc.). The transmembrane helices are joined by strands of
amino acids between transmembrane-2 and transmembrane-3,
transmembrane-4 and transmembrane-5, and transmembrane-6 and
transmembrane-7 on the exterior, or "extracellular" side, of the
cell membrane (these are referred to as "extracellular" regions 1,
2 and 3 (EC-1, EC-2 and EC-3), respectively). The transmembrane
helices are also joined by strands of amino acids between
transmembrane-1 and transmembrane-2, transmembrane-3 and
transmembrane-4, and transmembrane-5 and transmembrane-6 on the
interior, or "intracellular" side, of the cell membrane (these are
referred to as "intracellular" regions 1, 2 and 3 (IC-1, IC-2 and
IC-3), respectively). The "carboxy" ("C") terminus of the receptor
lies in the intracellular space within the cell, and the "amino"
("N") terminus of the receptor lies in the extracellular space
outside of the cell.
[0014] Generally, when a ligand binds with the receptor (often
referred to as "activation" of the receptor), there is a change in
the conformation of the receptor that facilitates coupling between
the intracellular region and an intracellular "G-protein." It has
been reported that GPCRs are "promiscuous" with respect to G
proteins, i.e., that a GPCR can interact with more than one G
protein. See, Kenakin, T., 43 Life Sciences 1095 (1988). Although
other G proteins exist, currently, Gq, Gs, Gi, Gz and Go are G
proteins that have been identified. Ligand-activated GPCR coupling
with the G-protein initiates a signaling cascade process (referred
to as "signal transduction"). Under normal conditions, signal
transduction ultimately results in cellular activation or cellular
inhibition. Although not wishing to be bound to theory, it is
thought that the IC-3 loop as well as the carboxy terminus of the
receptor interact with the G protein.
[0015] Gi-coupled GPCRs lower intracellular cAMP levels. The
Melanophore technology (see infra) is useful for identifying
Gi-coupled GPCRs.
[0016] Under physiological conditions, GPCRs exist in the cell
membrane in equilibrium between two different conformations: an
"inactive" state and an "active" state. A receptor in an inactive
state is unable to link to the intracellular signaling transduction
pathway to initiate signal transduction leading to a biological
response. Changing the receptor conformation to the active state
allows linkage to the transduction pathway (via the G-protein) and
produces a biological response.
[0017] A receptor may be stabilized in an active state by a ligand
or a compound such as a drug. Recent discoveries, including but not
exclusively limited to modifications to the amino acid sequence of
the receptor, provide means other than ligands or drugs to promote
and stabilize the receptor in the active state conformation. These
means effectively stabilize the receptor in an active state by
simulating the effect of a ligand binding to the receptor.
Stabilization by such ligand-independent means is termed
"constitutive receptor activation."
SUMMARY OF THE INVENTION
[0018] RUP25, RUP38, RUP19 and RUP11 belong to a sub-family of
human GPCRs on the basis of homology at the nucleotide level. See,
Tables B and C, infra. Polynucleotide sequence and polypeptide
sequence for human (h), rat (r), or mouse (m) RUP25, RUP38, RUP19,
or RUP11 is provided in the Sequence Listing (also see, Tables E
and F infra for corresponding SEQ. ID. NOs.).
[0019] Agonist engagement of Gi-coupled GPCRs is known to lead to
lowered levels of intracellular cAMP. Lower levels of cAMP in
adipocytes lead to diminished hormone sensitive lipase activity.
(See, supra.) The present invention is based in part on the
discovery by Applicant that GPCRs RUP25, RUP38, and RUP19 are
coupled to Gi and expressed endogenously by adipocytes. RUP38 and
RUP19 are further shown by Applicant to have limited tissue
distribution beyond adipose. RUP11 is also disclosed to be coupled
to Gi.
[0020] Applicant discloses herein that RUP25 is a nicotinic acid
and an antilipolytic GPCR. Applicant further discloses that RUP38
and RUP19 are antilipolytic GPCRs. RUP11 is also disclosed to be
antilipolytic. The present invention is directed in part to methods
of identifying whether a candidate compound is a modulator of
RUP25, RUP38, RUP19 or RUP11. The present invention also relates to
methods of using said modulator of RUP25, RUP38, RUP19 or RUP11.
Preferred said modulator is an agonist. Agonists of RUP25, RUP38,
RUP19 or RUP11 are useful as therapeutic agents for the prevention
or treatment of metabolic-related disorders, including
dyslipidemia, atherosclerosis, coronary heart disease, stroke,
insulin resistance, and type 2 diabetes.
[0021] Nicotinic acid is disclosed by Applicant to be an agonist
for RUP25 but not for RUP38 or RUP19. (-)-Nicotine is also
disclosed to be an agonist for RUP25. Exposure of cells expressing
RUP25 to nicotinic acid is shown by Applicant to lower the level of
intracellular cAMP. Exposure of isolated rat epididymal adipocyte
RUP25 to nicotinic acid is shown by Applicant to inhibit lipolysis.
Exposure of RUP25 within adipocyte primary cultures derived from
human subcutaneous fat to nicotinic acid is shown by Applicant also
to inhibit lipolysis. In vivo administration of nicotinic acid to
rats is shown by Applicant to lower plasma free fatty acids
(FFA).
[0022] Applicant has identified
(5-Hydroxy-1-methyl-3-propyl-1H-pyrazol-4-yl)-pyridin-3-yl-methanone
to be an agonist for RUP25 but not for RUP38. Exposure of cells
expressing RUP25 to
(5-hydroxy-1-methyl-3-propyl-1H-pyrazol-4-yl)-pyridin-3-yl-metha-
none is shown by Applicant to lower the level of intracellular
cAMP.
[0023] Applicant has identified
1-Isopropyl-1H-benzotriazole-5-carboxylic acid to be an agonist for
hRUP38 but not for RUP25. Exposure of cells expressing RUP38 to
1-Isopropyl-1H-benzotriazole-5-carboxylic acid is shown by
Applicant to lower the level of intracellular cAMP. Exposure of
RUP38 within adipocyte primary cultures derived from human
subcutaneous fat to 1-Isopropyl-1H-benzotriazole-5-carboxylic acid
is shown by Applicant to inhibit lipolysis.
[0024] Applicant has identified 3-(5-Bromo-2-ethoxy-phenyl)-acrylic
acid to also be an agonist for RUP38 but not for either RUP25 or
RUP19. Exposure of cells expressing RUP38 to
3-(5-Bromo-2-ethoxy-phenyl)-acrylic acid is shown by Applicant to
lower the level of intracellular cAMP.
[0025] Applicant, supra, provides direct in vitro evidence for
RUP25 and RUP38 being antilipolytic and direct in vivo evidence in
the rat for RUP25 being antilipolytic. Applicant also notes
illustrative clinical evidence that nicotinic acid receptor is
antilipolytic. Said evidence is consistent with the disclosure by
Applicant in the present application that RUP25 is a nicotinic acid
and an antilipolytic GPCR. Said evidence is consistent with the
disclosure by Applicant in the present application that RUP38 and
RUP19 are antilipolytic GPCRs. Said evidence is consistent with the
disclosure by Applicant in the present application that RUP11 is an
antilipolytic GPCR.
[0026] RUP38, RUP19 and RUP11 are further disclosed herein as being
antilipolytic GPCRs responsive to agonists other than nicotinic
acid. The failure of nicotinic acid to serve as an agonist for
RUP38, RUP19 or RUP11 indicates that RUP38, RUP19 and RUP11
represent novel antilipolytic pathways not engaged by conventional
nicotinic acid therapy.
[0027] See, Table M (Example 24) for a brief Summary and other
additional Examples, infra.
[0028] In a first aspect, the invention features a method of
identifying whether a candidate compound is a modulator of a
nicotinic acid GPCR, said receptor comprising an amino acid
sequence selected from the group consisting of:
[0029] (a) SEQ. ID. NO.:36 (hRUP25);
[0030] (b) SEQ. ID. NO.:137 (mRUP25); and
[0031] (c) SEQ. ID. NO.:139 (rRUP25);
[0032] or an allelic variant, a biologically active mutant, or a
biologically active fragment of said amino acid sequence;
comprising the steps of:
[0033] (a') contacting the candidate compound with the
receptor;
[0034] (b') determining whether the receptor functionality is
modulated;
wherein a change in receptor functionality is indicative of the
candidate compound being a modulator of a nicotinic acid GPCR.
[0035] In some embodiments, said nicotinic acid GPCR is
endogenous.
[0036] In some preferred embodiments, said nicotinic acid GPCR is
recombinant.
[0037] Preferred said identified modulator binds to said GPCR.
[0038] In some embodiments, said contacting is carried out in the
presence of a known ligand of the GPCR. In some embodiments, said
known ligand is an agonist of the GPCR. In some embodiments, said
agonist is nicotinic acid or an analog or derivative thereof. In
some embodiments, said agonist is (-)-nicotine or an analog or
derivative thereof.
[0039] The invention also relates to a method of identifying
whether a candidate compound is a modulator of lipolysis,
comprising the steps of: [0040] (a) contacting the candidate
compound with a GPCR comprising an amino acid sequence selected
from the group consisting of: [0041] (i) SEQ. ID. NO.:36 (hRUP25);
[0042] (ii) SEQ. ID. NO.:137 (mRUP25); and [0043] (iii) SEQ. ID.
NO.:139 (rRUP25); [0044] or an allelic variant, a biologically
active mutant, or a biologically active fragment of said amino acid
sequence; and [0045] (b) determining whether the receptor
functionality is modulated; wherein a change in receptor
functionality is indicative of the candidate compound being a
modulator of lipolysis.
[0046] In some embodiments, said GPCR is endogenous.
[0047] In some preferred embodiments, said GPCR is recombinant.
[0048] Preferred said identified modulator binds to said GPCR.
[0049] In some embodiments, said contacting is carried out in the
presence of a known ligand of the GPCR. In some embodiments, said
known ligand is an agonist of the GPCR. In some embodiments, said
agonist is nicotinic acid or an analog or derivative thereof. In
some embodiments, said agonist is (-)-nicotine or an analog or
derivative thereof.
[0050] The invention also relates to a method of determining
whether a candidate compound is a modulator of a nicotinic acid
GPCR, comprising the steps of: [0051] (a) culturing nicotinic acid
GPCR-expressing host cells under conditions that would allow
expression of a recombinant nicotinic acid GPCR, said host cells
being transfected with a polynucleotide encoding said recombinant
nicotinic acid GPCR comprising an amino acid sequence selected from
the group consisting of: [0052] (i) SEQ. ID. NO.:36 (hRUP25);
[0053] (ii) SEQ. ID. NO.:137 (mRUP25); and [0054] (iii) SEQ. ID.
NO.:139 (rRUP25); [0055] or an allelic variant, a biologically
active mutant, or a biologically active fragment of said amino acid
sequence; [0056] (b) contacting the nicotinic acid GPCR-expressing
host cells of step (a) with the candidate compound; [0057] (c)
contacting control host cells with the candidate compound of step
(b), wherein said control host cells do not express recombinant
nicotinic acid GPCR protein; [0058] (d) measuring the modulating
effect of the candidate compound which interacts with the
recombinant nicotinic acid GPCR from the host cells of step (a) and
control host cells of step (c); and [0059] (e) comparing the
modulating effect of the test compound on the host cells and
control host cells.
[0060] The invention also relates to a method of determining
whether a candidate compound is a modulator of a nicotinic acid
GPCR, comprising the steps of: [0061] (a) culturing nicotinic acid
GPCR-expressing host cells under conditions that would allow
expression of a recombinant nicotinic acid GPCR, said host cells
being transfected with a polynucleotide encoding said recombinant
nicotinic acid GPCR comprising an amino acid sequence selected from
the group consisting of: [0062] (i) SEQ. ID. NO.:36 (hRUP25);
[0063] (ii) SEQ. ID. NO.:137 (mRUP25); and [0064] (iii) SEQ. ID.
NO.:139 (rRUP25); [0065] or an allelic variant, a biologically
active mutant, or a biologically active fragment of said amino acid
sequence; [0066] (b) contacting a first population of nicotinic
acid GPCR-expressing cells of step (a) with a known ligand of said
nicotinic receptor GPCR; [0067] (c) contacting a second population
of nicotinic acid GPCR-expressing cells of step (a) with the
candidate compound and with the known nicotinic acid GPCR ligand;
[0068] (d) contacting control host cells with the candidate
compound of step (c), wherein said control host cells do not
express recombinant nicotinic acid GPCR protein; [0069] (e)
measuring the modulating effect of the candidate compound, which
interacts with recombinant nicotinic acid GPCR, in the presence and
absence of the known nicotinic acid GPCR ligand, from the cells of
step (b), step (c) and step (d); and [0070] (f) comparing the
modulating effect of the candidate compound as determined from step
(b), step (c) and step (d).
[0071] In some embodiments, said ligand is an agonist of said
nicotinic acid GPCR. In a particular embodiment, said agonist is
nicotinic acid or an analog or derivative thereof. In other
particular embodiment, said agonist is (-)-nicotine or an analog or
derivative thereof.
[0072] The invention also relates to a method of determining
whether a candidate compound is a modulator of a nicotinic acid
GPCR, comprising the steps of: [0073] (a) culturing nicotinic acid
GPCR-expressing host cells under conditions that would allow
expression of a recombinant nicotinic acid GPCR, said host cells
being transfected with a polynucleotide encoding said recombinant
nicotinic acid GPCR comprising an amino acid sequence selected from
the group consisting of: [0074] (i) SEQ. ID. NO.:36 (hRUP25);
[0075] (ii) SEQ. ID. NO.:137 (mRUP25); and [0076] (iii) SEQ. ID.
NO.:139 (rRUP25); [0077] or an allelic variant, a biologically
active mutant, or a biologically active fragment of said amino acid
sequence; [0078] (b) contacting a first population of the nicotinic
acid GPCR-expressing host cells of step (a) with the candidate
compound; [0079] (c) not contacting a second population of the
nicotinic acid GPCR-expressing cells of step (a) with the candidate
compound of step (b); [0080] (d) contacting control host cells to
the candidate compound of step (b), wherein said control host cells
do not express recombinant nicotinic acid GPCR protein; [0081] (e)
measuring the modulating effect of the candidate compound, which
interacts with recombinant nicotinic acid GPCR protein, from the
cells of step (b) and step (c) and from the cells of step (d); and
[0082] (f) comparing the modulating effect of the candidate
compound as determined from step (b) and step (c) and from step
(d).
[0083] In some embodiments, the nicotinic acid GPCR has an amino
acid sequence selected from the group consisting of:
SEQ. ID. NO.:36 (hRUP25);
[0084] (a) SEQ. ID. NO.:137 (mRUP25); and [0085] (b) SEQ. ID.
NO.:139 (rRUP25); [0086] or an allelic variant, a biologically
active mutant, or a biologically active fragment of said amino acid
sequence.
[0087] In some embodiments, the nicotinic acid GPCR comprises a
biologically active fragment of said amino acid sequence.
[0088] In some embodiments, said biologically active mutant is CART
or EFA. In preferred embodiments, said CART mutant has the amino
acid sequence of SEQ. ID. NO.:36 further substituted at amino acid
position 230 with lysine in place of isoleucine. In preferred
embodiments, said EFA mutant has the amino acid sequence of SEQ.
ID. NO.:159.
[0089] In preferred embodiments, said G protein is Gi.
[0090] In other preferred embodiments, said determining is through
the use of a Melanophore assay.
[0091] In other preferred embodiments, said determining is through
the measurement of the level of a second messenger selected from
the group consisting of cyclic AMP (cAMP), cyclic GMP (cGMP),
inositol triphosphate (IP.sub.3), diacylglycerol (DAG), and
Ca.sup.2+. In further preferred embodiments, said second messenger
is cAMP. In more preferred embodiments, the level of the cAMP is
reduced. In some embodiments, said measurement of cAMP is carried
out with membrane comprising said GPCR.
[0092] In other preferred embodiments, said determining is through
the measurement of an activity up-regulated or down-regulated by a
reduction in intracellular cAMP level. In further preferred
embodiments, said down-regulated activity is intracellular
lipolysis. In other further preferred embodiments, said
down-regulated activity is hormone sensitive lipase activity. In
other further preferred embodiments, said up-regulated activity is
adiponectin secretion.
[0093] In other preferred embodiments, said determining is through
CRE-reporter assay. In preferred embodiments, said reporter is
luciferase. In some embodiments, said reporter is
.beta.-galactosidase.
[0094] In other embodiments, said recombinant host cell further
comprises promiscuous G alpha 15/16 or chimeric Gq/Gi alpha subunit
and said determining is through measurement of intracellular
Ca.sup.2+. In preferred embodiments, said Ca.sup.2+ measurement is
carried out by FLIPR.
[0095] In other embodiments, said recombinant host cell further
comprises promiscuous G alpha 15/16 or chimeric Gq/Gi alpha subunit
and said determining is through measurement of intracellular
IP.sub.3.
[0096] In other preferred embodiments, said determining is through
the measurement of GTP.gamma.S binding to membrane comprising said
GPCR. In further preferred embodiments, said GTP.gamma.S is labeled
with [.sup.35S].
[0097] In other preferred embodiments, said method further
comprises the step of comparing the modulation of the receptor
caused by the candidate compound to a second modulation of the
receptor caused by contacting the receptor with a known modulator
of the receptor. In some preferred embodiments, said known
modulator is an agonist. In some preferred embodiments, said
agonist is nicotinic acid or an analog or derivative thereof. In
some preferred embodiments, said agonist is (-)-nicotine or an
analog or derivative thereof.
[0098] In a second aspect, the invention features a modulator of a
nicotinic acid GPCR identified according to the method of the first
aspect, provided that the modulator is not identical to a compound
having a formula selected from the group consisting of: ##STR1##
wherein:
[0099] R.sub.1 is selected from the group consisting of halogen,
hydroxyl, acetylamino, ammo, alkoxy, carboalkoxy, alkylthio,
monoalkylamino, dialkylamino, N-alkylcarbamyl, N,N-dialkylcarbamyl,
alkylsulfonyl, said alkyl groups containing from 1 to 4 carbons,
trifluoromethyl, trifluoromethoxy, trifluoromethylthio,
methoxymethyl, carboxy, carbamyl, alkanoyloxy containing up to 4
carbon atoms, phenyl, p-chlorophenyl, p-methylphenyl and
p-aminophenyl;
[0100] R.sub.2 is selected from the group consisting of halogen,
alkannoyloxy containing from 1-4 carbon atoms, carboalkoxy
containing from 2 to 5 carbon atoms, carbamyl, N-alkyl carbamyl and
N,N-dialkylcarbamyl wherein said alkyl groups contain from 1-4
carbon atoms and trifluoromethyl;
[0101] n is a whole number from 0 to 4; and
[0102] N-oxides thereof; ##STR2##
[0103] R.sub.3 and R.sub.4 are hydrogen, alkyl containing from 1 to
4 carbon atoms or cycloalkyl containing from 3 to 7 carbon
atoms;
[0104] n is a whole number from 0 to 4; and
[0105] N-oxides thereof; ##STR3## wherein:
[0106] R.sub.5 and R.sub.6 are each selected from the group
consisting of H, halogen, hydroxyl, amino, alkyloxy, alkylthio,
monoalkylamino, dialkylamino, N-alkylcarbamyl, N,N-dialkylcarbamyl,
alkylsulfoxy, alkylsulfony, said alkyl groups containing from 1 to
4 carbons, trifluoromethyl, trifluoromethoxy, trifluoromethylthio,
carboxy, carbamyl, alkanoyloxy containing up to 4 carbon atoms,
phenyl, p-chlorophenyl, p-methylphenyl and p-aminophenyl;
[0107] n is a whole number from 0 to 4; and
[0108] N-oxides thereof; ##STR4## wherein:
[0109] at least one of R.sub.7, R.sub.8 and R.sub.9 is C.sub.1-6
alkyl and the others are hydrogen atoms; R.sub.10 is hydroxy or
C.sub.1-6 alkoxy, or a salt of the compounds wherein R.sub.4 is
hydoxy with a pharmaceutically acceptable base; and a 4-N-oxide
thereof. The position of the N-oxide is designated by the following
numbering and a structure for a 4-N-oxide has the following
structure: ##STR5## One particular 4-N-oxide is
5-Methylpyrazine-2-carboxylic acid-4-oxide (Acipimox.TM.) and has
the structure: ##STR6## wherein:
[0110] at least one of R.sub.7, R.sub.8 and R.sub.9 is C.sub.1-6
alkyl and the others are hydrogen atoms; each of R.sub.11 and
R.sub.12, which may be the same or different, is hydrogen or
C.sub.1-6 alkyl; and a 4-N-oxide thereof; the position of the
N-oxide is the same as described above herein; ##STR7##
wherein:
[0111] at least one of R.sub.13 represents an alkyl group of 7-11
carbon atoms and R.sub.14 represents H or a lower alkyl group of up
to two carbon atoms, and a pharmaceutically acceptable carrier; (g)
Pyrazine-2-carboxylic acid amide and has the structure:
##STR8##
[0112] 5-chloro-pyrazine-2-carboxylic acid amide and has the
structure: ##STR9##
[0113] 5-amino-pyrazine-2-carboxylic acid amide and has the
structure: ##STR10##
[0114] 5-benzyl-pyrazine-2-carboxylic acid amide and has the
structure: ##STR11##
[0115] 6-chloro-pyrazine-2-carboxylic acid amide and has the
structure: ##STR12##
[0116] 6-methoxy-pyrazine-2-carboxylic acid amide and has the
structure: ##STR13##
[0117] 3-chloro-pyrazine-2-carboxylic acid amide and has the
structure: ##STR14##
[0118] 3-methoxy-pyrazine-2-carboxylic acid amide and has the
structure: ##STR15##
[0119] pyrazine-2-carboxylic acid ethylamide and has the structure:
##STR16##
[0120] morpholin-4-yl-pyrazine-2-ylmethanone and has the structure:
##STR17##
[0121] 5-methyl-pyrazine-2-carboxylic acid
(6-methyl-pyrazin-2-yl)-amide and has the structure: ##STR18##
[0122] 5-methyl-pyrazine-2-carboxylic acid
(5-methyl-pyrazin-2-yl)-amide and has the structure: ##STR19##
[0123] 5-methyl-pyrazine-2-carboxylic acid
(3-methyl-pyrazin-2-yl)-amide and has the structure: ##STR20##
[0124] (5-methyl-pyrazin-2-yl)-morpholin-4-yl-methanone and has the
structure: ##STR21##
[0125] 5-methyl-pyrazine-2-carboxylic acid hydroxyamide and has the
structure: ##STR22##
[0126] pyrazine-2-carboxylic acid and has the structure:
##STR23##
[0127] 5-amino-pyrazine-2-carboxylic acid and has the structure:
##STR24##
[0128] 5-benzyl-pyrazine-2-carboxylic acid and has the structure:
##STR25##
[0129] 6-chloro-pyrazine-2-carboxylic acid and has the structure:
##STR26##
[0130] 6-methoxy-pyrazine-2-carboxylic acid and has the structure:
##STR27##
[0131] 3-hydroxy-pyrazine-2-carboxylic acid and has the structure:
##STR28##
[0132] 5-methyl-pyrazine-2-carboxylic acid 2-hydroxy-ethyl ester
and has the structure: ##STR29##
[0133] 5-methyl-pyrazine-2-carboxylic acid allyl ester and has the
structure: ##STR30##
[0134] 5-methyl-pyrazine-2-carboxylic acid phenyl ester and has the
structure: ##STR31##
[0135] 5-methyl-pyrazine-2-carboxylic acid ethoxycarbonylmethyl
ester and has the structure: ##STR32##
[0136] pyrazine-2-carboxylic acid methyl ester and has the
structure: ##STR33##
[0137] 2-methyl-5-(1H-tetrazol-5-yl)-pyrazine and has the
structure: ##STR34##
[0138] and 4-N-oxides thereof as described above herein; (h)
5-(3-(5-Methyl)isoxazolyl)tetrazole and has the structure:
##STR35## (i) 5-(5-(3-Methyl)isoxazolyl)tetrazole and has the
structure: ##STR36## (j) 5-(3-Quinolyl)tetrazole and has the
structure: ##STR37## (k) Nicotinic acid and has the structure:
##STR38## (l) Pyridazine-4-carboxylic acid and has the structure:
##STR39## (m) 3-Pyridine acetic acid and has the structure:
##STR40## (n) 5-Methylnicotinic acid and has the structure:
##STR41## (o) 6-Methylnicotinic acid and has the structure:
##STR42## (p) Nicotinic acid-1-oxide and has the structure:
##STR43## (q) 2-Hydroxynicotinic acid and has the structure:
##STR44## (r) Furane-3-carboxylic acid and has the structure:
##STR45## (s) 5-Methylpyrazole-3-carboxylic acid and has the
structure: ##STR46## t) 3-Methylisoxazole-5-carboxylic acid and has
the structure: ##STR47##
[0139] In preferred embodiments, said modulator is selected from
the group consisting of agonist, partial agonist, inverse agonist
and antagonist. More preferably, said modulator is an agonist. In
some embodiments, said modulator is a partial agonist.
[0140] In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding assay
carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP25 polypeptide having the amino acid
sequence of SEQ. ID. NO.:36. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[0141] In some embodiments, said modulator is selective for the
GPCR.
[0142] In some embodiments, said modulator is antilipolytic.
[0143] In some embodiments, said modulator is orally bioavailable.
In some embodiments, said oral bioavailability is at least 1%, at
least 5%, at least 10%, at least 15%, at least 20%, at least 25%,
at least 30%, at least 35%, at least 40%, or at least 45 % relative
to intraperitoneal administration. In some embodiments, said oral
bioavailability is at least 1%, at least 5%, at least 10%, or at
least 15% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, at least 15%, at least 20%, at least 25%, at least
30%, at least 35%, at least 40%, or at least 45% relative to
intravenous administration. In some embodiments, said oral
bioavailability is at least 1%, at least 5%, at least 10%, or at
least 15% relative to intravenous administration.
[0144] In highly less preferred embodiments, said modulator is an
antibody or derivative thereof.
[0145] In a third aspect, the invention features the method of the
first aspect, wherein said candidate compound is an agonist of
hRUP38 GPCR comprising the amino acid sequence of SEQ. ID. NO.:135
and wherein said method further comprises the step of comparing the
modulation of hRUP25 GPCR comprising the amino acid sequence of
SEQ. ID. NO.:36 caused by said agonist to a second modulation of
hRUP25 GPCR comprising a variant of said amino acid sequence caused
by contacting the variant hRUP25 GPCR with said agonist.
[0146] In preferred embodiments, said variant amino acid sequence
is identical to the amino acid sequence of SEQ. ID. NO.:36, further
comprising a single amino acid substitution selected from the group
consisting of:
[0147] (a) A for V at amino acid position 27 of SEQ. ID.
NO.:36;
[0148] (b) V for L at amino acid position 83 of SEQ. ID.
NO.:36;
[0149] (c) Y for N at amino acid position 86 of SEQ. ID.
NO.:36;
[0150] (d) S for W at amino acid position 91 of SEQ. ID.
NO.:36;
[0151] (e) N for K at amino acid position 94 of SEQ. ID.
NO.:36;
[0152] (f) V for M at amino acid position 103 of SEQ. ID.
NO.:36;
[0153] (g) F for L at amino acid position 107 of SEQ. ID.
NO.:36;
[0154] (h) W for R at amino acid position 142 of SEQ. ID.
NO.:36;
[0155] (i) V for 1 at amino acid position 156 of SEQ. ID.
NO.:36;
[0156] (j) L for M at amino acid position 167 of SEQ. ID.
NO.:36;
[0157] (k) L for P at amino acid position 168 of SEQ. ID.
NO.:36;
[0158] (l) P for G at amino acid position 173 of SEQ. ID.
NO.:36;
[0159] (m) V for L at amino acid position 176 of SEQ. ID.
NO.:36;
[0160] (n) I for S at amino acid position 178 of SEQ. ID.
NO.:36;
[0161] (O)R for Q at amino acid position 187 of SEQ. ID.
NO.:36;
[0162] (p) L for F at amino acid position 198 of SEQ. ID. NO.:36;
and
[0163] (q) N for P at amino acid position 363 of SEQ. ID.
NO.:36.
[0164] In particularly preferred embodiments, said method is used
to identify whether said substituted amino acid additionally found
at the identical position within SEQ. ID. NO.:135 is necessary for
modulation of said hRUP38 GPCR by said agonist, comprising the
steps of:
[0165] (a) determining the level of modulation of said hRUP25 GPCR
by said agonist; and
[0166] (b) determining the level of modulation of said variant
hRUP25 GPCR by said agonist; wherein if said level of modulation
for (b) is greater than said level of modulation for (a), then said
substituted amino acid is necessary for modulation of said hRUP38
GPCR by said agonist.
[0167] In a fourth aspect, the invention features a method of
modulating the activity of a nicotinic acid GPCR, said receptor
comprising an amino acid sequence selected from the group
consisting of:
[0168] (a) SEQ. ID. NO.:36 (hRUP25);
[0169] (b) SEQ. ID. NO.:137 (mRUP25); and
[0170] (c) SEQ. ID. NO.:139 (rRUP25);
or an allelic variant, a biologically active mutant, or a
biologically active fragment of said amino acid sequence;
comprising the step of contacting the receptor with the modulator
of the second aspect.
[0171] In some embodiments, the nicotinic acid GPCR has an amino
acid sequence selected from the group consisting of:
[0172] (a) SEQ. ID. NO.:36 (hRUP25);
[0173] (b) SEQ. ID. NO.:137 (mRUP25); and
[0174] (c) SEQ. ID. NO.:139 (rRUP25);
or an allelic variant, a biologically active mutant, or a
biologically active fragment of said amino acid sequence.
[0175] In some embodiments, the nicotinic acid GPCR comprises an
active fragment of said amino acid sequence.
[0176] In some embodiments, the nicotinic acid GPCR is
endogenous.
[0177] In some embodiments, the nicotinic acid GPCR is
recombinant.
[0178] In some embodiments, said biologically active mutant is CART
or EFA. In preferred embodiments, said CART mutant has the amino
acid sequence of SEQ. ID. NO.:36 further substituted at amino acid
position 230 with lysine in place of isoleucine. In preferred
embodiments, said EFA mutant has the amino acid sequence of SEQ.
ID. NO.:159.
[0179] In preferred embodiments, said G protein is Gi.
[0180] In some preferred embodiments, said modulator is an
agonist.
[0181] In preferred embodiments, said modulator is selective for
the GPCR.
[0182] In other preferred embodiments, said contacting comprises
administration of the modulator to a membrane comprising the
receptor.
[0183] In other preferred embodiments, said contacting comprises
administration of the modulator to a cell or tissue comprising the
receptor.
[0184] In other preferred embodiments, said contacting comprises
administration of the modulator to an individual comprising the
receptor. In more preferred embodiments, said individual is a
mammal. In other more preferred embodiments, said mammal is a
horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat, non-human
primate or human. Yet more preferred is mouse, rat or human. Most
preferred is human.
[0185] In some preferred embodiments, said modulator is selective
for the GPCR.
[0186] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[0187] In some preferred embodiments, said modulator is
antilipolytic.
[0188] In some preferred embodiments, said modulator is an
agonist.
[0189] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP25 polypeptide having the amino acid
sequence of SEQ. ID. NO.:36. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[0190] In some preferred embodiments, said administration is
oral.
[0191] In preferred embodiments, said modulator is an agonist and
said individual is in need of prevention of or treatment for a
metabolic-related disorder selected from the group consisting
of:
[0192] (a) dyslipidemia;
[0193] (b) atherosclerosis;
[0194] (c) coronary heart disease;
[0195] (d) stroke;
[0196] (e) insulin resistance; and
[0197] (f) type 2 diabetes.
[0198] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[0199] In other embodiments, said modulator is an inverse agonist
and said metabolic-related disorder relates to a low level of
plasma free fatty acids.
[0200] In other preferred embodiments, said modulator is an agonist
and said individual is in need of a change in lipid metabolism
selected from the group consisting of:
[0201] (a) a decrease in the level of plasma triglycerides;
[0202] (b) a decrease in the level of plasma free fatty acids;
[0203] (c) a decrease in the level of plasma cholesterol;
[0204] (d) a decrease in the level of LDL-cholesterol;
[0205] (e) an increase in the level of HDL-cholesterol;
[0206] (f) a decrease in the total cholesterol/HDL-cholesterol
ratio; and
[0207] (g) an increase in the level of plasma adiponectin.
[0208] In other preferred embodiments, said needed change in lipid
metabolism is a decrease in the postprandial increase in plasma
free fatty acids due to a high fat meal or an inhibition of the
progression from impaired glucose tolerance to insulin
resistance.
[0209] In some embodiments, the modulator is an inverse agonist and
the needed change in lipid metabolism is an increase in the level
of plasma free fatty acids.
[0210] In other preferred embodiments, said modulator is an agonist
and said individual is a mouse genetically predisposed to a
metabolic-related disorder selected from the group consisting
of:
[0211] (a) dyslipidemia;
[0212] (b) atherosclerosis;
[0213] (c) coronary heart disease;
[0214] (d) stroke;
[0215] (e) insulin resistance; and
[0216] (f) type 2 diabetes.
[0217] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure.
[0218] In further preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[0219] In further preferred embodiments, said method is used to
identify whether said agonist has therapeutic efficacy for the
treatment of a metabolic-related disorder selected from the group
consisting of:
[0220] (a) dyslipidemia;
[0221] (b) atherosclerosis;
[0222] (c) coronary heart disease;
[0223] (d) stroke;
[0224] (e) insulin resistance; and
[0225] (f) type 2 diabetes;
comprising the steps of:
[0226] (a') administering or not administering said agonist to the
mouse; and
[0227] (b') determining whether the disorder is prevented, delayed,
or made less severe on administering said agonist compared to not
administering said agonist; wherein said determination is
indicative of said agonist having therapeutic efficacy.
[0228] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[0229] In other preferred embodiments, said modulator is an agonist
and said individual is a rat genetically predisposed to a
metabolic-related disorder selected from the group consisting
of:
[0230] (a) dyslipidemia;
[0231] (b) atherosclerosis;
[0232] (c) coronary heart disease;
[0233] (d) stroke;
[0234] (e) insulin resistance; and
[0235] (f) type 2 diabetes.
[0236] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In further preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[0237] In further preferred embodiments, said method is used to
identify whether said agonist has therapeutic efficacy for the
treatment of a metabolic-related disorder selected from the group
consisting of:
[0238] (a) dyslipidemia;
[0239] (b) atherosclerosis;
[0240] (c) coronary heart disease;
[0241] (d) stroke;
[0242] (e) insulin resistance; and
[0243] (f) type 2 diabetes; comprising the steps of:
[0244] (a') administering or not administering said agonist to the
rat; and
[0245] (b') determining whether the disorder is prevented, delayed,
or made less severe on administering said agonist compared to not
administering said agonist; wherein said determination is
indicative of said agonist having therapeutic efficacy.
[0246] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[0247] In a fifth aspect, the invention features a method of
preventing or treating a disorder of lipid metabolism in an
individual comprising contacting a therapeutically effective amount
of the modulator of the second aspect with a nicotinic acid GPCR,
said receptor comprising an amino acid sequence selected from the
group consisting of:
[0248] (a) SEQ. ID. NO.:36 (hRUP25);
[0249] (b) SEQ. ID. NO.:137 (mRUP25); and
[0250] (c) SEQ. ID. NO.:139 (rRUP25);
or an allelic variant or biologically active fragment of said amino
acid sequence.
[0251] In some preferred embodiments, said modulator is selective
for the GPCR.
[0252] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[0253] In some preferred embodiments, said modulator is
antilipolytic.
[0254] In some preferred embodiments, said modulator is an
agonist.
[0255] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP25 polypeptide having the amino acid
sequence of SEQ. ID. NO.:36. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[0256] In some preferred embodiments, said contacting comprises
oral administration of said modulator to said individual.
[0257] In preferred embodiment, said modulator is an agonist and
said disorder of lipid metabolism is selected from the group
consisting of:
[0258] (a) elevated level of plasma triglycerides;
[0259] (b) elevated level of plasma free fatty acids;
[0260] (c) elevated level of plasma cholesterol;
[0261] (d) elevated level of LDL-cholesterol;
[0262] (e) reduced level of HDL-cholesterol;
[0263] (f) elevated total cholesterol/HDL-cholesterol ratio;
and
[0264] (g) reduced level of plasma adiponectin.
[0265] In a sixth aspect, the invention features a method of
preventing or treating a metabolic-related disorder in an
individual comprising contacting a therapeutically effective amount
of the modulator of the second aspect with a nicotinic acid GPCR,
said receptor comprising an amino acid sequence selected from the
group consisting of:
[0266] (a) SEQ. ID. NO.:36 (hRUP25);
[0267] (b) SEQ. ID. NO.:137 (mRUP25); and
[0268] (c) SEQ. ID. NO.:139 (rRUP25);
or an allelic variant or biologically active fragment of said amino
acid sequence.
[0269] In some preferred embodiments, said modulator is selective
for the GPCR.
[0270] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[0271] In some preferred embodiments, said modulator is
antilipolytic.
[0272] In some preferred embodiments, said modulator is an
agonist.
[0273] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP25 polypeptide having the amino acid
sequence of SEQ. ID. NO.:36. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[0274] In some preferred embodiments, said contacting comprises
oral administration of said modulator to said individual.
[0275] In preferred embodiment, said modulator is an agonist and
said metabolic-related disorder is selected from the group
consisting of:
[0276] (a) dyslipidemia;
[0277] (b) atherosclerosis;
[0278] (c) coronary heart disease;
[0279] (d) stroke;
[0280] (e) insulin resistance; and
[0281] (f) type 2 diabetes.
[0282] In a seventh aspect, the invention features a method of
preparing a composition which comprises identifying a modulator of
a nicotinic acid GPCR and then admixing a carrier and the
modulator, wherein the modulator is identifiable by the method of
the first aspect and provided that the modulator is not identical
to a compound having a formula selected from the group consisting
of: ##STR48## wherein:
[0283] R.sub.1 is selected from the group consisting of halogen,
hydroxyl, acetylamino, amino, alkoxy, carboalkoxy, alkylthio,
monoalkylamino, dialkylamino, N-alkylcarbamyl, N,N-dialkylcarbamyl,
alkylsulfonyl, said alkyl groups containing from 1 to 4 carbons,
trifluoromethyl, trifluoromethoxy, trifluoromethylthio,
methoxymethyl, carboxy, carbamyl, alkanoyloxy containing up to 4
carbon atoms, phenyl, p-chlorophenyl, p-methylphenyl and
p-aminophenyl;
[0284] R.sub.2 is selected from the group consisting of halogen,
alkannoyloxy containing from 1-4 carbon atoms, carboalkoxy
containing from 2 to 5 carbon atoms, carbamyl, N-alkyl carbamyl and
N,N-dialkylcarbamyl wherein said alkyl groups contain from 1-4
carbon atoms and trifluoromethyl;
[0285] n is a whole number from 0 to 4; and
[0286] N-oxides thereof; ##STR49##
[0287] R.sub.3 and R.sub.4 are hydrogen, alkyl containing from 1 to
4 carbon atoms or cycloalkyl containing from 3 to 7 carbon
atoms;
[0288] n is a whole number from 0 to 4; and
[0289] N-oxides thereof; ##STR50## wherein:
[0290] R.sub.5 and R.sub.6 are each selected from the group
consisting of H, halogen, hydroxyl, amino, alkyloxy, alkylthio,
monoalkylamino, dialkylamino, N-alkylcarbamyl, N,N-dialkylcarbamyl,
alkylsulfoxy, alkylsulfony, said alkyl groups containing from 1 to
4 carbons, trifluoromethyl, trifluoromethoxy, trifluoromethylthio,
carboxy, carbamyl, alkanoyloxy containing up to 4 carbon atoms,
phenyl, p-chlorophenyl, p-methylphenyl and p-aminophenyl;
[0291] n is a whole number from 0 to 4; and
[0292] N-oxides thereof; ##STR51## wherein:
[0293] at least one of R.sub.7, R.sub.8 and R.sub.9 is C.sub.1-6
alkyl and the others are hydrogen atoms; R.sub.10 is hydroxy or
C.sub.1-6 alkoxy, or a salt of the compounds wherein R.sub.4 is
hydoxy with a pharmaceutically acceptable base; and a 4-N-oxide
thereof. The position of the N-oxide is designated by the following
numbering and a structure for a 4-N-oxide has the following
structure: ##STR52## One particular 4-N-oxide is
5-Methylpyrazine-2-carboxylic acid-4-oxide (Acipimox.TM.) and has
the structure: ##STR53## wherein:
[0294] at least one of R.sub.7, R.sub.8 and R.sub.9 is C.sub.1-6
alkyl and the others are hydrogen atoms; each of R.sub.11 and
R.sub.12, which may be the same or different, is hydrogen or
C.sub.1-6 alkyl; and a 4-N-oxide thereof; the position of the
N-oxide is the same as described above herein; ##STR54##
wherein:
[0295] at least one of R.sub.13 represents an alkyl group of 7-11
carbon atoms and R.sub.14 represents H or a lower alkyl group of up
to two carbon atoms, and a pharmaceutically acceptable carrier; (g)
Pyrazine-2-carboxylic acid amide and has the structure:
##STR55##
[0296] 5-chloro-pyrazine-2-carboxylic acid amide and has the
structure: ##STR56##
[0297] 5-amino-pyrazine-2-carboxylic acid amide and has the
structure: ##STR57##
[0298] 5-benzyl-pyrazine-2-carboxylic acid amide and has the
structure: ##STR58##
[0299] 6-chloro-pyrazine-2-carboxylic acid amide and has the
structure: ##STR59##
[0300] 6-methoxy-pyrazine-2-carboxylic acid amide and has the
structure: ##STR60##
[0301] 3-chloro-pyrazine-2-carboxylic acid amide and has the
structure: ##STR61##
[0302] 3-methoxy-pyrazine-2-carboxylic acid amide and has the
structure: ##STR62##
[0303] pyrazine-2-carboxylic acid ethylamide and has the structure:
##STR63##
[0304] morpholin-4-yl-pyrazine-2-ylmethanone and has the structure:
##STR64##
[0305] 5-methyl-pyrazine-2-carboxylic acid
(6-methyl-pyrazin-2-yl)-amide and has the structure: ##STR65##
[0306] 5-methyl-pyrazine-2-carboxylic acid
(5-methyl-pyrazin-2-yl)-amide and has the structure: ##STR66##
[0307] 5-methyl-pyrazine-2-carboxylic acid
(3-methyl-pyrazin-2-yl)-amide and has the structure: ##STR67##
[0308] (5-methyl-pyrazin-2-yl)-morpholin-4-yl-methanone and has the
structure: ##STR68##
[0309] 5-methyl-pyrazine-2-carboxylic acid hydroxyamide and has the
structure: ##STR69##
[0310] pyrazine-2-carboxylic acid and has the structure:
##STR70##
[0311] 5-amino-pyrazine-2-carboxylic acid and has the structure:
##STR71##
[0312] 5-benzyl-pyrazine-2-carboxylic acid and has the structure:
##STR72##
[0313] 6-chloro-pyrazine-2-carboxylic acid and has the structure:
##STR73##
[0314] 6-methoxy-pyrazine-2-carboxylic acid and has the structure:
##STR74##
[0315] 3-hydroxy-pyrazine-2-carboxylic acid and has the structure:
##STR75##
[0316] 5-methyl-pyrazine-2-carboxylic acid 2-hydroxy-ethyl ester
and has the structure: ##STR76##
[0317] 5-methyl-pyrazine-2-carboxylic acid allyl ester and has the
structure: ##STR77##
[0318] 5-methyl-pyrazine-2-carboxylic acid phenyl ester and has the
structure: ##STR78##
[0319] 5-methyl-pyrazine-2-carboxylic acid ethoxycarbonylmethyl
ester and has the structure: ##STR79##
[0320] pyrazine-2-carboxylic acid methyl ester and has the
structure: ##STR80##
[0321] 2-methyl-5-(1H-tetrazol-5-yl)-pyrazine and has the
structure: ##STR81##
[0322] and 4-N-oxides thereof as described above herein; (h)
5-(3-(5-Methyl)isoxazolyl)tetrazole and has the structure:
##STR82## (i) 5-(5-(3-Methyl)isoxazolyl)tetrazole and has the
structure: ##STR83## (j) 5-(3-Quinolyl)tetrazole and has the
structure: ##STR84## (k) Nicotinic acid and has the structure:
##STR85## (l) Pyridazine-4-carboxylic acid and has the structure:
##STR86## (m) 3-Pyridine acetic acid and has the structure:
##STR87## (n) 5-Methylnicotinic acid and has the structure:
##STR88## (O) 6-Methylnicotinic acid and has the structure:
##STR89## (p) Nicotinic acid-1-oxide and has the structure:
##STR90## (q) 2-Hydroxynicotinic acid and has the structure:
##STR91## (r) Furane-3-carboxylic acid and has the structure:
##STR92## (s) 5-Methylpyrazole-3-carboxylic acid and has the
structure: ##STR93## t) 3-Methylisoxazole-5-carboxylic acid and has
the structure: ##STR94##
[0323] In preferred embodiments, said modulator is selected from
the group consisting of agonist, partial agonist, inverse agonist
and antagonist. More preferably, said modulator is an agonist. In
some embodiments, said modulator is a partial agonist.
[0324] In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding assay
carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP25 polypeptide having the amino acid
sequence of SEQ. ID. NO.:36. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[0325] In some embodiments, said modulator is selective for the
GPCR.
[0326] In some embodiments, said modulator is antilipolytic.
[0327] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to either intraperitoneal or intravenous
administration. In some preferred embodiments, said oral
bioavailability is at least 1%, at least 5%, at least 10%, or at
least 15% relative to either intraperitoneal or intravenous
administration.
[0328] In an eighth aspect, the invention features a pharmaceutical
or physiologically acceptable composition comprising, consisting
essentially of, or consisting of the modulator of the second
aspect. In preferred embodiments, said modulator is an agonist.
[0329] In some preferred embodiments, said modulator is selective
for the GPCR.
[0330] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[0331] In some preferred embodiments, said modulator is
antilipolytic.
[0332] In some preferred embodiments, said modulator is an
agonist.
[0333] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP25 polypeptide having the amino acid
sequence of SEQ. ID. NO.:36. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[0334] In a ninth aspect, the invention features a method of
changing lipid metabolism comprising providing or administering to
an individual in need of said change said pharmaceutical or
physiologically acceptable composition of the eighth aspect, said
needed change in lipid metabolism selected from the group
consisting of:
[0335] (a) a decrease in the level of plasma triglycerides;
[0336] (b) a decrease in the level of plasma free fatty acids;
[0337] (c) a decrease in the level of plasma cholesterol;
[0338] (d) a decrease in the level of LDL-cholesterol;
[0339] (e) an increase in the level of HDL-cholesterol;
[0340] (f) a decrease in the total cholesterol/HDL-cholesterol
ratio; and
[0341] (g) an increase in the level of plasma adiponectin.
[0342] In preferred embodiments, a therapeutically effective amount
of said pharmaceutical or physiologically acceptable composition is
provided or administered to said individual.
[0343] In some preferred embodiments, said providing or
administering of said pharmaceutical or physiologically acceptable
composition is oral.
[0344] In other preferred embodiments, said needed change in lipid
metabolism is a decrease in the postprandial increase in plasma
free fatty acids due to a high fat meal or an inhibition of the
progression from impaired glucose tolerance to insulin
resistance.
[0345] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[0346] In a tenth aspect, the invention features a method of
preventing or treating a metabolic-related disorder comprising
providing or administering to an individual in need of said
treatment said pharmaceutical or physiologically acceptable
composition of the eighth aspect, said metabolic-related disorder
selected from the group consisting of:
[0347] (a) dyslipidemia;
[0348] (b) atherosclerosis;
[0349] (c) coronary heart disease;
[0350] (d) stroke;
[0351] (e) insulin resistance; and
[0352] (f) type 2 diabetes.
[0353] In preferred embodiments, a therapeutically effective amount
of said pharmaceutical or physiologically acceptable composition is
provided or administered to said individual.
[0354] In some preferred embodiments, said providing or
administering of said pharmaceutical or physiologically acceptable
composition is oral.
[0355] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, stroke,
Syndrome X, and heart disease. Heart disease includes, but is not
limited to, cardiac insufficiency, coronary insufficiency, and high
blood pressure. In other preferred embodiments, said
metabolic-related disorder is hyperlipidemia.
[0356] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[0357] In an eleventh aspect, the invention features a method of
using the modulator of the second aspect for the preparation of a
medicament for the treatment of a disorder in lipid metabolism in
an individual.
[0358] In some preferred embodiments, said modulator is selective
for the GPCR.
[0359] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[0360] In some preferred embodiments, said modulator is
antilipolytic.
[0361] In some preferred embodiments, said modulator is an
agonist.
[0362] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP25 polypeptide having the amino acid
sequence of SEQ. ID. NO.:36. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[0363] In some preferred embodiments, said treatment comprises oral
administration of said medicament to said individual.
[0364] In preferred embodiments, said modulator is an agonist and
said disorder in lipid metabolism is selected from the group
consisting of:
[0365] (a) elevated level of plasma triglycerides;
[0366] (b) elevated level of plasma free fatty acids;
[0367] (c) elevated level of plasma cholesterol;
[0368] (d) elevated level of LDL-cholesterol;
[0369] (e) reduced level of HDL-cholesterol;
[0370] (f) elevated total cholesterol/HDL-cholesterol ratio;
and
[0371] (g) reduced level of plasma adiponectin.
[0372] In other preferred embodiments, said disorder in lipid
metabolism is an elevated postprandial increase in plasma free
fatty acids due to a high fat meal or a progression from impaired
glucose tolerance to insulin resistance.
[0373] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[0374] In a twelfth aspect, the invention features a method of
using the modulator of the second aspect for the preparation of a
medicament for the treatment of a metabolic-related disorder in an
individual.
[0375] In some preferred embodiments, said modulator is selective
for the GPCR.
[0376] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[0377] In some preferred embodiments, said modulator is
antilipolytic.
[0378] In some preferred embodiments, said modulator is an
agonist.
[0379] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP25 polypeptide having the amino acid
sequence of SEQ. ID. NO.:36. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[0380] In some preferred embodiments, said treatment comprises oral
administration of said medicament to said individual.
[0381] In preferred embodiments, said modulator is an agonist and
said metabolic-related disorder is selected from the group
consisting of:
[0382] (a) dyslipidemia;
[0383] (b) atherosclerosis;
[0384] (c) coronary heart disease;
[0385] (d) stroke;
[0386] (e) insulin resistance; and
[0387] (f) type 2 diabetes.
[0388] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[0389] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[0390] In a thirteenth aspect, the invention features a method of
identifying whether a candidate compound binds to a nicotinic acid
GPCR, said receptor comprising an amino acid sequence selected from
the group consisting of:
[0391] (a) SEQ. ID. NO.:36 (hRUP25);
[0392] (b) SEQ. ID. NO.:137 (mRUP25); and
[0393] (c) SEQ. ID. NO.:139 (rRUP25);
or an allelic variant or a biologically active fragment of said
amino acid sequence; comprising the steps of:
[0394] (a') contacting the receptor with a labeled reference
compound known to bind to the GPCR in the presence or absence of
the candidate compound; and
[0395] (b') determining whether the binding of said labeled
reference compound to the receptor is inhibited in the presence of
the candidate compound;
wherein said inhibition is indicative of the candidate compound
binding to a nicotinic acid GPCR.
[0396] In some embodiments, the nicotinic acid GPCR comprises a
biologically active fragment of said amino acid sequence.
[0397] In some embodiments, the nicotinic acid GPCR is
endogenous.
[0398] In preferred embodiments, the nicotinic acid GPCR is
recombinant.
[0399] In preferred embodiments, said G protein is Gi.
[0400] In some preferred embodiments, said reference compound is
nicotinic acid.
[0401] In some preferred embodiments, said reference compound is
(-)-nicotine.
[0402] In some preferred embodiments, said reference compound is
the modulator of the second aspect.
[0403] In other embodiments, said reference compound is an antibody
specific for the GPCR, or a derivative thereof.
[0404] In preferred embodiments, said reference compound comprises
a label selected from the group consisting of:
[0405] (a) radioisotope;
[0406] (b) enzyme; and
[0407] (c) fluorophore.
In some preferred embodiments, said label is .sup.3H.
[0408] In other embodiments, said method further comprises the step
of comparing the level of inhibition of binding of a labeled first
reference compound by the candidate compound to a second level of
inhibition of binding of said labeled first reference compound by a
second reference compound known to bind to the GPCR.
[0409] In a fourteenth aspect, the invention features a method of
making a mouse genetically predisposed to a metabolic-related
disorder selected from the group consisting of:
[0410] (a) dyslipidemia;
[0411] (b) atherosclerosis;
[0412] (c) coronary heart disease;
[0413] (d) stroke;
[0414] (e) insulin resistance; and
[0415] (f) type 2 diabetes;
comprising the step of knocking out the gene encoding the nicotinic
acid mRUP25 GPCR polypeptide of SEQ. ID. NO.:137.
[0416] In some preferred embodiments, said knocking out the gene
encoding the nicotinic acid mRUP25 GPCR polypeptide pf SEQ. ID.
NO.:137 is essentially restricted to adipocytes.
[0417] In a fifteenth aspect, the invention features the knockout
mouse according to the method of the fourteenth aspect.
[0418] In a sixteenth aspect, the invention features a method of
using the knockout mouse of the fifteenth aspect to identify
whether a candidate compound has therapeutic efficacy for the
treatment of a metabolic-related disorder selected from the group
consisting of:
[0419] (a) dyslipidemia;
[0420] (b) atherosclerosis;
[0421] (c) coronary heart disease;
[0422] (d) stroke;
[0423] (e) insulin resistance; and
[0424] (f) type 2 diabetes;
comprising the steps of:
[0425] (a') administering or not administering the compound to the
mouse; and
[0426] (b') determining whether the disorder is prevented, delayed,
or made less severe on administering the compound compared to not
administering the compound; wherein said determination is
indicative of the compound having therapeutic efficacy.
[0427] In a seventeenth aspect, the invention features a method of
making a rat genetically predisposed to a metabolic-related
disorder selected from the group consisting of:
[0428] (a) dyslipidemia;
[0429] (b) atherosclerosis;
[0430] (c) coronary heart disease;
[0431] (d) stroke;
[0432] (e) insulin resistance; and
[0433] (f) type 2 diabetes;
comprising the step of knocking out the gene encoding the nicotinic
acid rRUP25 GPCR polypeptide of SEQ. ID. NO.:139.
[0434] In some preferred embodiments, said knocking out the gene
encoding the nicotinic acid rRUP25 GPCR polypeptide pf SEQ. ID.
NO.:139 is essentially restricted to adipocytes.
[0435] In an eighteenth aspect, the invention features the knockout
rat according to the method of the seventeenth aspect.
[0436] In a nineteenth aspect, the invention features a method of
using the knockout rat of the eighteenth aspect to identify whether
a candidate compound has therapeutic efficacy for the treatment of
a metabolic-related disorder selected from the group consisting
of:
[0437] (a) dyslipidemia;
[0438] (b) atherosclerosis;
[0439] (c) coronary heart disease;
[0440] (d) stroke;
[0441] (e) insulin resistance; and
[0442] (f) type 2 diabetes;
comprising the steps of:
[0443] (a') administering or not administering the compound to the
rat; and
[0444] (b') determining whether the disorder is prevented, delayed,
or made less severe on administering the compound compared to not
administering the compound; wherein said determination is
indicative of the compound having therapeutic efficacy.
[0445] In a twentieth aspect, the invention features an isolated,
purified or recombinant RUP25 polynucleotide selected from the
group consisting of:
[0446] (a) a polynucleotide comprising a contiguous span of at
least 75 nucleotides of SEQ.ID. NO.:35, SEQ. ID. NO.:136 or SEQ.
ID. NO.:138, or an allelic variant of said polynucleotide;
[0447] (b) a polynucleotide comprising a contiguous span of at
least 150 nucleotides of SEQ. ID. NO.:35, SEQ. ID. NO.:136 or SEQ.
ID. NO.:138, or an allelic variant of said polynucleotide;
[0448] (c) a polynucleotide comprising a contiguous span of at
least 250 nucleotides of SEQ. ID. NO.:35, SEQ. ID. NO.:136 or SEQ.
ID. NO.:138, or an allelic variant of said polynucleotide;
[0449] (d) a polynucleotide comprising a contiguous span of at
least 350 nucleotides of SEQ. ID. NO.:35, SEQ. ID. NO.:136 or SEQ.
ID. NO.:138, or an allelic variant of said polynucleotide;
[0450] (e) a polynucleotide comprising a contiguous span of at
least 500 nucleotides of SEQ. ID. NO.:35, SEQ. ID. NO.:136 or SEQ.
ID. NO.:138, or an allelic variant of said polynucleotide;
[0451] (f) a polynucleotide comprising a contiguous span of at
least 750 nucleotides of SEQ. ID. NOs.:35, SEQ. ID. NO.:136 or SEQ.
ID. NO.:138, or an allelic variant of said polynucleotide;
[0452] (g) a polynucleotide comprising a contiguous span of at
least 1000 nucleotides of SEQ. ID. NO.:35, SEQ. ID. NO.:136 or SEQ.
ID. NO.:138, or an allelic variant of said polynucleotide;
[0453] (h) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 20 amino acids of SEQ. ID. NO.:36, SEQ.
ID. NO: 137 or SEQ. ID. NO.:139 or an allelic variant of said
polypeptide;
[0454] (i) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 30 amino acids of SEQ. ID. NO.:36, SEQ.
ID. NO.:137 or SEQ. ID. NO.:139 or an allelic variant of said
polypeptide;
[0455] (j) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 40 amino acids of SEQ. ID. NO.:36, SEQ.
ID. NO.:137 or SEQ. ID. NO.:139 or an allelic variant of said
polypeptide;
[0456] (k) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 50 amino acids of SEQ. ID. NO.:36, SEQ.
ID. NO.:137 or SEQ. ID. NO.:139 or an allelic variant of said
polypeptide;
[0457] (l) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 75 amino acids of SEQ. ID. NO.:36, SEQ.
ID. NO.:137 or SEQ. ID. NO.:139 or an allelic variant of said
polypeptide;
[0458] (m) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 100 amino acids of SEQ. ID. NO.:36,
SEQ. ID. NO.:137 or SEQ. ID. NO.:139 or an allelic variant of said
polypeptide;
[0459] (n) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 150 amino acids of SEQ. ID. NO.:36,
SEQ. ID. NO.:137 or SEQ. ID. NO.:139 or an allelic variant of said
polypeptide;
[0460] (O) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 200 amino acids of SEQ. ID. NO.:36,
SEQ. ID. NO.:137 or SEQ. ID. NO.:139 or an allelic variant of said
polypeptide;
[0461] (p) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 250 amino acids of SEQ. ID. NO.:36,
SEQ. ID. NO.:137 or SEQ. ID. NO.:139 or an allelic variant of said
polypeptide; and
[0462] (q) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 300 amino acids of SEQ.ID. NO.:36, SEQ.
ID. NO.:137 or SEQ. ID. NO.:139 or an allelic variant of said
polypeptide.
[0463] The invention also relates to an isolated, purified or
recombinant RUP25 polynucleotide wherein said polynucleotide is
selected from the group consisting of:
[0464] (a) a polynucleotide comprising a nucleotide sequence
selected from the group consisting of SEQ. ID. NO.:35, SEQ. ID.
NO.:136 and SEQ. ID. NO.:138 or an allelic variant of said
polynucleotide;
[0465] (b) a polynucleotide selected from the group consisting of
the polynucleotide of SEQ. ID. NO.:35, the polynucleotide of SEQ.
ID. NO.:136 and the polynucleotide of SEQ. ID. NO.:138, or an
allelic variant of said polynucleotide;
[0466] (c) a polynucleotide comprising a nucleotide sequence
encoding a polypeptide having an amino acid sequence selected from
the group consisting of SEQ. ID. NO.:36, SEQ. ID. NO.:137 and SEQ.
ID. NO.:139 or an allelic variant of said polypeptide; and
[0467] (d) a polynucleotide encoding a polypeptide having an amino
acid sequence selected from the group consisting of SEQ. ID.
NO.:36, SEQ. ID. NO.:137 and SEQ. ID. NO.:139 or an allelic variant
of said polypeptide.
[0468] In preferred embodiments, said isolated, purified or
recombinant polynucleotide comprises at least 8 contiguous
nucleotides of a polynucleotide of the present invention. In other
preferred embodiments, said isolated, purified or recombinant
polynucleotide comprises at least 10, 12, 15, 18, 20, 25, 28, 30,
35, 40, 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or
1000 contiguous nucleotides of a polynucleotide of the present
invention. Preferably said polynucleotide encodes full-length RUP25
polypeptide or a biologically active fragment thereof.
[0469] The polynucleotides of the present invention include genomic
polynucleotides comprising RUP25 polynucleotides of the
invention.
[0470] The present invention also relates to a polynucleotide
encoding a fusion protein, wherein said fusion protein comprises an
RUP25 polypeptide of the invention fused to a heterologous
polypeptide. In a preferred embodiment, said polypeptide of the
invention is constitutively active and said heterologous
polypeptide is a G protein. In other embodiments, said heterologous
polypeptide provides an antigenic epitope. In a preferred
embodiment, said heterologous polypeptide provides a hemaglutinin
(HA) antigenic epitope. Methods relating to a polynucleotide
encoding a fusion protein are well known to those of ordinary skill
in the art.
[0471] The polynucleotides of the present invention also include
variant polynucleotides at least 60%, 70%, 80%, 90%, 95%, 96%, 97%,
98% or 99% identical to an RUP25 polynucleotide of the invention.
In a particularly preferred embodiments, polynucleotide sequence
homologies are evaluated using the Basic Local Alignment Search
Tool ("BLAST"), which is well known in the art [See, e.g., Karlin
and Altschul, Proc Natl Acad Sci USA (1990) 87:2264-8; Altschul et
al., J Mol Biol (1990) 215:403-410; Altschul et al., Nature
Genetics (1993) 3:266-72; and Altschul et al., Nucleic Acids Res
(1997) 25:3389-3402; the disclosures of which are incorporated by
reference in their entirety].
[0472] In further preferred embodiments, the invention features the
complement of said polynucleotide.
[0473] In a twenty-first aspect, the invention features an
isolated, purified or recombinant RUP25 polypeptide selected from
the group consisting of:
[0474] (a) a polypeptide comprising a contiguous span of at least
20 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID.
NO.:139;
[0475] (b) a polypeptide comprising a contiguous span of at least
30 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID.
NO.:139;
[0476] (c) a polypeptide comprising a contiguous span of at least
40 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID.
NO.:139;
[0477] (d) a polypeptide comprising a contiguous span of at least
50 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID.
NO.:139;
[0478] (e) a polypeptide comprising a contiguous span of at least
75 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID.
NO.:139;
[0479] (f) a polypeptide comprising a contiguous span of at least
100 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID.
NO.:139;
[0480] (g) a polypeptide comprising a contiguous span of at least
150 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID.
NO.:139;
[0481] (h) a polypeptide comprising a contiguous span of at least
200 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID.
NO.:139;
[0482] (i) a polypeptide comprising a contiguous span of at least
250 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID.
NO.:139; and
[0483] (j) a polypeptide comprising a contiguous span of at least
300 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID.
NO.:139; or an allelic variant of said polypeptide.
[0484] The invention also relates to an isolated, purified or
recombinant RUP25 polypeptide wherein said polypeptide is selected
from the group consisting of:
[0485] (a) a polypeptide comprising an amino acid sequence selected
from the group consisting of SEQ. ID. NO.:36, SEQ. ID. NO.:137 and
SEQ. ID. NO.:139; and
[0486] (b) a polypeptide selected from the group consisting of the
polypeptide of SEQ. ID. NO.:36, the polypeptide of SEQ. ID. NO: 137
and the polypeptide of SEQ. ID. NO.:139; or an allelic variant, a
biologically active mutant, or a biologically active fragment of
said polypeptide.
[0487] In preferred embodiments, said isolated, purified or
recombinant polypeptide comprises at least 6 contiguous amino acids
of an RUP25 polypeptide of the invention. In further embodiments,
said isolated, purified or recombinant polypeptide comprises at
least 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 125, 150,
175, 200, 225, 250, 275 or 300 contiguous amino acids of a
polypeptide of the present invention. Preferably, said polypeptide
is full-length RUP25 polypeptide or an active fragment thereof.
[0488] The present invention also relates to a fusion protein,
wherein said fusion protein comprises an RUP25 polypeptide of the
invention fused to a heterologous polypeptide. In a preferred
embodiment, said polypeptide of the invention is constitutively
active and said heterologous polypeptide is a G protein. In other
preferred embodiments, said heterologous polypeptide provides an
antigenic epitope. In particularly preferred embodiment, said
heterologous polypeptide provides a hemaglutinin (HA) antigenic
epitope. Methods relating to a fusion protein are well known to
those of ordinary skill in the art.
[0489] The polypeptides of the present invention also include
variant polypeptides at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identical to an RUP25 polypeptide of the
invention. In a particularly preferred embodiments, polypeptide
sequence homologies are evaluated using the Basic Local Alignment
Search Tool ("BLAST"), which is well known in the art [See, e.g.,
Karlin and Altschul, Proc Natl Acad Sci USA (1990) 87:2264-8;
Altschul et al., J Mol Biol (1990) 215:403410; Altschul et al.,
Nature Genetics (1993) 3:266-72; and Altschul et al., Nucleic Acids
Res (1997) 25:3389-3402; the disclosures of which are incorporated
by reference in their entirety].
[0490] In an twenty-second aspect, the invention features a
composition comprising, consisting essentially of, or consisting of
the RUP25 polypeptide of the twenty-first aspect.
[0491] In a twenty-third aspect, the invention features a
recombinant vector, said vector comprising, consisting essentially
of, or consisting of the polynucleotide of the twentieth aspect. In
some preferred embodiments, said vector is a targeting vector used
in a method of inactivating a gene encoding a nicotinic acid GPCR
of the invention. In other preferred embodiments, said vector is
used in a method of transient or stable transfection.
[0492] In particularly preferred embodiment, said vector is an
expression vector for the expression of a nicotinic acid GPCR in a
recombinant host cell wherein said expression vector comprises,
consists essentially of, or consists of the polynucleotide of the
twentieth aspect.
[0493] Although a variety of expression vectors are available to
those in the art, for purposes of utilization for both the
endogenous and non-endogenous human, mouse and rat GPCRs, it is
most preferred that the vector utilized be pCMV. In some
alternative embodiments as relates to said human, mouse and rat
nicotinic acid GPCRs, it is preferred that the vector utilized be
an adenoviral expression vector.
[0494] In a twenty-fourth aspect, the invention features a
prokaryotic or eukaryotic host cell comprising, consisting
essentially of, or consisting of the recombinant vector of the
twenty-third aspect. In some preferred embodiments, said host cell
is a eukaryotic embryonic stem cell wherein said vector of the
twenty-third aspect has been used in a method to inactivate a gene
encoding a nicotinic acid GPCR of the invention within said cell.
In some other preferred embodiments, said host cell is a eukaryotic
embryonic somatic cell wherein said vector of the twenty-third
aspect has been used in a method to inactivate a gene encoding a
nicotinic acid GPCR of the invention within said cell. In other
preferred embodiments, said host cell is prokaryotic and has been
transformed using the vector of the twenty-third aspect. In further
preferred embodiments, said host cell is eukaryotic and has been
transiently transfected using the vector of the twenty-third
aspect. In other further preferred embodiments, said host cell is
eukaryotic and has been stably transfected using the vector of the
twenty-third aspect.
[0495] In particularly preferred embodiment, said host cell
expresses a recombinant nicotinic acid GPCR wherein said host cell
comprises, consists essentially of, or consists of the expression
vector of the twenty-third aspect.
[0496] A further embodiment includes a prokaryotic or eukaryotic
host cell recombinant for the polynucleotide of the twentieth
aspect.
[0497] In some embodiments the host cell is eukaryotic, more
preferably, mammalian, and more preferably selected from the group
consisting of 293, 293T, CHO, and COS-7 cells. In other
embodiments, the host cell is eukaryotic, more preferably
melanophore.
[0498] In a twenty-fifth aspect, the invention features a process
for the expression of a nicotinic acid GPCR in a recombinant host
cell comprising the steps of:
[0499] (a) transfecting the expression vector of the twenty-third
aspect into a suitable host cell; and
[0500] (b) culturing the host cells under conditions which allow
expression of the nicotinic acid GPCR protein from the expression
vectors.
[0501] In a twenty-sixth aspect, the invention features an antibody
that specifically binds to the polypeptide of the twenty-first
aspect. In some preferred embodiments, the antibody is monoclonal.
In some embodiments, the antibody is polyclonal.
[0502] In a twenty-seventh aspect, the invention features a method
of binding the polypeptide of the twenty-first aspect to the
antibody of the twenty-sixth aspect, comprising contacting said
antibody with said polypeptide under conditions in which said
antibody can specifically bind to said polypeptide.
[0503] In a twenty-eighth aspect, the invention features a method
of detecting a nicotinic acid GPCR polypeptide in a biological
sample obtained from an individual comprising the steps of: [0504]
(a) obtaining said biological sample from said individual; [0505]
(b) contacting said biological sample with the antibody of the
twenty-sixth aspect; and [0506] (c) detecting the presence or
absence of binding of said antibody to said biological sample;
wherein a detection of said binding is indicative of the receptor
polypeptide being expressed in said biological sample.
[0507] In preferred embodiments, said detecting is through the use
of an enzyme-labeled secondary reagent. In other preferred
embodiments, said detecting is through the use of a
fluorophore-labeled secondary reagent. In other preferred
embodiments, said detecting is through the use of a
radioisotope-labeled secondary reagent. In other embodiments, the
antibody is directly labeled with enzyme, fluorophore or
radioisotope.
[0508] In other preferred embodiments, said biological sample is
taken from adipose, skin or blood.
[0509] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[0510] In further embodiments, said individual has a disorder of
lipid metabolism selected from the group consisting of:
[0511] (a) elevated level of plasma triglycerides;
[0512] (b) elevated level of plasma free fatty acids;
[0513] (c) elevated level of plasma cholesterol;
[0514] (d) elevated level of LDL-cholesterol;
[0515] (e) reduced level of HDL-cholesterol;
[0516] (f) elevated total cholesterol/HDL-cholesterol ratio;
and
[0517] (g) reduced level of plasma adiponectin.
[0518] In other preferred embodiments, said disorder in lipid
metabolism is an elevated postprandial increase in plasma free
fatty acids due to a high fat meal or a progression from impaired
glucose tolerance to insulin resistance.
[0519] In further embodiments, said individual has a
metabolic-related disorder selected from the group consisting
of:
[0520] (a) dyslipidemia;
[0521] (b) atherosclerosis;
[0522] (c) coronary heart disease;
[0523] (d) stroke;
[0524] (e) insulin resistance; and
[0525] (f) type 2 diabetes.
[0526] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[0527] In other embodiments, said method further comprises the step
of comparing the level of detection of said binding for a first
individual to the level of detection of said binding for a second
individual.
[0528] In a twenty-ninth aspect, the invention features a method of
detecting expression of a gene encoding a nicotinic acid GPCR in a
biological sample obtained from an individual comprising the steps
of: [0529] (a) obtaining said biological sample from said
individual; [0530] (b) contacting said biological sample with the
complementary polynucleotide of the twentieth aspect, optionally
labeled, under conditions permissive for hybridization; and [0531]
(c) detecting the presence or absence of said hybridization between
said complementary polynucleotide and an RNA species within said
sample; wherein a detection of said hybridization is indicative of
expression of said GPCR gene in said biological sample.
[0532] In preferred embodiments, the biological sample is taken
from adipose, skin or blood.
[0533] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[0534] In preferred embodiments, said individual has a disorder of
lipid metabolism selected from the group consisting of:
[0535] (a) elevated level of plasma triglycerides;
[0536] (b) elevated level of plasma free fatty acids;
[0537] (c) elevated level of plasma cholesterol;
[0538] (d) elevated level of LDL-cholesterol;
[0539] (e) reduced level of HDL-cholesterol;
[0540] (f) elevated total cholesterol/HDL-cholesterol ratio;
and
[0541] (g) reduced level of plasma adiponectin.
[0542] In other preferred embodiments, said disorder in lipid
metabolism is an elevated postprandial increase in plasma free
fatty acids due to a high fat meal or a progression from impaired
glucose tolerance to insulin resistance.
[0543] In other preferred embodiments, said individual has a
metabolic-related disorder selected from the group consisting
of:
[0544] (a) dyslipidemia;
[0545] (b) atherosclerosis;
[0546] (c) coronary heart disease;
[0547] (d) stroke;
[0548] (e) insulin resistance; and
[0549] (f) type 2 diabetes.
[0550] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[0551] In other embodiments, said method further comprises the step
of comparing the level of detection of said hybridization for a
first individual to the level of detection of said hybridization
for a second individual.
[0552] In some preferred embodiments, said complementary
polynucleotide is a primer and said hybridization is detected by
detecting the presence of an amplification product comprising the
sequence of said primer. In more preferred embodiments, said method
is RT-PCR.
[0553] In a thirtieth aspect, the invention features a GPCR Fusion
Protein construct comprising a constitutively active GPCR and a G
protein, said receptor comprising an amino acid sequence selected
from the group consisting of:
[0554] (a) SEQ. ID. NO.:36 (hRUP25);
[0555] (b) SEQ. ID. NO.:137 (mRUP25); and
[0556] (c) SEQ. ID. NO.:139 (rRUP25);
or an allelic variant or a biologically active fragment of said
amino acid sequence.
[0557] The invention also relates to a GPCR Fusion Protein
construct wherein the isoleucine at amino acid position 230 of SEQ.
ID. NO.:36 is substituted by lysine.
[0558] In a thirty-first aspect, the invention features a method of
binding a known ligand of RUP25 nicotinic acid GPCR to a
polypeptide selected from the group consisting of:
[0559] (a) a polypeptide comprising a contiguous span of at least 6
amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID.
NO.:139;
[0560] (b) a polypeptide comprising a contiguous span of at least
10 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID.
NO.:139;
[0561] (c) a polypeptide comprising a contiguous span of at least
15 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID.
NO.:139;
[0562] (d) a polypeptide comprising a contiguous span of at least
20 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID.
NO.:139;
[0563] (e) a polypeptide comprising a contiguous span of at least
25 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID.
NO.:139;
[0564] (f) a polypeptide comprising a contiguous span of at least
30 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID.
NO.:139;
[0565] (g) a polypeptide comprising a contiguous span of at least
35 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID.
NO.:139;
[0566] (h) a polypeptide comprising a contiguous span of at least
40 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID.
NO.:139;
[0567] (i) a polypeptide comprising a contiguous span of at least
45 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID.
NO.:139; and
[0568] (j) a polypeptide comprising a contiguous span of at least
50 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID.
NO.:139;
or an allelic variant of said polypeptide;
comprising the step of contacting said known ligand with said
polypeptide under conditions which allow said binding to occur.
[0569] In some embodiments, said known ligand is a modulator of the
nicotinic acid GPCR. In some embodiments, said known ligand is an
agonist of the nicotinic acid GPCR. In other embodiments, said
agonist is nicotinic acid or an analog or derivative thereof. In
other embodiments, said agonist is (-)-nicotine or an analog or
derivative thereof. In some embodiments, said known ligand is the
modulator of the second aspect. In some embodiments, said known
ligand is an antibody specific for the GPCR, or a derivative
thereof.
[0570] In other preferred embodiments, said method is used to
identify whether a candidate compound inhibits said binding of said
known ligand to said polypeptide, comprising the steps of: [0571]
(a) contacting said polypeptide with said known ligand, optionally
labeled, in the presence or absence of said candidate compound;
[0572] (b) detecting the complex between said known ligand and said
polypeptide; and [0573] (c) determining whether less of said
complex is formed in the presence of the compound than in the
absence of the compound; wherein said determination is indicative
of the candidate compound being an inhibitor of said binding of
said known ligand to said polypeptide.
[0574] In some embodiments, said known ligand is a known modulator
of the nicotinic acid GPCR. In some embodiments, said known ligand
is a modulator of the nicotinic acid GPCR. In some embodiments,
said known ligand is an agonist of the nicotinic acid GPCR. In
embodiments, said agonist is nicotinic acid or an analog or
derivative thereof. In other embodiments, said agonist is
(-)-nicotine or an analog or derivative thereof. In some
embodiments, said known ligand is the modulator of the second
aspect. In some embodiments, said known ligand is an antibody
specific for the GPCR, or a derivative thereof.
[0575] In other preferred embodiments, said method is used to
identify whether a candidate compound is an inhibitor of said
binding of said known ligand to said polypeptide, comprising the
steps of:
[0576] (a) contacting said polypeptide with said known ligand,
optionally labeled, in the presence separately of a plurality of
concentrations of said candidate compound for a time sufficient to
allow equilibration of binding;
[0577] (b) measuring unbound ligand and bound ligand; and
[0578] (c) determining K.sub.i for the candidate compound;
[0579] wherein a K.sub.i value of less than 50 .mu.M is indicative
of the candidate compound being an inhibitor of said binding of
said known ligand to said polypeptide. Preferably said K.sub.i
value is less than 25 .mu.M, 10 .mu.M, 5 .mu.M, 1 .mu.M, 750 nM,
500 nM, 400 nM, 300 nM, 250 nM, 200 nM, 150 nM, 100 nM, 90 nM, 80
nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM or 10 nM. In preferred
embodiments, K.sub.i determination is made through nonlinear curve
fitting with the program SCTFIT [De Lean et al. (1982) Mol
Pharmacol 21:5-16; cited in Lorenzen et al. (2001) Mol Pharmacol
59:349-357, the disclosures of which are incorporated by reference
herein in their entireties].
[0580] In some embodiments, said known ligand is a modulator of the
nicotinic acid GPCR. In some embodiments, said known ligand is an
agonist of the nicotinic acid GPCR. In other embodiments, said
agonist is nicotinic acid or an analog or derivative thereof. In
other embodiments, said agonist is (-)-nicotine or an analog or
derivative thereof. In some embodiments, said known ligand is the
modulator of the second aspect. In some embodiments, said known
ligand is an antibody specific for the GPCR, or a derivative
thereof.
[0581] In a thirty-second aspect, the invention features a method
of binding an optionally labeled affinity reagent specific for a
nicotinic acid GPCR to said receptor in a biological sample, said
receptor comprising an amino acid sequence selected from the group
consisting of:
[0582] (a) SEQ. ID. NO.:36 (hRUP25);
[0583] (b) SEQ. ID. NO.:137 (mRUP25); and
[0584] (c) SEQ. ID. NO.:139 (rRUP25);
or an allelic variant, a biologically active mutant, or a
biologically active fragment of said amino acid sequence,
comprising the steps of:
[0585] (a') obtaining said biological sample;
[0586] (b') contacting the affinity reagent with said receptor in
said biological sample; and
[0587] (c') detecting the complex of said affinity reagent with
said receptor.
[0588] In some embodiments, the nicotinic acid GPCR has an amino
acid sequence selected from the group consisting of:
[0589] (a) SEQ. ID. NO.:36 (hRUP25);
[0590] (b) SEQ. ID. NO.:137 (mRUP25); and
[0591] (c) SEQ. ID. NO.:139 (rRUP25); [0592] or an allelic variant,
a biologically active mutant, or a biologically active fragment of
said amino acid sequence.
[0593] In some embodiments, the nicotinic acid GPCR comprises an
active fragment of said amino acid sequence.
[0594] In some embodiments, the nicotinic acid GPCR is
endogenous.
[0595] In some embodiments, the nicotinic acid GPCR is
recombinant.
[0596] In some embodiments, said biologically active mutant is CART
or EFA. In preferred embodiments, said CART mutant has the amino
acid sequence of SEQ. ID. NO.:36 further substituted at amino acid
position 230 with lysine in place of isoleucine. In preferred
embodiments, said EFA mutant has the amino acid sequence of SEQ.
ID. NO.:159.
[0597] In preferred embodiments, said G protein is Gi.
[0598] In some embodiments, said affinity reagent is a modulator of
the GPCR. In some embodiments, said affinity reagent is an agonist
of the GPCR. In some embodiments, said affinity reagent is
nicotinic acid or an analog or derivative thereof. In some
embodiments, said affinity reagent is (-)-nicotine or an analog or
derivative thereof. In some embodiments, said affinity reagent is
the modulator of the second aspect. In some embodiments, said known
ligand is an antibody specific for the GPCR, or a derivative
thereof.
[0599] In further preferred embodiments, said affinity reagent
comprises a label selected from the group consisting of:
[0600] (a) radioisotope;
[0601] (b) enzyme; and
[0602] (c) fluorophore.
[0603] In preferred embodiments, said radioisotope is .sup.3H.
[0604] In a thirty-third aspect, the invention features the method
of the thirty-second aspect further comprising the step of
comparing the level of detection of said complex in a first
biological sample to a second level of detection of said complex in
a second biological sample.
[0605] In a thirty-fourth aspect, the invention features the method
of the thirty-third aspect wherein the relationship between said
first and second biological samples is selected from the group
consisting of:
[0606] (a) said second biological sample is a replicate of said
first biological sample;
[0607] (b) said first biological sample was obtained prior to an
experimental intervention whereas said second biological sample was
obtained after the experimental intervention, from the same
individual;
[0608] (c) said second biological sample was obtained at a
different time point after an experimental intervention than was
said first biological sample, from the same individual;
[0609] (d) said second biological sample corresponds to a different
subcellular compartment than does said first biological sample;
[0610] (e) said second biological sample represents a different
cell type than does said first biological sample;
[0611] (f) said second biological sample corresponds to a different
tissue than does said first biological sample;
[0612] (g) said second biological sample was obtained from a
different individual than was said first biological sample;
[0613] (h) said second biological sample was obtained at a
different point in time than was said first biological sample, from
the same individual;
[0614] (i) said first biological samples was obtained from a normal
individual, whereas said second biological sample was obtained from
an individual having a metabolic-related disorder;
[0615] (j) said first biological sample was obtained from a normal
individual, whereas said second biological sample was obtained from
an individual having a disorder in lipid metabolism;
[0616] (k) said first biological sample was obtained before a
therapeutic intervention whereas said second biological sample was
obtained after the therapeutic intervention, from the same
individual;
[0617] (l) said second biological sample was obtained at a
different time point after therapeutic intervention than was said
first biological sample, from the same individual; and
[0618] (m) said first biological sample was not exposed to a
compound, whereas said second biological sample was exposed to said
compound.
[0619] In a thirty-fifth aspect, the invention features a method of
identifying whether a candidate compound is a modulator of an
antilipolytic GPCR, said receptor comprising the amino acid
sequence of SEQ. ID. NO.:135 (hRUP38);
or an allelic variant, a biologically active mutant, or a
biologically active fragment of said amino acid sequence;
comprising the steps of:
[0620] (a) contacting the candidate compound with the receptor;
[0621] (b) determining whether the receptor functionality is
modulated;
wherein a change in receptor functionality is indicative of the
candidate compound being a modulator of an antilipolytic GPCR.
[0622] In some embodiments, said antilipolytic GPCR is
endogenous.
[0623] In some preferred embodiments, said antilipolytic GPCR is
recombinant.
[0624] Preferred said identified modulator binds to said GPCR.
[0625] In some embodiments, said contacting is carried out in the
presence of a known ligand of the GPCR. In some embodiments, said
known ligand is an agonist of the GPCR.
[0626] The invention also relates to a method of identifying
whether a candidate compound is a modulator of lipolysis,
comprising the steps of: [0627] (a) contacting the candidate
compound with a GPCR comprising the amino acid sequence of SEQ. ID.
NO.:135 (hRUP38); or an allelic variant, a biologically active
mutant, or a biologically active fragment of said amino acid
sequence; and [0628] (b) determining whether the receptor
functionality is modulated; wherein a change in receptor
functionality is indicative of the candidate compound being a
modulator of lipolysis.
[0629] In some embodiments, said GPCR is endogenous.
[0630] In some preferred embodiments, said GPCR is recombinant.
[0631] Preferred said identified modulator binds to said GPCR.
[0632] In some embodiments, said contacting is carried out in the
presence of a known ligand of the GPCR. In some embodiments, said
known ligand is an agonist of the GPCR.
[0633] The invention also relates to a method of determining
whether a candidate compound is a modulator of an antilipolytic
GPCR, comprising the steps of: [0634] (a) culturing antilipolytic
GPCR-expressing host cells under conditions that would allow
expression of a recombinant antilipolytic GPCR, said host cells
being transfected with a polynucleotide encoding said recombinant
antilipolytic GPCR comprising the amino acid sequence of SEQ. ID.
NO.:135 (hRUP38); or an allelic variant, a biologically active
mutant, or a biologically active fragment of said amino acid
sequence; [0635] (b) contacting the antilipolytic GPCR-expressing
host cells of step (a) with the candidate compound; [0636] (c)
contacting control host cells with the candidate compound of step
(b), wherein said control host cells do not express recombinant
antilipolytic GPCR protein; [0637] (d) measuring the modulating
effect of the candidate compound which interacts with the
recombinant antilipolytic GPCR from the host cells of step (a) and
control host cells of step (c); and [0638] (e) comparing the
modulating effect of the test compound on the host cells and
control host cells.
[0639] The invention also relates to a method of determining
whether a candidate compound is a modulator of an antilipolytic
GPCR, comprising the steps of: [0640] (a) culturing antilipolytic
GPCR-expressing host cells under conditions that would allow
expression of a recombinant antilipolytic GPCR, said host cells
being transfected with a polynucleotide encoding said recombinant
antilipolytic GPCR comprising the amino acid sequence of SEQ. ID.
NO.:135 (hRUP38); or an allelic variant, a biologically active
mutant, or a biologically active fragment of said amino acid
sequence; [0641] (b) contacting a first population of antilipolytic
GPCR-expressing cells of step (a) with a known ligand of said
antilipolytic GPCR; [0642] (c) contacting a second population of
antilipolytic GPCR-expressing cells of step (a) with the candidate
compound and with the known antilipolytic GPCR ligand; [0643] (d)
contacting control host cells with the candidate compound of step
(c), wherein said control host cells do not express recombinant
antilipolytic GPCR protein; [0644] (e) measuring the modulating
effect of the candidate compound, which interacts with recombinant
antilipolytic GPCR, in the presence and absence of the known anti
lipolytic GPCR ligand, from the cells of step (b), step (c) and
step (d); and [0645] (f) comparing the modulating effect of the
candidate compound as determined from step (b), step (c) and step
(d).
[0646] In some embodiments, said known ligand is an agonist of the
GPCR.
[0647] The invention also relates to a method of determining
whether a candidate compound is a modulator of an antilipolytic
GPCR, comprising the steps of: [0648] (a) culturing antilipolytic
GPCR-expressing host cells under conditions that would allow
expression of a recombinant antilipolytic GPCR, said host cells
being transfected with a polynucleotide encoding said recombinant
antilipolytic GPCR comprising the amino acid sequence of SEQ. ID.
NO.:135 (hRUP38); or an allelic variant, a biologically active
mutant, or a biologically active fragment of said amino acid
sequence; [0649] (b) contacting a first population of the
antilipolytic GPCR-expressing host cells of step (a) with the
candidate compound; [0650] (c) not contacting a second population
of the antilipolytic GPCR-expressing cells of step (a) with the
candidate compound of step (b); [0651] (d) contacting control host
cells to the candidate compound of step (b), wherein said control
host cells do not express recombinant antilipolytic GPCR protein;
[0652] (e) measuring the modulating effect of the candidate
compound, which interacts with recombinant antilipolytic GPCR
protein, from the cells of step (b) and step (c) and from the cells
of step (d); and [0653] (f) comparing the modulating effect of the
candidate compound as determined from step (b) and step (c) and
from step (d).
[0654] In some embodiments, the antilipolytic GPCR has the amino
acid sequence of SEQ. ID. NO.:135 (hRUP38); or an allelic variant,
a biologically active mutant, or a biologically active fragment of
said amino acid sequence.
[0655] In some embodiments, the antilipolytic GPCR comprises a
biologically active fragment of said amino acid sequence.
[0656] In some embodiments, said biologically active mutant is CART
or EFA. In preferred embodiments, said CART mutant has the amino
acid sequence of SEQ. ID. NO.:135 further substituted at amino acid
position 230 with lysine in place of isoleucine.
[0657] In preferred embodiments, said G protein is Gi.
[0658] In other preferred embodiments, said determining is through
the use of a Melanophore assay.
[0659] In other preferred embodiments, said determining is through
the measurement of the level of a second messenger selected from
the group consisting of cyclic AMP (cAMP), cyclic GMP (cGMP),
inositol triphosphate (IP.sub.3), diacylglycerol (DAG), and
Ca.sup.2+. In further preferred embodiments, said second messenger
is cAMP. In more preferred embodiments, the level of the cAMP is
reduced. In some embodiments, said measurement of cAMP is carried
out with membrane comprising said GPCR.
[0660] In other preferred embodiments, said determining is through
the measurement of an activity up-regulated or down-regulated by a
reduction in intracellular cAMP level. In further preferred
embodiments, said down-regulated activity is intracellular
lipolysis. In other further preferred embodiments, said
down-regulated activity is hormone sensitive lipase activity. In
other further preferred embodiments, said up-regulated activity is
adiponectin secretion.
[0661] In other preferred embodiments, said determining is through
CRE-reporter assay. In preferred embodiments, said reporter is
luciferase. In some embodiments, said reporter is
.beta.-galactosidase.
[0662] In other embodiments, said recombinant host cell further
comprises promiscuous G alpha 15/16 or chimeric Gq/Gi alpha subunit
and said determining is through measurement of intracellular
Ca.sup.2+. In preferred embodiments, said Ca.sup.2+ measurement is
carried out by FLIPR.
[0663] In other embodiments, said recombinant host cell further
comprises promiscuous G alpha 15/16 or chimeric Gq/Gi alpha subunit
and said determining is through measurement of intracellular
IP.sub.3.
[0664] In other preferred embodiments, said determining is through
the measurement of GTP.gamma.S binding to membrane comprising said
GPCR. In further preferred embodiments, said GTP.gamma.S is labeled
with [.sup.35S].
[0665] In other preferred embodiments, said method further
comprises the step of comparing the modulation of the receptor
caused by the candidate compound to a second modulation of the
receptor caused by contacting the receptor with a known modulator
of the receptor. In some preferred embodiments, said known
modulator is an agonist.
[0666] In a thirty-sixth aspect, the invention features a modulator
of an antilipolytic GPCR identified according to the method of the
thirty-fifth aspect.
[0667] In some preferred embodiments, said modulator is selected
from the group consisting of agonist, partial agonist, inverse
agonist and antagonist. More preferably, said modulator is an
agonist. In some embodiments, said modulator is a partial
agonist.
[0668] In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding assay
carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP38 polypeptide having the amino acid
sequence of SEQ. ID. NO.:135. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 900 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 800 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 700 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 600 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 550 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 500 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 450 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 400 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 350 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 300 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 250 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 200 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 150 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 100 .mu.M in said assay. In some preferred
embodiments, said modulator is an agonist with an EC.sub.50 in said
assay of less than a value selected from the interval of 600 .mu.M
to 1000 .mu.M.
[0669] In some embodiments, said modulator is selective for the
GPCR.
[0670] In some embodiments, said modulator is antilipolytic.
[0671] In some embodiments, said modulator is orally bioavailable.
In some embodiments, said oral bioavailability is at least 1%, at
least 5%, at least 10%, at least 15%, at least 20%, at least 25%,
at least 30%, at least 35%, at least 40%, or at least 45% relative
to intraperitoneal administration. In some preferred embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intraperitoneal administration. In
some embodiments, said oral bioavailability is at least 1%, at
least 5%, at least 10%, at least 15%, at least 20%, at least 25%,
at least 30%, at least 35%, at least 40%, or at least 45% relative
to intravenous administration. In some preferred embodiments, said
oral bioavailability is at least 1%, at least 5%, at least 10%, or
at least 15% relative to intravenous administration.
[0672] In highly less preferred embodiments, said modulator is an
antibody or derivative thereof.
[0673] In a thirty-seventh aspect, the invention features the
method of the thirty-fifth aspect, wherein said candidate compound
is an agonist of hRUP25 GPCR comprising the amino acid sequence of
SEQ. ID. NO.:36 and wherein said method further comprises the step
of comparing the modulation of hRUP38 GPCR comprising the amino
acid sequence of SEQ. ID. NO.:135 caused by said agonist to a
second modulation of hRUP38 GPCR comprising a variant of said amino
acid sequence caused by contacting the variant hRUP38 GPCR with
said agonist.
[0674] In preferred embodiments, said variant amino acid sequence
is identical to hRUP38 polypeptide of SEQ. ID. NO.:135, further
comprising a single amino acid substitution selected from the group
consisting of:
[0675] (a) V for A at amino acid position 27 of SEQ. ID.
NO.:135;
[0676] (b) L for V at amino acid position 83 of SEQ. ID.
NO.:135;
[0677] (c) N for Y at amino acid position 86 of SEQ. ID.
NO.:135;
[0678] (d) W for S at amino acid position 91 of SEQ. ID.
NO.:135;
[0679] (e) K for N at amino acid position 94 of SEQ. ID.
NO.:135;
[0680] (f) M for V at amino acid position 103 of SEQ. ID.
NO.:135;
[0681] (g) L for F at amino acid position 107 of SEQ. ID.
NO.:135;
[0682] (h) R for W at amino acid position 142 of SEQ. ID.
NO.:135;
[0683] (i) I for V at amino acid position 156 of SEQ. ID.
NO.:135;
[0684] (j) M for L at amino acid position 167 of SEQ. ID.
NO.:135;
[0685] (k) P for L at amino acid position 168 of SEQ. ID.
NO.:135;
[0686] (l) G for P at amino acid position 173 of SEQ. ID.
NO.:135;
[0687] (m) L for V at amino acid position 176 of SEQ. ID.
NO.:135;
[0688] (n) S for 1 at amino acid position 178 of SEQ. ID.
NO.:135;
[0689] (O) Q for Rat amino acid position 187 of SEQ. ID.
NO.:135;
[0690] (p) F for L at amino acid position 198 of SEQ. ID. NO.:135;
and
[0691] (q) P for N at amino acid position 363 of SEQ. ID.
NO.:135.
[0692] In particularly preferred embodiments, said method is used
to identify whether said substituted amino acid additionally found
at the identical position within SEQ. ID. NO.:36 is necessary for
modulation of said hRUP25 GPCR by said agonist, comprising the
steps of:
[0693] (a) determining the level of modulation of said hRUP38 GPCR
by said agonist; and
[0694] (b) determining the level of modulation of said variant
hRUP38 GPCR by said agonist;
wherein if said level of modulation for (b) is greater than said
level of modulation for (a), then said substituted amino acid is
necessary for modulation of said hRUP25 GPCR by said agonist.
[0695] In a thirty-eighth aspect, the invention features a method
of modulating the activity of an antilipolytic GPCR, said receptor
comprising the amino acid sequence of SEQ. ID. NO.:135 (hRUP38); or
an allelic variant, a biologically active mutant, or a biologically
active fragment of said amino acid sequence; comprising the step of
contacting the receptor with the modulator of the thirty-sixth
aspect.
[0696] In some embodiments, the antilipolytic GPCR has the amino
acid sequence of SEQ. ID. NO.:135 (hRUP38); or an allelic variant,
a biologically active mutant, or a biologically active fragment of
said amino acid sequence.
[0697] In some embodiments, the antilipolytic GPCR comprises an
active fragment of said amino acid sequence.
[0698] In some embodiments, the antilipolytic GPCR is
endogenous.
[0699] In some embodiments, the antilipolytic GPCR is
recombinant.
[0700] In some embodiments, said biologically active mutant is CART
or EFA. In preferred embodiments, said CART mutant has the amino
acid sequence of SEQ. ID. NO.:135 further substituted at amino acid
position 230 with lysine in place of isoleucine.
[0701] In preferred embodiments, said G protein is Gi.
[0702] In some preferred embodiments, said modulator is an
agonist.
[0703] In preferred embodiments, said modulator is selective for
the GPCR.
[0704] In other preferred embodiments, said contacting comprises
administration of the modulator to a membrane comprising the
receptor.
[0705] In other preferred embodiments, said contacting comprises
administration of the modulator to a cell or tissue comprising the
receptor.
[0706] In other preferred embodiments, said contacting comprises
administration of the modulator to an individual comprising the
receptor. In more preferred embodiments, said individual is a
mammal. In other more preferred embodiments, said mammal is a
horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat, non-human
primate or human. Yet more preferred is mouse, rat or human. Most
preferred is human.
[0707] In some preferred embodiments, said modulator is selective
for the GPCR.
[0708] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[0709] In some preferred embodiments, said modulator is
antilipolytic.
[0710] In some preferred embodiments, said modulator is an
agonist.
[0711] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP38 polypeptide having the amino acid
sequence of SEQ. ID. NO.:135. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 900 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 800 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 700 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 600 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 550 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 500 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 450 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 400 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 350 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 300 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 250 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 200 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 150 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 100 .mu.M in said assay. In some preferred
embodiments, said modulator is an agonist with an EC.sub.50 in said
assay of less than a value selected from the interval of 600 .mu.M
to 1000 .mu.M.
[0712] In some preferred embodiments, said administration is
oral.
[0713] In preferred embodiments, said modulator is an agonist and
said individual is in need of prevention of or treatment for a
metabolic-related disorder selected from the group consisting
of:
[0714] (a) dyslipidemia;
[0715] (b) atherosclerosis;
[0716] (c) coronary heart disease;
[0717] (d) stroke;
[0718] (e) insulin resistance; and
[0719] (f) type 2 diabetes.
[0720] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[0721] In other embodiments, said modulator is an inverse agonist
and said metabolic-related disorder relates to a low level of
plasma free fatty acids.
[0722] In other preferred embodiments, said modulator is an agonist
and said individual is in need of a change in lipid metabolism
selected from the group consisting of:
[0723] (a) a decrease in the level of plasma triglycerides;
[0724] (b) a decrease in the level of plasma free fatty acids;
[0725] (c) a decrease in the level of plasma cholesterol;
[0726] (d) a decrease in the level of LDL-cholesterol;
[0727] (e) an increase in the level of HDL-cholesterol;
[0728] (f) a decrease in the total cholesterol/HDL-cholesterol
ratio; and
[0729] (g) an increase in the level of plasma adiponectin.
[0730] In other preferred embodiments, said needed change in lipid
metabolism is a decrease in the postprandial increase in plasma
free fatty acids due to a high fat meal or an inhibition of the
progression from impaired glucose tolerance to insulin
resistance.
[0731] In some embodiments, the modulator is an inverse agonist and
the needed change in lipid metabolism is an increase in the level
of plasma free fatty acids.
[0732] In other preferred embodiments, said modulator is an agonist
and said individual is a mouse genetically predisposed to a
metabolic-related disorder selected from the group consisting
of:
[0733] (a) dyslipidemia;
[0734] (b) atherosclerosis;
[0735] (c) coronary heart disease;
[0736] (d) stroke;
[0737] (e) insulin resistance; and
[0738] (f) type 2 diabetes.
[0739] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure.
[0740] In further preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[0741] In further preferred embodiments, said method is used to
identify whether said agonist has therapeutic efficacy for the
treatment of a metabolic-related disorder selected from the group
consisting of:
[0742] (a) dyslipidemia;
[0743] (b) atherosclerosis;
[0744] (c) coronary heart disease;
[0745] (d) stroke;
[0746] (e) insulin resistance; and
[0747] (f) type 2 diabetes;
comprising the steps of:
[0748] (a') administering or not administering said agonist to the
mouse; and
[0749] (b') determining whether the disorder is prevented, delayed,
or made less severe on administering said agonist compared to not
administering said agonist;
wherein said determination is indicative of said agonist having
therapeutic efficacy.
[0750] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[0751] In other preferred embodiments, said modulator is an agonist
and said individual is a rat genetically predisposed to a
metabolic-related disorder selected from the group consisting
of:
[0752] (a) dyslipidemia;
[0753] (b) atherosclerosis;
[0754] (c) coronary heart disease;
[0755] (d) stroke;
[0756] (e) insulin resistance; and
[0757] (f) type 2 diabetes.
[0758] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In further preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[0759] In further preferred embodiments, said method is used to
identify whether said agonist has therapeutic efficacy for the
treatment of a metabolic-related disorder selected from the group
consisting of:
[0760] (a) dyslipidemia;
[0761] (b) atherosclerosis;
[0762] (c) coronary heart disease;
[0763] (d) stroke;
[0764] (e) insulin resistance; and
[0765] (f) type 2 diabetes;
comprising the steps of:
[0766] (a') administering or not administering said agonist to the
rat; and
[0767] (b') determining whether the disorder is prevented, delayed,
or made less severe on administering said agonist compared to not
administering said agonist; wherein said determination is
indicative of said agonist having therapeutic efficacy.
[0768] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[0769] In a thirty-ninth aspect, the invention features a method of
preventing or treating a disorder of lipid metabolism in an
individual comprising contacting a therapeutically effective amount
of the modulator of the thirty-sixth aspect with an antilipolytic
GPCR, said receptor comprising the amino acid sequence of SEQ. ID.
NO.:135 (hRUP38); or an allelic variant or biologically active
fragment of said amino acid sequence.
[0770] In some preferred embodiments, said modulator is selective
for the GPCR.
[0771] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[0772] In some preferred embodiments, said modulator is
antilipolytic.
[0773] In some preferred embodiments, said modulator is an
agonist.
[0774] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP38 polypeptide having the amino acid
sequence of SEQ. ID. NO.:135. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 900 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 800 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 700 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 600 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 550 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 500 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 450 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 400 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 350 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 300 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 250 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 200 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 150 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 100 .mu.M in said assay. In some preferred
embodiments, said modulator is an agonist with an EC.sub.50 in said
assay of less than a value selected from the interval of 600 .mu.M
to 1000 .mu.M.
[0775] In some preferred embodiments, said contacting comprises
oral administration of said modulator to said individual.
[0776] In preferred embodiment, said modulator is an agonist and
said disorder of lipid metabolism is selected from the group
consisting of:
[0777] (a) elevated level of plasma triglycerides;
[0778] (b) elevated level of plasma free fatty acids;
[0779] (c) elevated level of plasma cholesterol;
[0780] (d) elevated level of LDL-cholesterol;
[0781] (e) reduced level of HDL-cholesterol;
[0782] (f) elevated total cholesterol/HDL-cholesterol ratio;
and
[0783] (g) reduced level of plasma adiponectin.
[0784] In a fortieth aspect, the invention features a method of
preventing or treating a metabolic-related disorder in an
individual comprising contacting a therapeutically effective amount
of the modulator of the thirty-sixth aspect with an antilipolytic
GPCR, said receptor comprising the amino acid sequence of SEQ. ID.
NO.:135 (hRUP38); or an allelic variant or biologically active
fragment of said amino acid sequence.
[0785] In some preferred embodiments, said modulator is selective
for the GPCR.
[0786] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[0787] In some preferred embodiments, said modulator is
antilipolytic.
[0788] In some preferred embodiments, said modulator is an
agonist.
[0789] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 Of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP38 polypeptide having the amino acid
sequence of SEQ. ID. NO.:135. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 900 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 800 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 700 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 600 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 550 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 500 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 450 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 400 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 350 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 300 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 250 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 200 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 150 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 100 .mu.M in said assay. In some preferred
embodiments, said modulator is an agonist with an EC.sub.50 in said
assay of less than a value selected from the interval of 600 .mu.M
to 1000 .mu.M.
[0790] In some preferred embodiments, said contacting comprises
oral administration of said modulator to said individual.
[0791] In preferred embodiment, said modulator is an agonist and
said metabolic-related disorder is selected from the group
consisting of:
[0792] (a) dyslipidemia;
[0793] (b) atherosclerosis;
[0794] (c) coronary heart disease;
[0795] (d) stroke;
[0796] (e) insulin resistance; and
[0797] (f) type 2 diabetes.
[0798] In a forty-first aspect, the invention features a method of
preparing a composition which comprises identifying a modulator of
an antilipolytic GPCR and then admixing a carrier and the
modulator, wherein the modulator is identifiable by the method of
the thirty-fifth aspect.
[0799] In some preferred embodiments, said modulator is selected
from the group consisting of agonist, partial agonist, inverse
agonist and antagonist. More preferably, said modulator is an
agonist. In some embodiments, said modulator is a partial
agonist.
[0800] In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding assay
carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP38 polypeptide having the amino acid
sequence of SEQ. ID. NO.:135. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 900 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 800 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 700 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 600 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 550 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 500 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 450 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 400 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 350 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 300 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 250 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 200 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 150 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 100 .mu.M in said assay. In some preferred
embodiments, said modulator is an agonist with an EC.sub.50 in said
assay of less than a value selected from the interval of 600 .mu.M
to 1000 .mu.M.
[0801] In some embodiments, said modulator is selective for the
GPCR.
[0802] In some embodiments, said modulator is antilipolytic.
[0803] In some embodiments, said modulator is orally bioavailable.
In some embodiments, said oral bioavailability is at least 1%, at
least 5%, at least 10%, at least 15%, at least 20%, at least 25%,
at least 30%, at least 35%, at least 40%, or at least 45% relative
to either intraperitoneal or intravenous administration. In some
preferred embodiments, said oral bioavailability is at least 1%, at
least 5%, at least 10%, or at least 15% relative to either
intraperitoneal or intravenous administration.
[0804] In a forty-second aspect, the invention features a
pharmaceutical or physiologically acceptable composition
comprising, consisting essentially of, or consisting of the
modulator of the thirty-sixth aspect. In preferred embodiments,
said modulator is an agonist.
[0805] In some preferred embodiments, said modulator is selective
for the GPCR.
[0806] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[0807] In some preferred embodiments, said modulator is
antilipolytic.
[0808] In some preferred embodiments, said modulator is an
agonist.
[0809] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP38 polypeptide having the amino acid
sequence of SEQ. ID. NO.:135. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 900 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 800 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 700 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 600 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 550 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 500 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 450 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 400 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 350 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 300 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 250 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 200 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 150 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 100 .mu.M in said assay. In some preferred
embodiments, said modulator is an agonist with an EC.sub.50 in said
assay of less than a value selected from the interval of 600 .mu.M
to 1000 .mu.M.
[0810] In a forty-third aspect, the invention features a method of
changing lipid metabolism comprising providing or administering to
an individual in need of said change said pharmaceutical or
physiologically acceptable composition of the forty-second aspect,
said needed change in lipid metabolism selected from the group
consisting of:
[0811] (a) a decrease in the level of plasma triglycerides;
[0812] (b) a decrease in the level of plasma free fatty acids;
[0813] (c) a decrease in the level of plasma cholesterol;
[0814] (d) a decrease in the level of LDL-cholesterol;
[0815] (e) an increase in the level of HDL-cholesterol;
[0816] (f) a decrease in the total cholesterol/HDL-cholesterol
ratio; and
[0817] (g) an increase in the level of plasma adiponectin.
[0818] In preferred embodiments, a therapeutically effective amount
of said pharmaceutical or physiologically acceptable composition is
provided or administered to said individual.
[0819] In some preferred embodiments, said providing or
administering of said pharmaceutical or physiologically acceptable
composition is oral.
[0820] In other preferred embodiments, said needed change in lipid
metabolism is a decrease in the postprandial increase in plasma
free fatty acids due to a high fat meal or an inhibition of the
progression from impaired glucose tolerance to insulin
resistance.
[0821] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[0822] In a forty-fourth aspect, the invention features a method of
preventing or treating a metabolic-related disorder comprising
providing or administering to an individual in need of said
treatment said pharmaceutical or physiologically acceptable
composition of the forty-second aspect, said metabolic-related
disorder selected from the group consisting of:
[0823] (a) dyslipidemia;
[0824] (b) atherosclerosis;
[0825] (c) coronary heart disease;
[0826] (d) stroke;
[0827] (e) insulin resistance; and
[0828] (f) type 2 diabetes.
[0829] In preferred embodiments, a therapeutically effective amount
of said pharmaceutical or physiologically acceptable composition is
provided or administered to said individual.
[0830] In some preferred embodiments, said providing or
administering of said pharmaceutical or physiologically acceptable
composition is oral.
[0831] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, stroke,
Syndrome X, and heart disease. Heart disease includes, but is not
limited to, cardiac insufficiency, coronary insufficiency, and high
blood pressure. In other preferred embodiments, said
metabolic-related disorder is hyperlipidemia.
[0832] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[0833] In an forty-fifth aspect, the invention features a method of
using the modulator of the thirty-sixth aspect for the preparation
of a medicament for the treatment of a disorder in lipid metabolism
in an individual.
[0834] In some preferred embodiments, said modulator is selective
for the GPCR.
[0835] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[0836] In some preferred embodiments, said modulator is
antilipolytic.
[0837] In some preferred embodiments, said modulator is an
agonist.
[0838] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP38 polypeptide having the amino acid
sequence of SEQ. ID. NO.:135. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 900 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 800 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 700 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 600 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 550 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 500 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 450 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 400 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 350 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 300 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 250 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 200 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 150 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 100 .mu.M in said assay. In some preferred
embodiments, said modulator is an agonist with an EC.sub.50 in said
assay of less than a value selected from the interval of 600 .mu.M
to 1000 .mu.M.
[0839] In some preferred embodiments, said treatment comprises oral
administration of said medicament to said individual.
[0840] In preferred embodiments, said modulator is an agonist and
said disorder in lipid metabolism is selected from the group
consisting of:
[0841] (a) elevated level of plasma triglycerides;
[0842] (b) elevated level of plasma free fatty acids;
[0843] (c) elevated level of plasma cholesterol;
[0844] (d) elevated level of LDL-cholesterol;
[0845] (e) reduced level of HDL-cholesterol;
[0846] (f) elevated total cholesterol/HDL-cholesterol ratio;
and
[0847] (g) reduced level of plasma adiponectin.
[0848] In other preferred embodiments, said disorder in lipid
metabolism is an elevated postprandial increase in plasma free
fatty acids due to a high fat meal or a progression from impaired
glucose tolerance to insulin resistance.
[0849] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[0850] In a forty-sixth aspect, the invention features a method of
using the modulator of the thirty-sixth aspect for the preparation
of a medicament for the treatment of a metabolic-related disorder
in an individual.
[0851] In some preferred embodiments, said modulator is selective
for the GPCR.
[0852] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[0853] In some preferred embodiments, said modulator is
antilipolytic.
[0854] In some preferred embodiments, said modulator is an
agonist.
[0855] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP38 polypeptide having the amino acid
sequence of SEQ. ID. NO.:135. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 900 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 800 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 700 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 600 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 550 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 500 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 450 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 400 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 350 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 300 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 250 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 200 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 150 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 100 .mu.M in said assay. In some preferred
embodiments, said modulator is an agonist with an EC.sub.50 in said
assay of less than a value selected from the interval of 600 .mu.M
to 1000 .mu.M.
[0856] In some preferred embodiments, said treatment comprises oral
administration of said medicament to said individual.
[0857] In preferred embodiments, said modulator is an agonist and
said metabolic-related disorder is selected from the group
consisting of:
[0858] (a) dyslipidemia;
[0859] (b) atherosclerosis;
[0860] (c) coronary heart disease;
[0861] (d) stroke;
[0862] (e) insulin resistance; and
[0863] (f) type 2 diabetes.
[0864] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[0865] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[0866] In a forty-seventh aspect, the invention features a method
of identifying whether a candidate compound is binds to an
antilipolytic GPCR, said receptor comprising the amino acid
sequence of SEQ. ID. NO.:135 (hRUP38); or an allelic variant or a
biologically active fragment of said amino acid sequence;
comprising the steps of: [0867] (a) contacting the receptor with a
labeled reference compound known to bind to the GPCR in the
presence or absence of the candidate compound; and [0868] (b)
determining whether the binding of said labeled reference compound
to the receptor is inhibited in the presence of the candidate
compound; wherein said inhibition is indicative of the candidate
compound binding to an antilipolytic GPCR.
[0869] In some embodiments, the antilipolytic GPCR comprises a
biologically active fragment of said amino acid sequence.
[0870] In some embodiments, the antilipolytic GPCR is
endogenous.
[0871] In some embodiments, the antilipolytic GPCR is
recombinant.
[0872] In preferred embodiments, said G protein is Gi.
[0873] In some preferred embodiments, said reference compound is
the modulator of the thirty-sixth aspect.
[0874] In other embodiments, said reference compound is an antibody
specific for the GPCR, or a derivative thereof.
[0875] In preferred embodiments, said reference compound comprises
a label selected from the group consisting of:
[0876] (a) radioisotope;
[0877] (b) enzyme; and
[0878] (c) fluorophore.
In some preferred embodiments, said label is .sup.3H.
[0879] In other embodiments, said method further comprises the step
of comparing the level of inhibition of binding of a labeled first
reference compound by the candidate compound to a second level of
inhibition of binding of said labeled first reference compound by a
second reference compound known to bind to the GPCR.
[0880] In a forty-eighth aspect, the invention features a method of
making a transgenic mouse, comprising the step of engineering said
mouse to carry as part of its own genetic material the gene
encoding the human antilipolytic GPCR polypeptide of SEQ. ID.
NO.:135 (hRUP38).
[0881] In some preferred embodiments, expression of said gene is
placed under the control of an essentially adipocyte specific
promoter.
[0882] In a forty-ninth aspect, the invention features the
transgenic mouse according to the method of the forty-eighth
aspect.
[0883] In a fiftieth aspect, the invention features a method of
using the transgenic mouse of the forty-ninth aspect to identify
whether an agonist of said human receptor has therapeutic efficacy
for the treatment of a disorder of lipid metabolism selected from
the group consisting of:
[0884] (a) elevated level of plasma triglycerides;
[0885] (b) elevated level of plasma free fatty acids;
[0886] (c) elevated level of plasma cholesterol;
[0887] (d) elevated level of LDL-cholesterol;
[0888] (e) reduced level of HDL-cholesterol;
[0889] (f) elevated total cholesterol/HDL-cholesterol ratio;
and
[0890] (g) reduced level of plasma adiponectin; comprising the
steps of:
[0891] (a') administering or not administering the agonist to the
mouse; and
[0892] (b') determining whether on administering the agonist there
is a change selected from the group consisting of: [0893] (i) a
decrease in the level of plasma triglycerides; [0894] (ii) a
decrease in the level of plasma free fatty acids; [0895] (iii) a
decrease in the level of plasma cholesterol; [0896] (iv) a decrease
in the level of LDL-cholesterol; [0897] (v) an increase in the
level of HDL-cholesterol; [0898] (vi) a decrease in the total
cholesterol/HDL-cholesterol ratio; and [0899] (vii) an increase in
the level of plasma adiponectin; wherein said change is indicative
of the agonist having therapeutic efficacy.
[0900] In a fifty-first aspect, the invention features a method of
using the transgenic mouse of the forty-ninth aspect to identify
whether an agonist of said human receptor has therapeutic efficacy
for the treatment of a metabolic-related disorder selected from the
group consisting of:
[0901] (a) dyslipidemia;
[0902] (b) atherosclerosis;
[0903] (c) coronary heart disease;
[0904] (d) stroke;
[0905] (e) insulin resistance; and
[0906] (f) type 2 diabetes;
comprising the steps of:
[0907] (a') administering or not administering the agonist to the
mouse; and
[0908] (b') determining whether on administering the agonist there
is a change selected from the group consisting of: [0909] (i) a
decrease in the level of plasma triglycerides; [0910] (ii) a
decrease in the level of plasma free fatty acids; [0911] (iii) a
decrease in the level of plasma cholesterol; [0912] (iv) a decrease
in the level of LDL-cholesterol; [0913] (v) an increase in the
level of HDL-cholesterol; [0914] (vi) a decrease in the total
cholesterol/HDL-cholesterol ratio; and [0915] (vii) an increase in
the level of plasma adiponectin; wherein said change is indicative
of the agonist having therapeutic efficacy.
[0916] In a fifty-second aspect, the invention features a method of
making a transgenic rat, comprising the step of engineering said
rat to carry as part of its own genetic material the gene encoding
the human antilipolytic GPCR polypeptide of SEQ. ID. NO.:135
(hRUP38).
[0917] In some preferred embodiments, expression of said gene is
placed under the control of an essentially adipocyte specific
promoter.
[0918] In a fifty-third aspect, the invention features the
transgenic rat according to the method of the fifty-second
aspect.
[0919] In a fifty-fourth aspect, the invention features a method of
using the transgenic rat of the fifty-third aspect to identify
whether an agonist of said human receptor has therapeutic efficacy
for the treatment of a disorder of lipid metabolism selected from
the group consisting of: [0920] (a) elevated level of plasma
triglycerides; [0921] (b) elevated level of plasma free fatty
acids; [0922] (c) elevated level of plasma cholesterol; [0923] (d)
elevated level of LDL-cholesterol; [0924] (e) reduced level of
HDL-cholesterol; [0925] (f) elevated total
cholesterol/HDL-cholesterol ratio; and [0926] (g) reduced level of
plasma adiponectin; comprising the steps of:
[0927] (a') administering or not administering the agonist to the
rat; and
[0928] (b') determining whether on administering the agonist there
is a change selected from the group consisting of: [0929] (i) a
decrease in the level of plasma triglycerides; [0930] (ii) a
decrease in the level of plasma free fatty acids; [0931] (iii) a
decrease in the level of plasma cholesterol; [0932] (iv) a decrease
in the level of LDL-cholesterol; [0933] (v) an increase in the
level of HDL-cholesterol; [0934] (vi) a decrease in the total
cholesterol/HDL-cholesterol ratio; and [0935] (vii) an increase in
the level of plasma adiponectin; wherein said change is indicative
of the agonist having therapeutic efficacy.
[0936] In a fifty-fifth aspect, the invention features a method of
using the transgenic rat of the fifty-third aspect to identify
whether an agonist of said human receptor has therapeutic efficacy
for the treatment of a metabolic-related disorder selected from the
group consisting of: [0937] (a) dyslipidemia; [0938] (b)
atherosclerosis; [0939] (c) coronary heart disease; [0940] (d)
stroke; [0941] (e) insulin resistance; and [0942] (f) type 2
diabetes; comprising the steps of:
[0943] (a') administering or not administering the agonist to the
rat; and
[0944] (b') determining whether on administering the agonist there
is a change selected from the group consisting of: [0945] (i) a
decrease in the level of plasma triglycerides; [0946] (ii) a
decrease in the level of plasma free fatty acids;
[0947] (iii) a decrease in the level of plasma cholesterol; [0948]
(iv) a decrease in the level of LDL-cholesterol; [0949] (v) an
increase in the level of HDL-cholesterol; [0950] (vi) a decrease in
the total cholesterol/HDL-cholesterol ratio; and [0951] (vii) an
increase in the level of plasma adiponectin; wherein said change is
indicative of the agonist having therapeutic efficacy.
[0952] In a fifty-sixth aspect, the invention features an isolated,
purified or recombinant RUP38 polynucleotide selected from the
group consisting of:
[0953] (a) a polynucleotide comprising a contiguous span of at
least 75 nucleotides of SEQ.ID. NO.:134, or an allelic variant of
said polynucleotide;
[0954] (b) a polynucleotide comprising a contiguous span of at
least 150 nucleotides of SEQ. ID. NO.:134, or an allelic variant of
said polynucleotide;
[0955] (c) a polynucleotide comprising a contiguous span of at
least 250 nucleotides of SEQ. ID. NO.:134, or an allelic variant of
said polynucleotide;
[0956] (d) a polynucleotide comprising a contiguous span of at
least 350 nucleotides of SEQ. ID. NO.:134, or an allelic variant of
said polynucleotide;
[0957] (e) a polynucleotide comprising a contiguous span of at
least 500 nucleotides of SEQ. ID. NO.:134, or an allelic variant of
said polynucleotide;
[0958] (f) a polynucleotide comprising a contiguous span of at
least 750 nucleotides of SEQ. ID. NOs.:134, or an allelic variant
of said polynucleotide;
[0959] (g) a polynucleotide comprising a contiguous span of at
least 1000 nucleotides of SEQ. ID. NO.:134, or an allelic variant
of said polynucleotide;
[0960] (h) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 20 amino acids of SEQ. ID. NO.:135 or
an allelic variant of said polypeptide;
[0961] (i) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 30 amino acids of SEQ. ID. NO.:135, or
an allelic variant of said polypeptide;
[0962] (j) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 40 amino acids of SEQ. ID. NO.:135, or
an allelic variant of said polypeptide;
[0963] (k) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 50 amino acids of SEQ. ID. NO.:135 or
an allelic variant of said polypeptide;
[0964] (l) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 75 amino acids of SEQ. ID. NO.:135 or
an allelic variant of said polypeptide;
[0965] (m) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 100 amino acids of SEQ. ID. NO.:135 or
an allelic variant of said polypeptide;
[0966] (n) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 150 amino acids of SEQ. ID. NO.:135 or
an allelic variant of said polypeptide;
[0967] (O) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 200 amino acids of SEQ. ID. NO.:135 or
an allelic variant of said polypeptide;
[0968] (p) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 250 amino acids of SEQ. ID. NO.:135 or
an allelic variant of said polypeptide; and
[0969] (q) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 300 amino acids of SEQ.ID. NO.:135 or
an allelic variant of said polypeptide.
[0970] The invention also relates to an isolated, purified or
recombinant RUP25 polynucleotide wherein said polynucleotide is
selected from the group consisting of:
[0971] (a) a polynucleotide comprising the nucleotide sequence of
SEQ. ID. NO.:134 or an allelic variant of said nucleotide
sequence;
[0972] (b) the polynucleotide of SEQ. ID. NO.:134, or an allelic
variant of said polynucleotide;
[0973] (c) a polynucleotide comprising a nucleotide sequence
encoding a polypeptide having the amino acid sequence of SEQ. ID.
NO.:135 or an allelic variant of said amino acid sequence; and
[0974] (d) a polynucleotide encoding a polypeptide having the amino
acid sequence of SEQ. ID. NO.:135, or an allelic variant of said
polypeptide.
[0975] In preferred embodiments, said isolated, purified or
recombinant polynucleotide comprises at least 8 contiguous
nucleotides of a polynucleotide of the present invention. In other
preferred embodiments, said isolated, purified or recombinant
polynucleotide comprises at least 10, 12, 15, 18, 20, 25, 28, 30,
35, 40, 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or
1000 contiguous nucleotides of a polynucleotide of the present
invention. Preferably said polynucleotide encodes full-length RUP38
polypeptide or a biologically active fragment thereof.
[0976] The polynucleotides of the present invention include genomic
polynucleotides comprising RUP38 polynucleotides of the
invention.
[0977] The present invention also relates to a polynucleotide
encoding a fusion protein, wherein said fusion protein comprises an
RUP38 polypeptide of the invention fused to a heterologous
polypeptide. In a preferred embodiment, said polypeptide of the
invention is constitutively active and said heterologous
polypeptide is a G protein. In other embodiments, said heterologous
polypeptide provides an antigenic epitope. In a preferred
embodiment, said heterologous polypeptide provides a hemaglutinin
(HA) antigenic epitope. Methods relating to a polynucleotide
encoding a fusion protein are well known to those of ordinary skill
in the art.
[0978] The polynucleotides of the present invention also include
variant polynucleotides at least 60%, 70%, 80%, 90%, 95%, 96%, 97%,
98% or 99% identical to an RUP38 polynucleotide of the invention.
In a particularly preferred embodiments, polynucleotide sequence
homologies are evaluated using the Basic Local Alignment Search
Tool ("BLAST"), which is well known in the art [See, e.g., Karlin
and Altschul, Proc Natl Acad Sci USA (1990) 87:2264-8; Altschul et
al., J Mol Biol (1990) 215:403-410; Altschul et al., Nature
Genetics (1993) 3:266-72; and Altschul et al., Nucleic Acids Res
(1997) 25:3389-3402; the disclosures of which are incorporated by
reference in their entirety].
[0979] In further preferred embodiments, the invention features the
complement of said polynucleotide.
[0980] In a fifty-seventh aspect, the invention features an
isolated, purified or recombinant RUP38 polypeptide selected from
the group consisting of:
[0981] (a) a polypeptide comprising a contiguous span of at least
20 amino acids of SEQ. ID. NO.:135 or an allelic variant of said
contiguous span of amino acids;
[0982] (b) a polypeptide comprising a contiguous span of at least
30 amino acids of SEQ. ID. NO.:135 or an allelic variant of said
contiguous span of amino acids;
[0983] (c) a polypeptide comprising a contiguous span of at least
40 amino acids of SEQ. ID. NO.:135 or an allelic variant of said
contiguous span of amino acids;
[0984] (d) a polypeptide comprising a contiguous span of at least
50 amino acids of SEQ. ID. NO.:135 or an allelic variant of said
contiguous span of amino acids;
[0985] (e) a polypeptide comprising a contiguous span of at least
75 amino acids of SEQ. ID. NO.:135 or an allelic variant of said
contiguous span of amino acids;
[0986] (f) a polypeptide comprising a contiguous span of at least
100 amino acids of SEQ. ID. NO.:135 or an allelic variant of said
contiguous span of amino acids;
[0987] (g) a polypeptide comprising a contiguous span of at least
150 amino acids of SEQ. ID. NO.:135 or an allelic variant of said
contiguous span of amino acids;
[0988] (h) a polypeptide comprising a contiguous span of at least
200 amino acids of SEQ. ID. NO.:135 or an allelic variant of said
contiguous span of amino acids;
[0989] (i) a polypeptide comprising a contiguous span of at least
250 amino acids of SEQ. ID. NO.:135 or an allelic variant of said
contiguous span of amino acids; and
[0990] (j) a polypeptide comprising a contiguous span of at least
300 amino acids of SEQ. ID. NO.:135 or an allelic variant of said
contiguous span of amino acids.
[0991] The invention also relates to an isolated, purified or
recombinant RUP38 polypeptide wherein said polypeptide is selected
from the group consisting of:
[0992] (a) a polypeptide comprising the amino acid sequence of SEQ.
ID. NO.:135 or an allelic variant or a biologically active mutant
of said amino acid sequence; and
[0993] (b) the polypeptide having the amino acid sequence of SEQ.
ID. NO.:135 or an allelic variant or a biologically active mutant
of said amino acid sequence; or a biologically active fragment of
said polypeptide.
[0994] In preferred embodiments, said isolated, purified or
recombinant polypeptide comprises at least 6 contiguous amino acids
of an RUP38 polypeptide of the invention. In further embodiments,
said isolated, purified or recombinant polypeptide comprises at
least 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 125, 150,
175, 200, 225, 250, 275 or 300 contiguous amino acids of a
polypeptide of the present invention. Preferably, said polypeptide
is full-length RUP38 polypeptide or an active fragment thereof.
[0995] The present invention also relates to a fusion protein,
wherein said fusion protein comprises an RUP38 polypeptide of the
invention fused to a heterologous polypeptide. In a preferred
embodiment, said polypeptide of the invention is constitutively
active and said heterologous polypeptide is a G protein. In other
preferred embodiments, said heterologous polypeptide provides an
antigenic epitope. In particularly preferred embodiment, said
heterologous polypeptide provides a hemaglutinin (HA) antigenic
epitope. Methods relating to a fusion protein are well known to
those of ordinary skill in the art.
[0996] The polypeptides of the present invention also include
variant polypeptides at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identical to an RUP38 polypeptide of the
invention. In a particularly preferred embodiments, polypeptide
sequence homologies are evaluated using the Basic Local Alignment
Search Tool ("BLAST"), which is well known in the art [See, e.g.,
Karlin and Altschul, Proc Natl Acad Sci USA (1990) 87:2264-8;
Altschul et al., J Mol Biol (1990) 215:403410; Altschul et al.,
Nature Genetics (1993) 3:266-72; and Altschul et al., Nucleic Acids
Res (1997) 25:3389-3402; the disclosures of which are incorporated
by reference in their entirety].
[0997] In an fifty-eighth aspect, the invention features a
composition comprising, consisting essentially of, or consisting of
the RUP38 polypeptide of the fifty-seventh aspect.
[0998] In a fifty-ninth aspect, the invention features a
recombinant vector, said vector comprising, consisting essentially
of, or consisting of the polynucleotide of the fifty-sixth aspect.
In some embodiments, said vector is a targeting vector used in a
method of inactivating a gene encoding an antilipolytic GPCR of the
invention. In some preferred embodiments, said vector is used in a
method of transient or stable transfection. In other preferred
embodiments, said vector is used in a method of transgenic
expression of an antilipolytic GPCR.
[0999] In particularly preferred embodiment, said vector is an
expression vector for the expression of a an antilipolytic GPCR in
a recombinant host cell wherein said expression vector comprises,
consists essentially of, or consists of the polynucleotide of the
fifty-sixth aspect. Although a variety of expression vectors are
available to those in the art, for purposes of utilization for both
the endogenous and non-endogenous human, mouse and rat GPCRs, it is
most preferred that the vector utilized be pCMV. In some
alternative embodiments as relates to said human, mouse and rat
antilipolytic GPCRs, it is preferred that the vector utilized be an
adenoviral expression vector.
[1000] In a sixtieth aspect, the invention features a prokaryotic
or eukaryotic host cell comprising, consisting essentially of, or
consisting of the recombinant vector of the fifty-ninth aspect. In
some embodiments, said host cell is a eukaryotic embryonic stem
cell wherein said vector of the fifty-ninth aspect has been used in
a method to inactivate a gene encoding an antilipolytic GPCR of the
invention within said cell. In some embodiments, said host cell is
a eukaryotic embryonic somatic cell wherein said vector of the
fifty-ninth aspect has been used in a method to inactivate a gene
encoding an antilipolytic GPCR of the invention within said cell.
In some preferred embodiments, said host cell is derived from a
mouse or rat made transgenic for a human RUP38 antilipolytic GPCR
of the invention. In some preferred embodiments, said host cell is
prokaryotic and has been transformed using the vector of the
fifty-ninth aspect. In further preferred embodiments, said host
cell is eukaryotic and has been transiently transfected using the
vector of the fifty-ninth aspect. In other further preferred
embodiments, said host cell is eukaryotic and has been stably
transfected using the vector of the fifty-ninth aspect.
[1001] In particularly preferred embodiment, said host cell
expresses a recombinant antilipolytic GPCR wherein said host cell
comprises, consists essentially of, or consists of the expression
vector of the fifty-ninth aspect.
[1002] A further embodiment includes a prokaryotic or eukaryotic
host cell recombinant for the polynucleotide of the fifty-sixth
aspect.
[1003] In some embodiments the host cell is eukaryotic, more
preferably, mammalian, and more preferably selected from the group
consisting of 293, 293T, CHO, and COS-7 cells. In other
embodiments, the host cell is eukaryotic, more preferably
melanophore.
[1004] In a sixty-first aspect, the invention features a process
for the expression of a antilipolytic GPCR in a recombinant host
cell comprising the steps of: [1005] (a) transfecting the
expression vector of the fifty-ninth aspect into a suitable host
cell; and [1006] (b) culturing the host cells under conditions
which allow expression of the antilipolytic GPCR protein from the
expression vectors.
[1007] In a sixty-second aspect, the invention features an antibody
that specifically binds to the polypeptide of the fifty-seventh
aspect. In some preferred embodiments, the antibody is monoclonal.
In some embodiments, the antibody is polyclonal.
[1008] In a sixty-third aspect, the invention features a method of
binding the polypeptide of the fifty-seventh aspect to the antibody
of the sixty-second aspect, comprising contacting said antibody
with said polypeptide under conditions in which said antibody can
specifically bind to said polypeptide.
[1009] In a sixty-fourth aspect, the invention features a method of
detecting an antilipolytic GPCR polypeptide in a biological sample
obtained from an individual comprising the steps of:
[1010] (a) obtaining said biological sample from said
individual;
[1011] (b) contacting said biological sample with the antibody of
the sixty-second aspect; and
[1012] (c) detecting the presence or absence of binding of said
antibody to said biological sample;
wherein a detection of said binding is indicative of the receptor
polypeptide being expressed in said biological sample.
[1013] In preferred embodiments, said detecting is through the use
of an enzyme-labeled secondary reagent. In other preferred
embodiments, said detecting is through the use of a
fluorophore-labeled secondary reagent. In other preferred
embodiments, said detecting is through the use of a
radioisotope-labeled secondary reagent. In other embodiments, the
antibody is directly labeled with enzyme, fluorophore or
radioisotope.
[1014] In other preferred embodiments, said biological sample is
taken from adipose, skin or blood.
[1015] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[1016] In further embodiments, said individual has a disorder of
lipid metabolism selected from the group consisting of:
[1017] (a) elevated level of plasma triglycerides;
[1018] (b) elevated level of plasma free fatty acids;
[1019] (c) elevated level of plasma cholesterol;
[1020] (d) elevated level of LDL-cholesterol;
[1021] (e) reduced level of HDL-cholesterol;
[1022] (f) elevated total cholesterol/HDL-cholesterol ratio;
and
[1023] (g) reduced level of plasma adiponectin.
[1024] In other preferred embodiments, said disorder in lipid
metabolism is an elevated postprandial increase in plasma free
fatty acids due to a high fat meal or a progression from impaired
glucose tolerance to insulin resistance.
[1025] In further embodiments, said individual has a
metabolic-related disorder selected from the group consisting
of:
[1026] (a) dyslipidemia;
[1027] (b) atherosclerosis;
[1028] (c) coronary heart disease;
[1029] (d) stroke;
[1030] (e) insulin resistance; and
[1031] (f) type 2 diabetes.
[1032] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[1033] In other embodiments, said method further comprises the step
of comparing the level of detection of said binding for a first
individual to the level of detection of said binding for a second
individual.
[1034] In a sixty-fifth aspect, the invention features a method of
detecting expression of a gene encoding an antilipolytic GPCR in a
biological sample obtained from an individual comprising the steps
of: [1035] (a) obtaining said biological sample from said
individual; [1036] (b) contacting said biological sample with the
complementary polynucleotide of the fifty-sixth aspect, optionally
labeled, under conditions permissive for hybridization; and [1037]
(c) detecting the presence or absence of said hybridization between
said complementary polynucleotide and an RNA species within said
sample; wherein a detection of said hybridization is indicative of
expression of said GPCR gene in said biological sample.
[1038] In preferred embodiments, the biological sample is taken
from adipose, skin or blood.
[1039] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[1040] In preferred embodiments, said individual has a disorder of
lipid metabolism selected from the group consisting of:
[1041] (a) elevated level of plasma triglycerides;
[1042] (b) elevated level of plasma free fatty acids;
[1043] (c) elevated level of plasma cholesterol;
[1044] (d) elevated level of LDL-cholesterol;
[1045] (e) reduced level of HDL-cholesterol;
[1046] (f) elevated total cholesterol/HDL-cholesterol ratio;
and
[1047] (g) reduced level of plasma adiponectin.
[1048] In other preferred embodiments, said disorder in lipid
metabolism is an elevated postprandial increase in plasma free
fatty acids due to a high fat meal or a progression from impaired
glucose tolerance to insulin resistance.
[1049] In other preferred embodiments, said individual has a
metabolic-related disorder selected from the group consisting
of:
[1050] (a) dyslipidemia;
[1051] (b) atherosclerosis;
[1052] (c) coronary heart disease;
[1053] (d) stroke;
[1054] (e) insulin resistance; and
[1055] (f) type 2 diabetes.
[1056] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[1057] In other embodiments, said method further comprises the step
of comparing the level of detection of said hybridization for a
first individual to the level of detection of said hybridization
for a second individual.
[1058] In some preferred embodiments, said complementary
polynucleotide is a primer and said hybridization is detected by
detecting the presence of an amplification product comprising the
sequence of said primer. In more preferred embodiments, said method
is RT-PCR.
[1059] In a sixty-sixth aspect, the invention features a GPCR
Fusion Protein construct comprising a constitutively active GPCR
and a G protein, said receptor comprising the amino acid sequence
of SEQ. ID. NO.:135 (hRUP38) or an allelic variant or a
biologically active fragment of said amino acid sequence.
[1060] The invention also relates to a GPCR Fusion Protein
construct wherein the isoleucine at amino acid position 230 of SEQ.
ID. NO.:135 is substituted by lysine.
[1061] In a sixty-seventh aspect, the invention features a method
of binding a known ligand of RUP38 antilipolytic GPCR to a
polypeptide selected from the group consisting of:
[1062] (a) a polypeptide comprising a contiguous span of at least 6
amino acids of SEQ. ID. NO.:135;
[1063] (b) a polypeptide comprising a contiguous span of at least
10 amino acids of SEQ. ID. NO.:135;
[1064] (c) a polypeptide comprising a contiguous span of at least
15 amino acids of SEQ. ID. NO.:135;
[1065] (d) a polypeptide comprising a contiguous span of at least
20 amino acids of SEQ. ID. NO.:135;
[1066] (e) a polypeptide comprising a contiguous span of at least
25 amino acids of SEQ. ID. NO.:135;
[1067] (f) a polypeptide comprising a contiguous span of at least
30 amino acids of SEQ. ID. NO.:135;
[1068] (g) a polypeptide comprising a contiguous span of at least
35 amino acids of SEQ. ID. NO.:135;
[1069] (h) a polypeptide comprising a contiguous span of at least
40 amino acids of SEQ. ID. NO.:135;
[1070] (i) a polypeptide comprising a contiguous span of at least
45 amino acids of SEQ. ID. NO.:135; and
[1071] (j) a polypeptide comprising a contiguous span of at least
50 amino acids of SEQ. ID. NO.:135; or an allelic variant of said
polypeptide;
comprising the step of contacting said known ligand with said
polypeptide under conditions which allow said binding to occur.
[1072] In some embodiments, said known ligand is a modulator of the
antilipolytic GPCR. In some embodiments, said known modulator is an
agonist of the antilipolytic GPCR. In some embodiments, said known
ligand is the modulator of the thirty-sixth aspect. In some
embodiments, said known ligand is an antibody specific for the
GPCR, or a derivative thereof.
[1073] In other preferred embodiments, said method is used to
identify whether a candidate compound inhibits said binding of said
known ligand to said polypeptide, comprising the steps of: [1074]
(a) contacting said polypeptide with said known ligand, optionally
labeled, in the presence or absence of said candidate compound;
[1075] (b) detecting the complex between said known ligand and said
polypeptide; and [1076] (c) determining whether less of said
complex is formed in the presence of the compound than in the
absence of the compound; wherein said determination is indicative
of the candidate compound being an inhibitor of said binding of
said known ligand to said polypeptide.
[1077] In some embodiments, said known ligand is a modulator of the
antilipolytic GPCR. In some embodiments, said known modulator is an
agonist. In some embodiments, said known ligand is the modulator of
the thirty-sixth aspect. In some embodiments, said known ligand is
an antibody specific for the GPCR, or a derivative thereof.
[1078] In other preferred embodiments, said method is used to
identify whether a candidate compound is an inhibitor of said
binding of said known ligand to said polypeptide, comprising the
steps of:
[1079] (a) contacting said polypeptide with said known ligand,
optionally labeled, in the presence separately of a plurality of
concentrations of said candidate compound for a time sufficient to
allow equilibration of binding;
[1080] (b) measuring unbound ligand and bound ligand; and
[1081] (c) determining K.sub.i for the candidate compound;
[1082] wherein a K.sub.i value of less than 50 uM is indicative of
the candidate compound being an inhibitor of said binding of said
known ligand to said polypeptide. Preferably said K.sub.i value is
less than 25 .mu.M, 10 .mu.M, 5 .mu.M, 1 .mu.M, 750 .mu.M, 500 nM,
400 nM, 300 .mu.M, 250 .mu.M, 200 nM, 150 nM, 100 nM, 90 nM, 80 nM,
70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM or 10 nM. In preferred
embodiments, K.sub.i determination is made through nonlinear curve
fitting with the program SCTFIT [De Lean et al. (1982) Mol
Pharmacol 21:5-16; cited in Lorenzen et al. (2001) Mol Pharmacol
59:349-357, the disclosures of which are incorporated by reference
herein in their entireties].
[1083] In some embodiments, said known ligand is a modulator of the
antilipolytic GPCR. In some embodiments, said known modulator is an
agonist. In some embodiments, said known ligand is the modulator of
the thirty-sixth aspect. In some embodiments, said known ligand is
an antibody specific for the GPCR, or a derivative thereof.
[1084] In a sixty-eighth aspect, the invention features a method of
binding an optionally labeled affinity reagent specific for an
antilipolytic GPCR to said receptor in a biological sample, said
receptor comprising the amino acid sequence of SEQ. ID. NO.:135
(hRUP38); or an allelic variant, a biologically active mutant, or a
biologically active fragment of said amino acid sequence,
comprising the steps of:
[1085] (a) obtaining said biological sample;
[1086] (b) contacting the affinity reagent with said receptor in
said biological sample; and
[1087] (c) detecting the complex of said affinity reagent with said
receptor.
[1088] In some embodiments, the antilipolytic GPCR has the amino
acid sequence of SEQ. ID. NO.:135 (hRUP38); or an allelic variant,
a biologically active mutant, or a biologically active fragment of
said amino acid sequence.
[1089] In some embodiments, the antilipolytic GPCR comprises a
biologically active fragment of said amino acid sequence.
[1090] In some embodiments, the antilipolytic GPCR is
endogenous.
[1091] In some embodiments, the antilipolytic GPCR is
recombinant.
[1092] In some embodiments, said biologically active mutant is CART
or EFA. In preferred embodiments, said CART mutant has the amino
acid sequence of SEQ. ID. NO.:135 further substituted at amino acid
position 230 with lysine in place of isoleucine.
[1093] In preferred embodiments, said G protein is Gi.
[1094] In some embodiments, said affinity reagent is a modulator of
the GPCR. In some embodiments, said affinity reagent is an agonist
of the GPCR. In some embodiments, said affinity reagent is the
modulator of the thirty-sixth aspect. In some embodiments, said
affinity reagent is an antibody specific for the GPCR, or a
derivative thereof.
[1095] In further preferred embodiments, said affinity reagent
comprises a label selected from the group consisting of:
[1096] (a) radioisotope;
[1097] (b) enzyme; and
[1098] (c) fluorophore.
[1099] In preferred embodiments, said radioisotope is .sup.3H.
[1100] In a sixty-ninth aspect, the invention features the method
of the sixty-eighth aspect further comprising the step of comparing
the level of detection of said complex in a first biological sample
to a second level of detection of said complex in a second
biological sample.
[1101] In a seventieth aspect, the invention features the method of
the sixty-ninth aspect wherein the relationship between said first
and second biological samples is selected from the group consisting
of:
[1102] (a) said second biological sample is a replicate of said
first biological sample;
[1103] (b) said first biological sample was obtained prior to an
experimental intervention
whereas said second biological sample was obtained after the
experimental intervention, from the same individual;
[1104] (c) said second biological sample was obtained at a
different time point after an experimental intervention than was
said first biological sample, from the same individual;
[1105] (d) said second biological sample corresponds to a different
subcellular compartment than does said first biological sample;
[1106] (e) said second biological sample represents a different
cell type than does said first biological sample;
[1107] (f) said second biological sample corresponds to a different
tissue than does said first biological sample;
[1108] (g) said second biological sample was obtained from a
different individual than was said first biological sample;
[1109] (h) said second biological sample was obtained at a
different point in time than was said first biological sample, from
the same individual;
[1110] (i) said first biological samples was obtained from a normal
individual, whereas said second biological sample was obtained from
an individual having a metabolic-related disorder;
[1111] (j) said first biological sample was obtained from a normal
individual, whereas said second biological sample was obtained from
an individual having a disorder in lipid metabolism;
[1112] (k) said first biological sample was obtained before a
therapeutic intervention whereas said second biological sample was
obtained after the therapeutic intervention, from the same
individual;
[1113] (l) said second biological sample was obtained at a
different time point after therapeutic intervention than was said
first biological sample, from the same individual; and
[1114] (m) said first biological sample was not exposed to a
compound, whereas said second biological sample was exposed to said
compound.
[1115] In a seventy-first aspect, the invention features a method
of identifying whether a candidate compound is a modulator of an
antilipolytic GPCR, said receptor comprising an amino acid sequence
selected from the group consisting of:
[1116] (a) SEQ. ID. NO.:24 (hRUP19);
[1117] (b) SEQ. ID. NO.:151 (mRUP19); and
[1118] (c) SEQ. ID. NO.:157 (rRUP19);
[1119] or an allelic variant, a biologically active mutant, or a
biologically active fragment of said amino acid sequence;
comprising the steps of:
[1120] (a') contacting the candidate compound with the
receptor;
[1121] (b') determining whether the receptor functionality is
modulated;
wherein a change in receptor functionality is indicative of the
candidate compound being a modulator of an antilipolytic GPCR.
[1122] In some embodiments, said antilipolytic GPCR is
endogenous.
[1123] In some preferred embodiments, said antilipolytic GPCR is
recombinant.
[1124] Preferred said identified modulator binds to said GPCR.
[1125] In some embodiments, said contacting is carried out in the
presence of a known ligand of the GPCR. In some embodiments, said
known ligand is an agonist of the GPCR.
[1126] The invention also relates to a method of identifying
whether a candidate compound is a modulator of lipolysis,
comprising the steps of: [1127] (a) contacting the candidate
compound with a GPCR comprising an amino acid sequence selected
from the group consisting of: [1128] (i) SEQ. ID. NO.:24 (hRUP19);
[1129] (ii) SEQ. ID. NO.:151 (mRUP19); and [1130] (iii) SEQ. ID.
NO.:157 (rRUP19); [1131] or an allelic variant, a biologically
active mutant, or a biologically active fragment of said amino acid
sequence; and [1132] (b) determining whether the receptor
functionality is modulated; wherein a change in receptor
functionality is indicative of the candidate compound being a
modulator of lipolysis.
[1133] In some embodiments, said GPCR is endogenous.
[1134] In some preferred embodiments, said GPCR is recombinant.
[1135] Preferred said identified modulator binds to said GPCR.
[1136] In some embodiments, said contacting is carried out in the
presence of a known ligand of the GPCR. In some embodiments, said
known ligand is an agonist of the GPCR.
[1137] The invention also relates to a method of determining
whether a candidate compound is a modulator of an antilipolytic
GPCR, comprising the steps of: [1138] (a) culturing antilipolytic
GPCR-expressing host cells under conditions that would allow
expression of a recombinant antilipolytic GPCR, said host cells
being transfected with a polynucleotide encoding said recombinant
antilipolytic GPCR comprising an amino acid sequence selected from
the group consisting of: [1139] (i) SEQ. ID. NO.:24 (hRUP19);
[1140] (ii) SEQ. ID. NO.:151 (mRUP19); and [1141] (iii) SEQ. ID.
NO.:157 (rRUP19); [1142] or an allelic variant, a biologically
active mutant, or a biologically active fragment of said amino acid
sequence; [1143] (b) contacting the antilipolytic GPCR-expressing
host cells of step (a) with the candidate compound; [1144] (c)
contacting control host cells with the candidate compound of step
(b), wherein said control host cells do not express recombinant
antilipolytic GPCR protein; [1145] (d) measuring the modulating
effect of the candidate compound which interacts with the
recombinant antilipolytic GPCR from the host cells of step (a) and
control host cells of step (c); and [1146] (e) comparing the
modulating effect of the test compound on the host cells and
control host cells.
[1147] The invention also relates to a method of determining
whether a candidate compound is a modulator of an antilipolytic
GPCR, comprising the steps of: [1148] (a) culturing antilipolytic
GPCR-expressing host cells under conditions that would allow
expression of a recombinant antilipolytic GPCR, said host cells
being transfected with a polynucleotide encoding said recombinant
antilipolytic GPCR comprising an amino acid sequence selected from
the group consisting of: [1149] (i) SEQ. ID. NO.:24 (hRUP19);
[1150] (ii) SEQ. ID. NO.:151 (mRUP19); and [1151] (iii) SEQ. ID.
NO.:157 (rRUP19); [1152] or an allelic variant, a biologically
active mutant, or a biologically active fragment of said amino acid
sequence; [1153] (b) contacting a first population of antilipolytic
GPCR-expressing cells of step (a) with a known ligand of said
antilipolytic GPCR; [1154] (c) contacting a second population of
antilipolytic GPCR-expressing cells of step (a) with the candidate
compound and with the known antilipolytic GPCR ligand; [1155] (d)
contacting control host cells with the candidate compound of step
(c), wherein said control host cells do not express recombinant
antilipolytic GPCR protein; [1156] (e) measuring the modulating
effect of the candidate compound, which interacts with recombinant
antilipolytic GPCR, in the presence and absence of the known
antilipolytic GPCR ligand, from the cells of step (b), step (c) and
step (d); and [1157] (f) comparing the modulating effect of the
candidate compound as determined from step (b), step (c) and step
(d).
[1158] In some embodiments, said ligand is an agonist of the
GPCR.
[1159] The invention also relates to a method of determining
whether a candidate compound is a modulator of an antilipolytic
GPCR, comprising the steps of: [1160] (a) culturing antilipolytic
GPCR-expressing host cells under conditions that would allow
expression of a recombinant antilipolytic GPCR, said host cells
being transfected with a polynucleotide encoding said recombinant
antilipolytic GPCR comprising an amino acid sequence selected from
the group consisting of: [1161] (i) SEQ. ID. NO.:24 (hRUP19);
[1162] (ii) SEQ. ID. NO.:151 (mRUP19); and [1163] (iii) SEQ. ID.
NO.:157 (rRUP19); [1164] or an allelic variant, a biologically
active mutant, or a biologically active fragment of said amino acid
sequence; [1165] (b) contacting a first population of the
antilipolytic GPCR-expressing host cells of step (a) with the
candidate compound; [1166] (c) not contacting a second population
of the antilipolytic GPCR-expressing cells of step (a) with the
candidate compound of step (b); [1167] (d) contacting control host
cells to the candidate compound of step (b), wherein said control
host cells do not express recombinant antilipolytic GPCR protein;
[1168] (e) measuring the modulating effect of the candidate
compound, which interacts with recombinant antilipolytic GPCR
protein, from the cells of step (b) and step (c) and from the cells
of step (d); and [1169] (f) comparing the modulating effect of the
candidate compound as determined from step (b) and step (c) and
from step (d).
[1170] In some embodiments, the antilipolytic GPCR has an amino
acid sequence selected from the group consisting of:
[1171] (a) SEQ. ID. NO.:24 (hRUP19);
[1172] (b) SEQ. ID. NO.:151 (mRUP19); and
[1173] (c) SEQ. ID. NO.:157 (rRUP19); [1174] or an allelic variant,
a biologically active mutant, or a biologically active fragment of
said amino acid sequence.
[1175] In some embodiments, the antilipolytic GPCR comprises a
biologically active fragment of said amino acid sequence.
[1176] In some embodiments, said biologically active mutant is CART
or EFA. In preferred embodiments, said CART mutant has the amino
acid sequence of SEQ. ID. NO.:24 further substituted at amino acid
position 219 with lysine in place of threonine.
[1177] In preferred embodiments, said G protein is Gi.
[1178] In other preferred embodiments, said determining is through
the use of a Melanophore assay.
[1179] In other preferred embodiments, said determining is through
the measurement of the level of a second messenger selected from
the group consisting of cyclic AMP (cAMP), cyclic GMP (cGMP),
inositol triphosphate (IP.sub.3), diacylglycerol (DAG), and
Ca.sup.2+. In further preferred embodiments, said second messenger
is cAMP. In more preferred embodiments, the level of the cAMP is
reduced. In some embodiments, said measurement of cAMP is carried
out with membrane comprising said GPCR.
[1180] In other preferred embodiments, said determining is through
the measurement of an activity up-regulated or down-regulated by a
reduction in intracellular cAMP level. In further preferred
embodiments, said down-regulated activity is intracellular
lipolysis. In other further preferred embodiments, said
down-regulated activity is hormone sensitive lipase activity. In
other further preferred embodiments, said up-regulated activity is
adiponectin secretion.
[1181] In other preferred embodiments, said determining is through
CRE-reporter assay. In preferred embodiments, said reporter is
luciferase. In some embodiments, said reporter is
.beta.-galactosidase.
[1182] In other preferred embodiments, said recombinant host cell
further comprises promiscuous G alpha 15/16 or chimeric Gq/Gi alpha
subunit and said determining is through measurement of
intracellular Ca.sup.2+. In preferred embodiments, said Ca.sup.2+
measurement is carried out by FLIPR.
[1183] In other preferred embodiments, said recombinant host cell
further comprises promiscuous G alpha 15/16 or chimeric Gq/Gi alpha
subunit and said determining is through measurement of
intracellular IP.sub.3.
[1184] In other preferred embodiments, said determining is through
the measurement of GTP.gamma.S binding to membrane comprising said
GPCR. In further preferred embodiments, said GTP.gamma.S is labeled
with [.sup.35S].
[1185] In other preferred embodiments, said method further
comprises the step of comparing the modulation of the receptor
caused by the candidate compound to a second modulation of the
receptor caused by contacting the receptor with a known modulator
of the receptor. In some preferred embodiments, said known
modulator is an agonist.
[1186] In a seventy-second aspect, the invention features a
modulator of an antilipolytic GPCR identified according to the
method of the seventy-first aspect.
[1187] In some preferred embodiments, said modulator is selected
from the group consisting of agonist, partial agonist, inverse
agonist and antagonist. More preferably, said modulator is an
agonist. In some embodiments, said modulator is a partial
agonist.
[1188] In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 1000 .mu.M in GTP-YS binding assay carried
out with membrane from stably transfected CHO cells expressing
recombinant hRUP19 polypeptide having the amino acid sequence of
SEQ. ID. NO.:24. In some embodiments, said modulator is an agonist
with an EC.sub.50 of less than 900 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 800 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 700 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[1189] In some embodiments, said modulator is selective for the
GPCR.
[1190] In some embodiments, said modulator is antilipolytic.
[1191] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some preferred
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some preferred
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intravenous
administration.
[1192] In highly less preferred embodiments, said modulator is an
antibody or derivative thereof.
[1193] In a seventy-third aspect, the invention features a method
of modulating the activity of an antilipolytic GPCR, said receptor
comprising an amino acid sequence selected from the group
consisting of:
[1194] (a) SEQ. ID. NO.:24 (hRUP19);
[1195] (b) SEQ. ID. NO.:151 (mRUP19); and
[1196] (c) SEQ. ID. NO.:157 (rRUP19);
or an allelic variant, a biologically active mutant, or a
biologically active fragment of said amino acid sequence;
comprising the step of contacting the receptor with the modulator
of the seventy-second aspect.
[1197] In some embodiments, the antilipolytic GPCR has an amino
acid sequence selected from the group consisting of:
[1198] (a) SEQ. ID. NO.:24 (hRUP19);
[1199] (b) SEQ. ID. NO.:151 (mRUP19); and
[1200] (c) SEQ. ID. NO.:157 (rRUP19);
or an allelic variant, a biologically active mutant, or a
biologically active fragment of said amino acid sequence.
[1201] In some embodiments, the antilipolytic GPCR comprises an
active fragment of said amino acid sequence.
[1202] In some embodiments, the antilipolytic GPCR is
recombinant.
[1203] In some embodiments, the antilipolytic GPCR is
endogenous.
[1204] In some embodiments, said biologically active mutant is CART
or EFA. In preferred embodiments, said CART mutant has the amino
acid sequence of SEQ. ID. NO.:24 further substituted at amino acid
position 219 with lysine in place of threonine.
[1205] In preferred embodiments, said G protein is Gi.
[1206] In some preferred embodiments, said modulator is an
agonist.
[1207] In preferred embodiments, said modulator is selective for
the GPCR.
[1208] In other preferred embodiments, said contacting comprises
administration of the modulator to a membrane comprising the
receptor.
[1209] In other preferred embodiments, said contacting comprises
administration of the modulator to a cell or tissue comprising the
receptor.
[1210] In other preferred embodiments, said contacting comprises
administration of the modulator to an individual comprising the
receptor. In more preferred embodiments, said individual is a
mammal. In other more preferred embodiments, said mammal is a
horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat, non-human
primate or human. Yet more preferred is mouse, rat or human. Most
preferred is human.
[1211] In some preferred embodiments, said modulator is selective
for the GPCR.
[1212] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[1213] In some preferred embodiments, said modulator is
antilipolytic.
[1214] In some preferred embodiments, said modulator is an
agonist.
[1215] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP19 polypeptide having the amino acid
sequence of SEQ. ID. NO.:24. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[1216] In some preferred embodiments, said administration is
oral.
[1217] In preferred embodiments, said modulator is an agonist and
said individual is in need of prevention of or treatment for a
metabolic-related disorder selected from the group consisting
of:
[1218] (a) dyslipidemia;
[1219] (b) atherosclerosis;
[1220] (c) coronary heart disease;
[1221] (d) stroke;
[1222] (e) insulin resistance; and
[1223] (f) type 2 diabetes.
[1224] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[1225] In other embodiments, said modulator is an inverse agonist
and said metabolic-related disorder relates to a low level of
plasma free fatty acids.
[1226] In other preferred embodiments, said modulator is an agonist
and said individual is in need of a change in lipid metabolism
selected from the group consisting of:
[1227] (a) a decrease in the level of plasma triglycerides;
[1228] (b) a decrease in the level of plasma free fatty acids;
[1229] (c) a decrease in the level of plasma cholesterol;
[1230] (d) a decrease in the level of LDL-cholesterol;
[1231] (e) an increase in the level of HDL-cholesterol;
[1232] (f) a decrease in the total cholesterol/HDL-cholesterol
ratio; and
[1233] (g) an increase in the level of plasma adiponectin.
[1234] In other preferred embodiments, said needed change in lipid
metabolism is a decrease in the postprandial increase in plasma
free fatty acids due to a high fat meal or an inhibition of the
progression from impaired glucose tolerance to insulin
resistance.
[1235] In some embodiments, the modulator is an inverse agonist and
the needed change in lipid metabolism is an increase in the level
of plasma free fatty acids.
[1236] In other preferred embodiments, said modulator is an agonist
and said individual is a mouse genetically predisposed to a
metabolic-related disorder selected from the group consisting
of:
[1237] (a) dyslipidemia;
[1238] (b) atherosclerosis;
[1239] (c) coronary heart disease;
[1240] (d) stroke;
[1241] (e) insulin resistance; and
[1242] (f) type 2 diabetes.
[1243] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure.
[1244] In further preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[1245] In further preferred embodiments, said method is used to
identify whether said agonist has therapeutic efficacy for the
treatment of a metabolic-related disorder selected from the group
consisting of:
[1246] (a) dyslipidemia;
[1247] (b) atherosclerosis;
[1248] (c) coronary heart disease;
[1249] (d) stroke;
[1250] (e) insulin resistance; and
[1251] (f) type 2 diabetes;
comprising the steps of:
[1252] (a') administering or not administering said agonist to the
mouse; and
[1253] (b') determining whether the disorder is prevented, delayed,
or made less severe on administering said agonist compared to not
administering said agonist; wherein said determination is
indicative of said agonist having therapeutic efficacy.
[1254] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[1255] In other preferred embodiments, said modulator is an agonist
and said individual is a rat genetically predisposed to a
metabolic-related disorder selected from the group consisting
of:
[1256] (a) dyslipidemia;
[1257] (b) atherosclerosis;
[1258] (c) coronary heart disease;
[1259] (d) stroke;
[1260] (e) insulin resistance; and
[1261] (f) type 2 diabetes.
[1262] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In further preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[1263] In further preferred embodiments, said method is used to
identify whether said agonist has therapeutic efficacy for the
treatment of a metabolic-related disorder selected from the group
consisting of:
[1264] (a) dyslipidemia;
[1265] (b) atherosclerosis;
[1266] (c) coronary heart disease;
[1267] (d) stroke;
[1268] (e) insulin resistance; and
[1269] (f) type 2 diabetes;
comprising the steps of:
[1270] (a') administering or not administering said agonist to the
rat; and
[1271] (b') determining whether the disorder is prevented, delayed,
or made less severe on administering said agonist compared to not
administering said agonist; wherein said determination is
indicative of said agonist having therapeutic efficacy.
[1272] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[1273] In a seventy-fourth aspect, the invention features a method
of preventing or treating a disorder of lipid metabolism in an
individual comprising contacting a therapeutically effective amount
of the modulator of the seventy-second aspect with an antilipolytic
GPCR, said receptor comprising an amino acid sequence selected from
the group consisting of:
[1274] (a) SEQ. ID. NO.:24 (hRUP19);
[1275] (b) SEQ. ID. NO.:151 (mRUP19); and
[1276] (c) SEQ. ID. NO.:157 (rRUP19);
or an allelic variant or biologically active fragment of said amino
acid sequence.
[1277] In some preferred embodiments, said modulator is selective
for the GPCR.
[1278] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[1279] In some preferred embodiments, said modulator is
antilipolytic.
[1280] In some preferred embodiments, said modulator is an
agonist.
[1281] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP19 polypeptide having the amino acid
sequence of SEQ. ID. NO.:24. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[1282] In some preferred embodiments, said contacting comprises
oral administration of said modulator to said individual.
[1283] In preferred embodiment, said modulator is an agonist and
said disorder of lipid metabolism is selected from the group
consisting of:
[1284] (a) elevated level of plasma triglycerides;
[1285] (b) elevated level of plasma free fatty acids;
[1286] (c) elevated level of plasma cholesterol;
[1287] (d) elevated level of LDL-cholesterol;
[1288] (e) reduced level of HDL-cholesterol;
[1289] (f) elevated total cholesterol/HDL-cholesterol ratio;
and
[1290] (g) reduced level of plasma adiponectin.
[1291] In a seventy-fifth aspect, the invention features a method
of preventing or treating a metabolic-related disorder in an
individual comprising contacting a therapeutically effective amount
of the modulator of the seventy-second aspect with an antilipolytic
GPCR, said receptor comprising an amino acid sequence selected from
the group consisting of:
[1292] (a) SEQ. ID. NO.:24 (hRUP19);
[1293] (b) SEQ. ID. NO.:151 (mRUP19); and
[1294] (c) SEQ. ID. NO.:157 (rRUP19);
or an allelic variant or biologically active fragment of said amino
acid sequence.
[1295] In some preferred embodiments, said modulator is selective
for the GPCR.
[1296] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[1297] In some preferred embodiments, said modulator is
antilipolytic.
[1298] In some preferred embodiments, said modulator is an
agonist.
[1299] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP19 polypeptide having the amino acid
sequence of SEQ. ID. NO.:24. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[1300] In some preferred embodiments, said contacting comprises
oral administration of said modulator to said individual.
[1301] In preferred embodiment, said modulator is an agonist and
said metabolic-related disorder is selected from the group
consisting of:
[1302] (a) dyslipidemia;
[1303] (b) atherosclerosis;
[1304] (c) coronary heart disease;
[1305] (d) stroke;
[1306] (e) insulin resistance; and
[1307] (f) type 2 diabetes.
[1308] In a seventy-sixth aspect, the invention features a method
of preparing a composition which comprises identifying a modulator
of a antilipolytic GPCR and then admixing a carrier and the
modulator, wherein the modulator is identifiable by the method of
the seventy-first aspect.
[1309] In some preferred embodiments, said modulator is selected
from the group consisting of agonist, partial agonist, inverse
agonist and antagonist. More preferably, said modulator is an
agonist. In some embodiments, said modulator is a partial
agonist.
[1310] In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding assay
carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP19 polypeptide having the amino acid
sequence of SEQ. ID. NO.:24. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[1311] In some embodiments, said modulator is selective for the
GPCR.
[1312] In some embodiments, said modulator is antilipolytic.
[1313] In some embodiments, said modulator is orally bioavailable.
In some embodiments, said oral bioavailability is at least 1%, at
least 5%, at least 10%, at least 15%, at least 20%, at least 25%,
at least 30%, at least 35%, at least 40%, or at least 45% relative
to either intraperitoneal or intravenous administration. In some
preferred embodiments, said oral bioavailability is at least 1%, at
least 5%, at least 10%, or at least 15% relative to either
intraperitoneal or intravenous administration.
[1314] In an seventy-seventh aspect, the invention features a
pharmaceutical or physiologically acceptable composition
comprising, consisting essentially of, or consisting of the
modulator of the seventy-second aspect. In preferred embodiments,
said modulator is an agonist.
[1315] In some preferred embodiments, said modulator is selective
for the GPCR.
[1316] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[1317] In some preferred embodiments, said modulator is
antilipolytic.
[1318] In some preferred embodiments, said modulator is an
agonist.
[1319] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP19 polypeptide having the amino acid
sequence of SEQ. ID. NO.:24. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[1320] In a seventy-eighth aspect, the invention features a method
of changing lipid metabolism comprising providing or administering
to an individual in need of said change said pharmaceutical or
physiologically acceptable composition of the seventy-seventh
aspect, said needed change in lipid metabolism selected from the
group consisting of:
[1321] (a) a decrease in the level of plasma triglycerides;
[1322] (b) a decrease in the level of plasma free fatty acids;
[1323] (c) a decrease in the level of plasma cholesterol;
[1324] (d) a decrease in the level of LDL-cholesterol;
[1325] (e) an increase in the level of HDL-cholesterol;
[1326] (f) a decrease in the total cholesterol/HDL-cholesterol
ratio; and
[1327] (g) an increase in the level of plasma adiponectin.
[1328] In preferred embodiments, a therapeutically effective amount
of said pharmaceutical or physiologically acceptable composition is
provided or administered to said individual.
[1329] In some preferred embodiments, said providing or
administering of said pharmaceutical or physiologically acceptable
composition is oral.
[1330] In other preferred embodiments, said needed change in lipid
metabolism is a decrease in the postprandial increase in plasma
free fatty acids due to a high fat meal or an inhibition of the
progression from impaired glucose tolerance to insulin
resistance.
[1331] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[1332] In a seventy-ninth aspect, the invention features a method
of preventing or treating a metabolic-related disorder comprising
providing or administering to an individual in need of said
treatment said pharmaceutical or physiologically acceptable
composition of the seventy-seventh aspect, said metabolic-related
disorder selected from the group consisting of:
[1333] (a) dyslipidemia;
[1334] (b) atherosclerosis;
[1335] (c) coronary heart disease;
[1336] (d) stroke;
[1337] (e) insulin resistance; and
[1338] (f) type 2 diabetes.
[1339] In preferred embodiments, a therapeutically effective amount
of said pharmaceutical or physiologically acceptable composition is
provided or administered to said individual.
[1340] In some preferred embodiments, said providing or
administering of said pharmaceutical or physiologically acceptable
composition is oral.
[1341] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, stroke,
Syndrome X, and heart disease. Heart disease includes, but is not
limited to, cardiac insufficiency, coronary insufficiency, and high
blood pressure. In other preferred embodiments, said
metabolic-related disorder is hyperlipidemia.
[1342] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[1343] In an eightieth aspect, the invention features a method of
using the modulator of the seventy-second aspect for the
preparation of a medicament for the treatment of a disorder in
lipid metabolism in an individual.
[1344] In some preferred embodiments, said modulator is selective
for the GPCR.
[1345] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[1346] In some preferred embodiments, said modulator is
antilipolytic.
[1347] In some preferred embodiments, said modulator is an
agonist.
[1348] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP19 polypeptide having the amino acid
sequence of SEQ. ID. NO.:24. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[1349] In some preferred embodiments, said treatment comprises oral
administration of said medicament to said individual.
[1350] In preferred embodiments, said modulator is an agonist and
said disorder in lipid metabolism is selected from the group
consisting of:
[1351] (a) elevated level of plasma triglycerides;
[1352] (b) elevated level of plasma free fatty acids;
[1353] (c) elevated level of plasma cholesterol;
[1354] (d) elevated level of LDL-cholesterol;
[1355] (e) reduced level of HDL-cholesterol;
[1356] (f) elevated total cholesterol/HDL-cholesterol ratio;
and
[1357] (g) reduced level of plasma adiponectin.
[1358] In other preferred embodiments, said disorder in lipid
metabolism is an elevated postprandial increase in plasma free
fatty acids due to a high fat meal or a progression from impaired
glucose tolerance to insulin resistance.
[1359] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[1360] In an eighty-first aspect, the invention features a method
of using the modulator of the seventy-second aspect for the
preparation of a medicament for the treatment of a
metabolic-related disorder in an individual.
[1361] In some preferred embodiments, said modulator is selective
for the GPCR.
[1362] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[1363] In some preferred embodiments, said modulator is
antilipolytic.
[1364] In some preferred embodiments, said modulator is an
agonist.
[1365] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP19 polypeptide having the amino acid
sequence of SEQ. ID. NO.:24. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[1366] In some preferred embodiments, said treatment comprises oral
administration of said medicament to said individual.
[1367] In preferred embodiments, said modulator is an agonist and
said metabolic-related disorder is selected from the group
consisting of:
[1368] (a) dyslipidemia;
[1369] (b) atherosclerosis;
[1370] (c) coronary heart disease;
[1371] (d) stroke;
[1372] (e) insulin resistance; and
[1373] (f) type 2 diabetes.
[1374] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[1375] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[1376] In an eighty-second aspect, the invention features a method
of identifying whether a candidate compound binds to an
antilipolytic GPCR, said receptor comprising an amino acid sequence
selected from the group consisting of:
[1377] (a) SEQ. ID. NO.:24 (hRUP19);
[1378] (b) SEQ. ID. NO.:151 (mRUP19); and
[1379] (c) SEQ. ID. NO.:157 (rRUP19);
or an allelic variant or a biologically active fragment of said
amino acid sequence; comprising the steps of:
[1380] (a') contacting the receptor with a labeled reference
compound known to bind to the GPCR in the presence or absence of
the candidate compound; and
[1381] (b') determining whether the binding of said labeled
reference compound to the receptor is inhibited in the presence of
the candidate compound; wherein said inhibition is indicative of
the candidate compound binding to an antilipolytic GPCR.
[1382] In some embodiments, the antilipolytic GPCR comprises a
biologically active fragment of said amino acid sequence.
[1383] In some embodiments, the antilipolytic GPCR is
endogenous.
[1384] In some embodiments, the antilipolytic GPCR is
recombinant.
[1385] In preferred embodiments, said G protein is Gi.
[1386] In some preferred embodiments, said reference compound is
the modulator of the seventy-second aspect.
[1387] In other embodiments, said reference compound is an antibody
specific for the GPCR, or a derivative thereof.
[1388] In preferred embodiments, said reference compound comprises
a label selected from the group consisting of:
[1389] (a) radioisotope;
[1390] (b) enzyme; and
[1391] (c) fluorophore.
In some preferred embodiments, said label is .sup.3H.
[1392] In other embodiments, said method further comprises the step
of comparing the level of inhibition of binding of a labeled first
reference compound by the candidate compound to a second level of
inhibition of binding of said labeled first reference compound by a
second reference compound known to bind to the GPCR.
[1393] In an eighty-third aspect, the invention features a method
of making a mouse genetically predisposed to a metabolic-related
disorder selected from the group consisting of:
[1394] (a) dyslipidemia;
[1395] (b) atherosclerosis;
[1396] (c) coronary heart disease;
[1397] (d) stroke;
[1398] (e) insulin resistance; and
[1399] (f) type 2 diabetes;
comprising the step of knocking out the gene encoding the
antilipolytic mRUP19 GPCR polypeptide of SEQ. ID. NO.:151.
[1400] In some preferred embodiments, said knocking out the gene
encoding the antilipolytic mRUP19 GPCR polypeptide of SEQ. ID.
NO.:151 is essentially restricted to adipocytes.
[1401] In an eighty-fourth aspect, the invention features the
knockout mouse according to the method of the eighty-third
aspect.
[1402] In an eighty-fifth aspect, the invention features a method
of using the knockout mouse of the eighty-fourth aspect to identify
whether a candidate compound has therapeutic efficacy for the
treatment of a metabolic-related disorder selected from the group
consisting of:
[1403] (a) dyslipidemia;
[1404] (b) atherosclerosis;
[1405] (c) coronary heart disease;
[1406] (d) stroke;
[1407] (e) insulin resistance; and
[1408] (f) type 2 diabetes;
comprising the steps of:
[1409] (a') administering or not administering the compound to the
mouse; and
[1410] (b') determining whether the disorder is prevented, delayed,
or made less severe on administering the compound compared to not
administering the compound; wherein said determination is
indicative of the compound having therapeutic efficacy.
[1411] In an eighty-sixth aspect, the invention features a method
of making a rat genetically predisposed to a metabolic-related
disorder selected from the group consisting of:
[1412] (a) dyslipidemia;
[1413] (b) atherosclerosis;
[1414] (c) coronary heart disease;
[1415] (d) stroke;
[1416] (e) insulin resistance; and
[1417] (f) type 2 diabetes;
comprising the step of knocking out the gene encoding the
antilipolytic rRUP19 GPCR polypeptide of SEQ. ID. NO.:157.
[1418] In some preferred embodiments, said knocking out the gene
encoding the antilipolytic rRUP19 GPCR polypeptide of SEQ. ID.
NO.:157 is essentially restricted to adipocytes.
[1419] In an eighty-seventh aspect, the invention features the
knockout rat according to the method of the eighty-sixth
aspect.
[1420] In an eighty-eighth aspect, the invention features a method
of using the knockout rat of the eighty-seventh aspect to identify
whether a candidate compound has therapeutic efficacy for the
treatment of a metabolic-related disorder selected from the group
consisting of:
[1421] (a) dyslipidemia;
[1422] (b) atherosclerosis;
[1423] (c) coronary heart disease;
[1424] (d) stroke;
[1425] (e) insulin resistance; and
[1426] (f) type 2 diabetes;
comprising the steps of:
[1427] (a') administering or not administering the compound to the
rat; and
[1428] (b') determining whether the disorder is prevented, delayed,
or made less severe on administering the compound compared to not
administering the compound; wherein said determination is
indicative of the compound having therapeutic efficacy.
[1429] In an eighty-ninth aspect, the invention features an
isolated, purified or recombinant RUP19 polynucleotide selected
from the group consisting of:
[1430] (a) a polynucleotide comprising a contiguous span of at
least 75 nucleotides of SEQ.ID. NO.:23, SEQ. ID. NO.:150 or SEQ.
ID. NO.:156, or an allelic variant of said polynucleotide;
[1431] (b) a polynucleotide comprising a contiguous span of at
least 150 nucleotides of SEQ. ID. NO.:23, SEQ. ID. NO.:150 or SEQ.
ID. NO.:156, or an allelic variant of said polynucleotide;
[1432] (c) a polynucleotide comprising a contiguous span of at
least 250 nucleotides of SEQ. ID. NO.:23, SEQ. ID. NO.:150 or SEQ.
ID. NO.:156, or an allelic variant of said polynucleotide;
[1433] (d) a polynucleotide comprising a contiguous span of at
least 350 nucleotides of SEQ. ID. NO.:23, SEQ. ID. NO.:150 or SEQ.
ID. NO.:156, or an allelic variant of said polynucleotide;
[1434] (e) a polynucleotide comprising a contiguous span of at
least 500 nucleotides of SEQ. ID. NO.:23, SEQ. ID. NO.:150 or SEQ.
ID. NO.:156, or an allelic variant of said polynucleotide;
[1435] (f) a polynucleotide comprising a contiguous span of at
least 750 nucleotides of SEQ. ID. NOs.:23, SEQ. ID. NO.:150 or 156,
or an allelic variant of said polynucleotide;
[1436] (g) a polynucleotide comprising a contiguous span of at
least 1000 nucleotides of SEQ. ID. NO.:23, SEQ. ID. NO.:150 or SEQ.
ID. NO.:156, or an allelic variant of said polynucleotide;
[1437] (h) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 20 amino acids of SEQ. ID. NO.:24, SEQ.
ID. NO:151 or SEQ. ID. NO.:157 or an allelic variant of said
polypeptide;
[1438] (i) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 30 amino acids of SEQ. ID. NO.:24, SEQ.
ID. NO.:151 or SEQ. ID. NO.:157 or an allelic variant of said
polypeptide;
[1439] (j) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 40 amino acids of SEQ. ID. NO.:24, SEQ.
ID. NO.:151 or SEQ. ID. NO.:157 or an allelic variant of said
polypeptide;
[1440] (k) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 50 amino acids of SEQ. ID. NO.:24, SEQ.
ID. NO.:151 or SEQ. ID. NO.:157 or an allelic variant of said
polypeptide;
[1441] (l) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 75 amino acids of SEQ. ID. NO.:24, SEQ.
ID. NO.:151 or SEQ. ID. NO.:157 or an allelic variant of said
polypeptide;
[1442] (m) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 100 amino acids of SEQ. ID. NO.:24,
SEQ. ID. NO.:151 or SEQ. ID. NO.:157 or an allelic variant of said
polypeptide;
[1443] (n) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 150 amino acids of SEQ. ID. NO.:24,
SEQ. ID. NO.:151 or SEQ. ID. NO.:157 or an allelic variant of said
polypeptide;
[1444] (O) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 200 amino acids of SEQ. ID. NO.:24,
SEQ. ID. NO.:151 or SEQ. ID. NO.:157 or an allelic variant of said
polypeptide;
[1445] (p) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 250 amino acids of SEQ. ID. NO.:24,
SEQ. ID. NO.:151 or SEQ. ID. NO.:157 or an allelic variant of said
polypeptide; and
[1446] (q) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 300 amino acids of SEQ.ID. NO.:24, SEQ.
ID. NO.:151 or SEQ. ID. NO.:157 or an allelic variant of said
polypeptide.
[1447] The invention also relates to an isolated, purified or
recombinant RUP19 polynucleotide wherein said polynucleotide is
selected from the group consisting of:
[1448] (a) a polynucleotide comprising a nucleotide sequence
selected from the group consisting of SEQ. ID. NO.:23, SEQ. ID.
NO.:150 and SEQ. ID. NO.:156 or an allelic variant of said
polynucleotide;
[1449] (b) a polynucleotide selected from the group consisting of
the polynucleotide of SEQ. ID. NO.:23, the polynucleotide of SEQ.
ID. NO.:150 and the polynucleotide of SEQ. ID. NO.:156, or an
allelic variant of said polynucleotide;
[1450] (c) a polynucleotide comprising a nucleotide sequence
encoding a polypeptide having an amino acid sequence selected from
the group consisting of SEQ. ID. NO.:24, SEQ. ID. NO.:151 and SEQ.
ID. NO.:157 or an allelic variant of said polypeptide; and
[1451] (d) a polynucleotide encoding a polypeptide having an amino
acid sequence selected from the group consisting of SEQ. ID.
NO.:24, SEQ. ID. NO.:151 and SEQ. ID. NO.:157 or an allelic variant
of said polypeptide.
[1452] In preferred embodiments, said isolated, purified or
recombinant polynucleotide comprises at least 8 contiguous
nucleotides of a polynucleotide of the present invention. In other
preferred embodiments, said isolated, purified or recombinant
polynucleotide comprises at least 10, 12, 15, 18, 20, 25, 28, 30,
35, 40, 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or
1000 contiguous nucleotides of a polynucleotide of the present
invention. Preferably said polynucleotide encodes full-length RUP19
polypeptide or a biologically active fragment thereof.
[1453] The polynucleotides of the present invention include genomic
polynucleotides comprising RUP19 polynucleotides of the
invention.
[1454] The present invention also relates to a polynucleotide
encoding a fusion protein, wherein said fusion protein comprises an
RUP19 polypeptide of the invention fused to a heterologous
polypeptide. In a preferred embodiment, said polypeptide of the
invention is constitutively active and said heterologous
polypeptide is a G protein. In other embodiments, said heterologous
polypeptide provides an antigenic epitope. In a preferred
embodiment, said heterologous polypeptide provides a hemaglutinin
(HA) antigenic epitope. Methods relating to a polynucleotide
encoding a fusion protein are well known to those of ordinary skill
in the art.
[1455] The polynucleotides of the present invention also include
variant polynucleotides at least 60%, 70%, 80%, 90%, 95%, 96%, 97%,
98% or 99% identical to an RUP19 polynucleotide of the invention.
In a particularly preferred embodiments, polynucleotide sequence
homologies are evaluated using the Basic Local Alignment Search
Tool ("BLAST"), which is well known in the art [See, e.g., Karlin
and Altschul, Proc Natl Acad Sci USA (1990) 87:2264-8; Altschul et
al., J Mol Biol (1990) 215:403-410; Altschul et al., Nature
Genetics (1993) 3:266-72; and Altschul et al., Nucleic Acids Res
(1997) 25:3389-3402; the disclosures of which are incorporated by
reference in their entirety].
[1456] In further preferred embodiments, the invention features the
complement of said polynucleotide.
[1457] In a ninetieth aspect, the invention features an isolated,
purified or recombinant RUP19 polypeptide selected from the group
consisting of:
[1458] (a) a polypeptide comprising a contiguous span of at least
20 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID.
NO.:157;
[1459] (b) a polypeptide comprising a contiguous span of at least
30 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID.
NO.:157;
[1460] (c) a polypeptide comprising a contiguous span of at least
40 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID.
NO.:157;
[1461] (d) a polypeptide comprising a contiguous span of at least
50 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID.
NO.:157;
[1462] (e) a polypeptide comprising a contiguous span of at least
75 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID.
NO.:157;
[1463] (f) a polypeptide comprising a contiguous span of at least
100 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID.
NO.:157;
[1464] (g) a polypeptide comprising a contiguous span of at least
150 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID.
NO.:157;
[1465] (h) a polypeptide comprising a contiguous span of at least
200 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID.
NO.:157;
[1466] (i) a polypeptide comprising a contiguous span of at least
250 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID.
NO.:157; and
[1467] (j) a polypeptide comprising a contiguous span of at least
300 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID.
NO.:157; or an allelic variant of said polypeptide.
[1468] The invention also relates to an isolated, purified or
recombinant RUP19 polypeptide wherein said polypeptide is selected
from the group consisting of:
[1469] (a) a polypeptide comprising an amino acid sequence selected
from the group consisting of SEQ. ID. NO.:24, SEQ. ID. NO.:151 and
SEQ. ID. NO.:157; and
[1470] (b) a polypeptide selected from the group consisting of the
polypeptide of SEQ. ID. NO.:24, the polypeptide of SEQ. ID. NO:151
and the polypeptide of SEQ. ID. NO.:157; or an allelic variant, a
biologically active mutant, or a biologically active fragment of
said polypeptide.
[1471] In preferred embodiments, said isolated, purified or
recombinant polypeptide comprises at least 6 contiguous amino acids
of an RUP19 polypeptide of the invention. In further embodiments,
said isolated, purified or recombinant polypeptide comprises at
least 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 125, 150,
175, 200, 225, 250, 275 or 300 contiguous amino acids of a
polypeptide of the present invention. Preferably, said polypeptide
is full-length RUP19 polypeptide or an active fragment thereof.
[1472] The present invention also relates to a fusion protein,
wherein said fusion protein comprises an RUP19 polypeptide of the
invention fused to a heterologous polypeptide. In a preferred
embodiment, said polypeptide of the invention is constitutively
active and said heterologous polypeptide is a G protein. In other
preferred embodiments, said heterologous polypeptide provides an
antigenic epitope. In particularly preferred embodiment, said
heterologous polypeptide provides a hemaglutinin (HA) antigenic
epitope. Methods relating to a fusion protein are well known to
those of ordinary skill in the art.
[1473] The polypeptides of the present invention also include
variant polypeptides at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identical to an RUP19 polypeptide of the
invention. In a particularly preferred embodiments, polypeptide
sequence homologies are evaluated using the Basic Local Alignment
Search Tool ("BLAST"), which is well known in the art [See, e.g.,
Karlin and Altschul, Proc Natl Acad Sci USA (1990) 87:2264-8;
Altschul et al., J Mol Biol (1990) 215:403-410; Altschul et al.,
Nature Genetics (1993) 3:266-72; and Altschul et al., Nucleic Acids
Res (1997) 25:3389-3402; the disclosures of which are incorporated
by reference in their entirety].
[1474] In an ninety-first aspect, the invention features a
composition comprising, consisting essentially of, or consisting of
the RUP19 polypeptide of the ninetieth aspect.
[1475] In a ninety-second aspect, the invention features a
recombinant vector, said vector comprising, consisting essentially
of, or consisting of the polynucleotide of the eighty-ninth aspect.
In preferred embodiments, said vector is a targeting vector used in
a method of inactivating a gene encoding an antilipolytic GPCR of
the invention. In other preferred embodiments, said vector is used
in a method of transient or stable transfection.
[1476] In particularly preferred embodiment, said vector is an
expression vector for the expression of a antilipolytic GPCR in a
recombinant host cell wherein said expression vector comprises,
consists essentially of, or consists of the polynucleotide of the
eighty-ninth aspect.
[1477] Although a variety of expression vectors are available to
those in the art, for purposes of utilization for both the
endogenous and non-endogenous human, mouse and rat GPCRs, it is
most preferred that the vector utilized be pCMV. In some
alternative embodiments as relates to said human, mouse and rat
antilipolytic GPCRs, it is preferred that the vector utilized be an
adenoviral expression vector.
[1478] In a ninety-third aspect, the invention features a
prokaryotic or eukaryotic host cell comprising, consisting
essentially of, or consisting of the recombinant vector of the
ninety-second aspect. In some preferred embodiments, said host cell
is a eukaryotic embryonic stem cell wherein said vector of the
ninety-second aspect has been used in a method to inactivate a gene
encoding an antilipolytic GPCR of the invention within said cell.
In some other preferred embodiments, said host cell is a eukaryotic
embryonic somatic cell wherein said vector of the ninety-second
aspect has been used in a method to inactivate a gene encoding an
antilipolytic GPCR of the invention within said cell. In other
preferred embodiments, said host cell is prokaryotic and has been
transformed using the vector of the ninety-second aspect. In
further preferred embodiments, said host cell is eukaryotic and has
been transiently transfected using the vector of the ninety-second
aspect. In other further preferred embodiments, said host cell is
eukaryotic and has been stably transfected using the vector of the
ninety-second aspect.
[1479] In particularly preferred embodiment, said host cell
expresses a recombinant antilipolytic GPCR wherein said host cell
comprises, consists essentially of, or consists of the expression
vector of the ninety-second aspect.
[1480] A further embodiment includes a prokaryotic or eukaryotic
host cell recombinant for the polynucleotide of the eighty-ninth
aspect.
[1481] In some embodiments the host cell is eukaryotic, more
preferably, mammalian, and more preferably selected from the group
consisting of 293, 293T, CHO, and COS-7 cells. In other
embodiments, the host cell is eukaryotic, more preferably
melanophore.
[1482] In a ninety-fourth aspect, the invention features a process
for the expression of an antilipolytic GPCR in a recombinant host
cell comprising the steps of: [1483] (a) transfecting the
expression vector of the ninety-second aspect into a suitable host
cell; and [1484] (b) culturing the host cells under conditions
which allow expression of the antilipolytic GPCR protein from the
expression vectors.
[1485] In a ninety-fifth aspect, the invention features an antibody
that specifically binds to the polypeptide of the ninetieth aspect.
In some preferred embodiments, the antibody is monoclonal. In some
embodiments, the antibody is polyclonal.
[1486] In a ninety-sixth aspect, the invention features a method of
binding the polypeptide of the ninetieth aspect to the antibody of
the ninety-fifth aspect, comprising contacting said antibody with
said polypeptide under conditions in which said antibody can
specifically bind to said polypeptide.
[1487] In a ninety-seventh aspect, the invention features a method
of detecting an antilipolytic GPCR polypeptide in a biological
sample obtained from an individual comprising the steps of:
[1488] (a) obtaining said biological sample from said
individual;
[1489] (b) contacting said biological sample with the antibody of
the ninety-fifth aspect; and
[1490] (c) detecting the presence or absence of binding of said
antibody to said biological sample;
wherein a detection of said binding is indicative of the receptor
polypeptide being expressed in said biological sample.
[1491] In preferred embodiments, said detecting is through the use
of an enzyme-labeled secondary reagent. In other preferred
embodiments, said detecting is through the use of a
fluorophore-labeled secondary reagent. In other preferred
embodiments, said detecting is through the use of a
radioisotope-labeled secondary reagent. In other embodiments, the
antibody is directly labeled with enzyme, fluorophore or
radioisotope.
[1492] In other preferred embodiments, said biological sample is
taken from adipose, skin or blood.
[1493] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[1494] In further embodiments, said individual has a disorder of
lipid metabolism selected from the group consisting of:
[1495] (a) elevated level of plasma triglycerides;
[1496] (b) elevated level of plasma free fatty acids;
[1497] (c) elevated level of plasma cholesterol;
[1498] (d) elevated level of LDL-cholesterol;
[1499] (e) reduced level of HDL-cholesterol;
[1500] (f) elevated total cholesterol/HDL-cholesterol ratio;
and
[1501] (g) reduced level of plasma adiponectin.
[1502] In other preferred embodiments, said disorder in lipid
metabolism is an elevated postprandial increase in plasma free
fatty acids due to a high fat meal or a progression from impaired
glucose tolerance to insulin resistance.
[1503] In further embodiments, said individual has a
metabolic-related disorder selected from the group consisting
of:
[1504] (a) dyslipidemia;
[1505] (b) atherosclerosis;
[1506] (c) coronary heart disease;
[1507] (d) stroke;
[1508] (e) insulin resistance; and
[1509] (f) type 2 diabetes.
[1510] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[1511] In other embodiments, said method further comprises the step
of comparing the level of detection of said binding for a first
individual to the level of detection of said binding for a second
individual.
[1512] In a ninety-eighth aspect, the invention features a method
of detecting expression of a gene encoding antilipolytic GPCR in a
biological sample obtained from an individual comprising the steps
of: [1513] (a) obtaining said biological sample from said
individual; [1514] (b) contacting said biological sample with the
complementary polynucleotide of the eighty-ninth aspect, optionally
labeled, under conditions permissive for hybridization; and [1515]
(c) detecting the presence or absence of said hybridization between
said complementary polynucleotide and an RNA species within said
sample; wherein a detection of said hybridization is indicative of
expression of said GPCR gene in said biological sample.
[1516] Methods of labeling a nucleic acid probe are well known to
those of ordinary skill in the art.
[1517] In preferred embodiments, the biological sample is taken
from adipose, skin or blood.
[1518] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[1519] In preferred embodiments, said individual has a disorder of
lipid metabolism selected from the group consisting of:
[1520] (a) elevated level of plasma triglycerides;
[1521] (b) elevated level of plasma free fatty acids;
[1522] (c) elevated level of plasma cholesterol;
[1523] (d) elevated level of LDL-cholesterol;
[1524] (e) reduced level of HDL-cholesterol;
[1525] (f) elevated total cholesterol/HDL-cholesterol ratio;
and
[1526] (g) reduced level of plasma adiponectin.
[1527] In other preferred embodiments, said disorder in lipid
metabolism is an elevated postprandial increase in plasma free
fatty acids due to a high fat meal or a progression from impaired
glucose tolerance to insulin resistance.
[1528] In other preferred embodiments, said individual has a
metabolic-related disorder selected from the group consisting
of:
[1529] (a) dyslipidemia;
[1530] (b) atherosclerosis;
[1531] (c) coronary heart disease;
[1532] (d) stroke;
[1533] (e) insulin resistance; and
[1534] (f) type 2 diabetes.
[1535] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[1536] In other embodiments, said method further comprises the step
of comparing the level of detection of said hybridization for a
first individual to the level of detection of said hybridization
for a second individual.
[1537] In some preferred embodiments, said complementary
polynucleotide is a primer and said hybridization is detected by
detecting the presence of an amplification product comprising the
sequence of said primer. In more preferred embodiments, said method
is RT-PCR.
[1538] In a ninety-ninth aspect, the invention features a GPCR
Fusion Protein construct comprising a constitutively active GPCR
and a G protein, said receptor comprising an amino acid sequence
selected from the group consisting of:
[1539] (a) SEQ. ID. NO.:24 (hRUP19);
[1540] (b) SEQ. ID. NO.:151 (mRUP19); and
[1541] (c) SEQ. ID. NO.:157 (rRUP19);
or an allelic variant or a biologically active fragment of said
amino acid sequence.
[1542] The invention also relates to a GPCR Fusion Protein
construct wherein the threonine at amino acid position 219 of SEQ.
ID. NO.:24 is substituted by lysine.
[1543] In a one hundredth aspect, the invention features a method
of binding a known ligand of RUP19 antilipolytic GPCR to a
polypeptide selected from the group consisting of:
[1544] (a) a polypeptide comprising a contiguous span of at least 6
amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID.
NO.:157;
[1545] (b) a polypeptide comprising a contiguous span of at least
10 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID.
NO.:157;
[1546] (c) a polypeptide comprising a contiguous span of at least
15 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID.
NO.:157;
[1547] (d) a polypeptide comprising a contiguous span of at least
20 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID.
NO.:157;
[1548] (e) a polypeptide comprising a contiguous span of at least
25 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID.
NO.:157;
[1549] (f) a polypeptide comprising a contiguous span of at least
30 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID.
NO.:157;
[1550] (g) a polypeptide comprising a contiguous span of at least
35 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID.
NO.:157;
[1551] (h) a polypeptide comprising a contiguous span of at least
40 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID.
NO.:157;
[1552] (i) a polypeptide comprising a contiguous span of at least
45 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID.
NO.:157; and
[1553] (j) a polypeptide comprising a contiguous span of at least
50 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID.
NO.:157;
or an allelic variant of said polypeptide;
comprising the step of contacting said known ligand with said
polypeptide under conditions which allow said binding to occur.
[1554] In a some embodiments, said known ligand is a modulator of
the GPCR. In some embodiments, said known ligand is an agonist of
the GPCR. In some embodiments, said known ligand is the modulator
of the seventy-second aspect. In some embodiments, said known
ligand is an antibody specific for the GPCR, or a derivative
thereof.
[1555] In other preferred embodiments, said method is used to
identify whether a candidate compound inhibits said binding of said
known ligand to said polypeptide, comprising the steps of: [1556]
(a) contacting said polypeptide with said known ligand, optionally
labeled, in the presence or absence of said candidate compound;
[1557] (b) detecting the complex between said known ligand and said
polypeptide; and [1558] (c) determining whether less of said
complex is formed in the presence of the compound than in the
absence of the compound; wherein said determination is indicative
of the candidate compound being an inhibitor of said binding of
said known ligand to said polypeptide.
[1559] In a some embodiments, said known ligand is a modulator of
the GPCR. In some embodiments, said known ligand is an agonist of
the GPCR. In some embodiments, said known ligand is the modulator
of the seventy-second aspect. In some embodiments, said known
ligand is an antibody specific for the GPCR, or a derivative
thereof.
[1560] In other preferred embodiments, said method is used to
identify whether a candidate compound is an inhibitor of said
binding of said known ligand to said polypeptide, comprising the
steps of:
[1561] (a) contacting said polypeptide with said known ligand,
optionally labeled, in the presence separately of a plurality of
concentrations of said candidate compound for a time sufficient to
allow equilibration of binding;
[1562] (b) measuring unbound ligand and bound ligand; and
[1563] (c) determining K.sub.i for the candidate compound;
[1564] wherein a K.sub.i value of less than 50 .mu.M is indicative
of the candidate compound being an inhibitor of said binding of
said known ligand to said polypeptide. Preferably said K.sub.i
value is less than 25 .mu.M, 10 .mu.M, 5 .mu.M, 1 .mu.M, 750 nM,
500 nM, 400 nM, 300 nM, 250 nM, 200 nM, 150 nM, 100 nM, 90 nM, 80
nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 mM, 20 nM or 10 nM. In preferred
embodiments, K.sub.i determination is made through nonlinear curve
fitting with the program SCTFIT [De Lean et al. (1982) Mol
Pharmacol 21:5-16; cited in Lorenzen et al. (2001) Mol Pharmacol
59:349-357, the disclosures of which are incorporated by reference
herein in their entireties].
[1565] In a some embodiments, said known ligand is a modulator of
the GPCR. In some embodiments, said known ligand is an agonist of
the GPCR. In some embodiments, said known ligand is the modulator
of the seventy-second aspect. In some embodiments, said known
ligand is an antibody specific for the GPCR, or a derivative
thereof.
[1566] In a one hundred first aspect, the invention features a
method of binding an optionally labeled affinity reagent specific
for an antilipolytic GPCR to said receptor in a biological sample,
said receptor comprising an amino acid sequence selected from the
group consisting of:
[1567] (a) SEQ. ID. NO.:24 (hRUP19);
[1568] (b) SEQ. ID. NO.:151 (mRUP19); and
[1569] (c) SEQ. ID. NO.:157 (rRUP19);
or an allelic variant, a biologically active mutant, or a
biologically active fragment of said amino acid sequence,
comprising the steps of:
[1570] (a') obtaining said biological sample;
[1571] (b') contacting the affinity reagent with said receptor in
said biological sample; and
[1572] (c') detecting the complex of said affinity reagent with
said receptor.
[1573] In some embodiments, the antilipolytic GPCR has an amino
acid sequence selected from the group consisting of:
[1574] (a) SEQ. ID. NO.:24 (hRUP19);
[1575] (b) SEQ. ID. NO.:151 (mRUP19); and
[1576] (c) SEQ. ID. NO.:157 (rRUP19);
or an allelic variant, a biologically active mutant, or a
biologically active fragment of said amino acid sequence.
[1577] In some embodiments, the antilipolytic GPCR comprises an
active fragment of said amino acid sequence.
[1578] In some embodiments, the antilipolytic GPCR is
endogenous.
[1579] In some embodiments, the antilipolytic GPCR is
recombinant.
[1580] In some embodiments, said biologically active mutant is CART
or EFA. In preferred embodiments, said CART mutant has the amino
acid sequence of SEQ. ID. NO.:24 further substituted at amino acid
position 219 with lysine in place of threonine.
[1581] In preferred embodiments, said G protein is Gi.
[1582] In a some embodiments, said affinity reagent is a modulator
of the GPCR. In some embodiments, said affinity reagent is an
agonist of the GPCR. In some embodiments, said affinity reagent is
the modulator of the seventy-second aspect. In some embodiments,
said affinity reagent is an antibody specific for the GPCR, or a
derivative thereof.
[1583] In further preferred embodiments, said affinity reagent
comprises a label selected from the group consisting of:
[1584] (a) radioisotope;
[1585] (b) enzyme; and
[1586] (c) fluorophore.
In preferred embodiments, said radioisotope is .sup.3H.
[1587] In a one hundred second aspect, the invention features the
method of the one hundred first aspect further comprising the step
of comparing the level of detection of said complex in a first
biological sample to a second level of detection of said complex in
a second biological sample.
[1588] In a one hundred third aspect, the invention features the
method of the one hundred second aspect wherein the relationship
between said first and second biological samples is selected from
the group consisting of:
[1589] (a) said second biological sample is a replicate of said
first biological sample;
[1590] (b) said first biological sample was obtained prior to an
experimental intervention whereas said second biological sample was
obtained after the experimental intervention, from the same
individual;
[1591] (c) said second biological sample was obtained at a
different time point after an experimental intervention than was
said first biological sample, from the same individual;
[1592] (d) said second biological sample corresponds to a different
subcellular compartment than does said first biological sample;
[1593] (e) said second biological sample represents a different
cell type than does said first biological sample;
[1594] (f) said second biological sample corresponds to a different
tissue than does said first biological sample;
[1595] (g) said second biological sample was obtained from a
different individual than was said first biological sample;
[1596] (h) said second biological sample was obtained at a
different point in time than was said first biological sample, from
the same individual;
[1597] (i) said first biological samples was obtained from a normal
individual, whereas said second biological sample was obtained from
an individual having a metabolic-related disorder;
[1598] (j) said first biological sample was obtained from a normal
individual, whereas said second biological sample was obtained from
an individual having a disorder in lipid metabolism;
[1599] (k) said first biological sample was obtained before a
therapeutic intervention whereas said second biological sample was
obtained after the therapeutic intervention, from the same
individual;
[1600] (l) said second biological sample was obtained at a
different time point after therapeutic intervention than was said
first biological sample, from the same individual; and
[1601] (m) said first biological sample was not exposed to a
compound, whereas said second biological sample was exposed to said
compound.
[1602] In a one hundred fourth aspect, the invention features a
method of identifying whether a candidate compound is a modulator
of an antilipolytic GPCR, said receptor comprising the amino acid
sequence of SEQ. ID. NO.:8 (hRUP11);
or an allelic variant, a biologically active mutant, or a
biologically active fragment of said amino acid sequence;
comprising the steps of:
[1603] (a) contacting the candidate compound with the receptor;
[1604] (b) determining whether the receptor functionality is
modulated;
wherein a change in receptor functionality is indicative of the
candidate compound being a modulator of an antilipolytic GPCR.
[1605] In some embodiments, said antilipolytic GPCR is
endogenous.
[1606] In some preferred embodiments, said antilipolytic GPCR is
recombinant.
[1607] Preferred said identified modulator binds to said GPCR.
[1608] In some embodiments, said contacting is carried out in the
presence of a known ligand of the GPCR. In some embodiments, said
known ligand is an agonist of the GPCR.
[1609] The invention also relates to a method of identifying
whether a candidate compound is a modulator of lipolysis,
comprising the steps of: [1610] (a) contacting the candidate
compound with a GPCR comprising the amino acid sequence of SEQ. ID.
NO.:8 (hRUP11); or an allelic variant, a biologically active
mutant, or a biologically active fragment of said amino acid
sequence; and [1611] (b) determining whether the receptor
functionality is modulated; wherein a change in receptor
functionality is indicative of the candidate compound being a
modulator of lipolysis.
[1612] In some embodiments, said GPCR is endogenous.
[1613] In some preferred embodiments, said GPCR is recombinant.
[1614] Preferred said identified modulator binds to said GPCR.
[1615] In some embodiments, said contacting is carried out in the
presence of a known ligand of the GPCR. In some embodiments, said
known ligand is an agonist of the GPCR.
[1616] The invention also relates to a method of determining
whether a candidate compound is a modulator of an antilipolytic
GPCR, comprising the steps of: [1617] (a) culturing antilipolytic
GPCR-expressing host cells under conditions that would allow
expression of a recombinant antilipolytic GPCR, said host cells
being transfected with a polynucleotide encoding said recombinant
antilipolytic GPCR comprising the amino acid sequence of SEQ. ID.
NO.:8 (hRUP11); or an allelic variant, a biologically active
mutant, or a biologically active fragment of said amino acid
sequence; [1618] (b) contacting the antilipolytic GPCR-expressing
host cells of step (a) with the candidate compound; [1619] (c)
contacting control host cells with the candidate compound of step
(b), wherein said control host cells do not express recombinant
antilipolytic GPCR protein; [1620] (d) measuring the modulating
effect of the candidate compound which interacts with the
recombinant antilipolytic GPCR from the host cells of step (a) and
control host cells of step (c); and [1621] (e) comparing the
modulating effect of the test compound on the host cells and
control host cells.
[1622] The invention also relates to a method of determining
whether a candidate compound is a modulator of an antilipolytic
GPCR, comprising the steps of: [1623] (a) culturing antilipolytic
GPCR-expressing host cells under conditions that would allow
expression of a recombinant antilipolytic GPCR, said host cells
being transfected with a polynucleotide encoding said recombinant
antilipolytic GPCR comprising the amino acid sequence of SEQ. ID.
NO.:8 (hRUP11); or an allelic variant, a biologically active
mutant, or a biologically active fragment of said amino acid
sequence; [1624] (b) contacting a first population of antilipolytic
GPCR-expressing cells of step (a) with a known ligand of said
antilipolytic GPCR; [1625] (c) contacting a second population of
antilipolytic GPCR-expressing cells of step (a) with the candidate
compound and with the known antilipolytic GPCR ligand; [1626] (d)
contacting control host cells with the candidate compound of step
(c), wherein said control host cells do not express recombinant
antilipolytic GPCR protein; [1627] (e) measuring the modulating
effect of the candidate compound, which interacts with recombinant
antilipolytic GPCR, in the presence and absence of the known
antilipolytic GPCR ligand, from the cells of step (b), step (c) and
step (d); and [1628] (f) comparing the modulating effect of the
candidate compound as determined from step (b), step (c) and step
(d).
[1629] In some embodiments, said known ligand is an agonist of the
GPCR.
[1630] The invention also relates to a method of determining
whether a candidate compound is a modulator of an antilipolytic
GPCR, comprising the steps of: [1631] (a) culturing antilipolytic
GPCR-expressing host cells under conditions that would allow
expression of a recombinant antilipolytic GPCR, said host cells
being transfected with a polynucleotide encoding said recombinant
antilipolytic GPCR comprising the amino acid sequence of SEQ. ID.
NO.:8 (hRUP11); or an allelic variant, a biologically active
mutant, or a biologically active fragment of said amino acid
sequence; [1632] (b) contacting a first population of the
antilipolytic GPCR-expressing host cells of step (a) with the
candidate compound; [1633] (c) not contacting a second population
of the antilipolytic GPCR-expressing cells of step (a) with the
candidate compound of step (b); [1634] (d) contacting control host
cells to the candidate compound of step (b), wherein said control
host cells do not express recombinant antilipolytic GPCR protein;
[1635] (e) measuring the modulating effect of the candidate
compound, which interacts with recombinant antilipolytic GPCR
protein, from the cells of step (b) and step (c) and from the cells
of step (d); and [1636] (f) comparing the modulating effect of the
candidate compound as determined from step (b) and step (c) and
from step (d).
[1637] In some embodiments, the antilipolytic GPCR has the amino
acid sequence of SEQ. ID. NO.:8 (hRUP11); or an allelic variant, a
biologically active mutant, or a biologically active fragment of
said amino acid sequence.
[1638] In some embodiments, the antilipolytic GPCR comprises a
biologically active fragment of said amino acid sequence.
[1639] In some embodiments, said biologically active mutant is CART
or EFA. In preferred embodiments, said CART mutant has the amino
acid sequence of SEQ. ID. NO.:8 further substituted at amino acid
position 294 with lysine in place of methionine.
[1640] In preferred embodiments, said G protein is Gi.
[1641] In other preferred embodiments, said determining is through
the use of a Melanophore assay.
[1642] In other preferred embodiments, said determining is through
the measurement of the level of a second messenger selected from
the group consisting of cyclic AMP (cAMP), cyclic GMP (cGMP),
inositol triphosphate (IP.sub.3), diacylglycerol (DAG), and
Ca.sup.2+. In further preferred embodiments, said second messenger
is cAMP. In more preferred embodiments, the level of the cAMP is
reduced. In some embodiments, said measurement of cAMP is carried
out with membrane comprising said GPCR.
[1643] In other preferred embodiments, said determining is through
the measurement of an activity up-regulated or down-regulated by a
reduction in intracellular cAMP level. In further preferred
embodiments, said down-regulated activity is intracellular
lipolysis. In other further preferred embodiments, said
down-regulated activity is hormone sensitive lipase activity. In
other further preferred embodiments, said up-regulated activity is
adiponectin secretion.
[1644] In other preferred embodiments, said determining is through
CRE-reporter assay. In preferred embodiments, said reporter is
luciferase. In some embodiments, said reporter is
.beta.-galactosidase.
[1645] In other preferred embodiments, said recombinant host cell
further comprises promiscuous G alpha 15/16 or chimeric Gq/Gi alpha
subunit and said determining is through measurement of
intracellular Ca.sup.2+. In preferred embodiments, said Ca.sup.2+
measurement is carried out by FLIPR.
[1646] In other preferred embodiments, said recombinant host cell
further comprises promiscuous G alpha 15/16 or chimeric Gq/Gi alpha
subunit and said determining is through measurement of
intracellular IP.sub.3.
[1647] In other preferred embodiments, said determining is through
the measurement of GTP.gamma.S binding to membrane comprising said
GPCR. In further preferred embodiments, said GTP.gamma.S is labeled
with [.sup.35S].
[1648] In other preferred embodiments, said method further
comprises the step of comparing the modulation of the receptor
caused by the candidate compound to a second modulation of the
receptor caused by contacting the receptor with a known modulator
of the receptor. In some preferred embodiments, said known
modulator is an agonist.
[1649] In a one hundred fifth aspect, the invention features a
modulator of an antilipolytic GPCR identified according to the
method of the one hundred fourth aspect.
[1650] In some preferred embodiments, said modulator is selected
from the group consisting of agonist, partial agonist, inverse
agonist and antagonist. More preferably, said modulator is an
agonist. In some embodiments, said modulator is a partial
agonist.
[1651] In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding assay
carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP11 polypeptide having the amino acid
sequence of SEQ. ID. NO.:8. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[1652] In some embodiments, said modulator is selective for the
GPCR.
[1653] In some embodiments, said modulator is antilipolytic.
[1654] In some embodiments, said modulator is orally bioavailable.
In some embodiments, said oral bioavailability is at least 1%, at
least 5%, at least 10%, at least 15%, at least 20%, at least 25%,
at least 30%, at least 35%, at least 40%, or at least 45% relative
to intraperitoneal administration. In some preferred embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intraperitoneal administration. In
some embodiments, said oral bioavailability is at least 1%, at
least 5%, at least 10%, at least 15%, at least 20%, at least 25%,
at least 30%, at least 35%, at least 40%, or at least 45% relative
to intravenous administration. In some preferred embodiments, said
oral bioavailability is at least 1%, at least 5%, at least 10%, or
at least 15% relative to intravenous administration.
[1655] In highly less preferred embodiments, said modulator is an
antibody or derivative thereof.
[1656] In a one hundred sixth aspect, the invention features a
method of modulating the activity of an antilipolytic GPCR, said
receptor comprising the amino acid sequence of SEQ. ID. NO.:8
(hRUP11); or an allelic variant, a biologically active mutant, or a
biologically active fragment of said amino acid sequence;
comprising the step of contacting the receptor with the modulator
of the one hundred fifth aspect.
[1657] In some embodiments, the antilipolytic GPCR has the amino
acid sequence of SEQ. ID. NO.:8 (hRUP11); or an allelic variant, a
biologically active mutant, or a biologically active fragment of
said amino acid sequence.
[1658] In some embodiments, the antilipolytic GPCR comprises an
active fragment of said amino acid sequence.
[1659] In some embodiments, the antilipolytic GPCR is
endogenous.
[1660] In some embodiments, the antilipolytic GPCR is
recombinant.
[1661] In some embodiments, said biologically active mutant is CART
or EFA. In preferred embodiments, said CART mutant has the amino
acid sequence of SEQ. ID. NO.:8 further substituted at amino acid
position 294 with lysine in place of methionine.
[1662] In preferred embodiments, said G protein is Gi.
[1663] In some preferred embodiments, said modulator is an
agonist.
[1664] In preferred embodiments, said modulator is selective for
the GPCR.
[1665] In other preferred embodiments, said contacting comprises
administration of the modulator to a membrane comprising the
receptor.
[1666] In other preferred embodiments, said contacting comprises
administration of the modulator to a cell or tissue comprising the
receptor.
[1667] In other preferred embodiments, said contacting comprises
administration of the modulator to an individual comprising the
receptor. In more preferred embodiments, said individual is a
mammal. In other more preferred embodiments, said mammal is a
horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat, non-human
primate or human. Yet more preferred is mouse, rat or human. Most
preferred is human.
[1668] In some preferred embodiments, said modulator is selective
for the GPCR.
[1669] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[1670] In some preferred embodiments, said modulator is
antilipolytic.
[1671] In some preferred embodiments, said modulator is an
agonist.
[1672] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP11 polypeptide having the amino acid
sequence of SEQ. ID. NO.:8. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[1673] In some preferred embodiments, said administration is
oral.
[1674] In preferred embodiments, said modulator is an agonist and
said individual is in need of prevention of or treatment for a
metabolic-related disorder selected from the group consisting
of:
[1675] (a) dyslipidemia;
[1676] (b) atherosclerosis;
[1677] (c) coronary heart disease;
[1678] (d) stroke;
[1679] (e) insulin resistance; and
[1680] (f) type 2 diabetes.
[1681] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[1682] In other embodiments, said modulator is an inverse agonist
and said metabolic-related disorder relates to a low level of
plasma free fatty acids.
[1683] In other preferred embodiments, said modulator is an agonist
and said individual is in need of a change in lipid metabolism
selected from the group consisting of:
[1684] (a) a decrease in the level of plasma triglycerides;
[1685] (b) a decrease in the level of plasma free fatty acids;
[1686] (c) a decrease in the level of plasma cholesterol;
[1687] (d) a decrease in the level of LDL-cholesterol;
[1688] (e) an increase in the level of HDL-cholesterol;
[1689] (f) a decrease in the total cholesterol/HDL-cholesterol
ratio; and
[1690] (g) an increase in the level of plasma adiponectin.
[1691] In other preferred embodiments, said needed change in lipid
metabolism is a decrease in the postprandial increase in plasma
free fatty acids due to a high fat meal or an inhibition of the
progression from impaired glucose tolerance to insulin
resistance.
[1692] In some embodiments, the modulator is an inverse agonist and
the needed change in lipid metabolism is an increase in the level
of plasma free fatty acids.
[1693] In other preferred embodiments, said modulator is an agonist
and said individual is a mouse genetically predisposed to a
metabolic-related disorder selected from the group consisting
of:
[1694] (a) dyslipidemia;
[1695] (b) atherosclerosis;
[1696] (c) coronary heart disease;
[1697] (d) stroke;
[1698] (e) insulin resistance; and
[1699] (f) type 2 diabetes.
[1700] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure.
[1701] In further preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[1702] In further preferred embodiments, said method is used to
identify whether said agonist has therapeutic efficacy for the
treatment of a metabolic-related disorder selected from the group
consisting of:
[1703] (a) dyslipidemia;
[1704] (b) atherosclerosis;
[1705] (c) coronary heart disease;
[1706] (d) stroke;
[1707] (e) insulin resistance; and
[1708] (f) type 2 diabetes;
comprising the steps of:
[1709] (a') administering or not administering said agonist to the
mouse; and
[1710] (b') determining whether the disorder is prevented, delayed,
or made less severe on administering said agonist compared to not
administering said agonist; wherein said determination is
indicative of said agonist having therapeutic efficacy.
[1711] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[1712] In other preferred embodiments, said modulator is an agonist
and said individual is a rat genetically predisposed to a
metabolic-related disorder selected from the group consisting
of:
[1713] (a) dyslipidemia;
[1714] (b) atherosclerosis;
[1715] (c) coronary heart disease;
[1716] (d) stroke;
[1717] (e) insulin resistance; and
[1718] (f) type 2 diabetes.
[1719] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In further preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[1720] In further preferred embodiments, said method is used to
identify whether said agonist has therapeutic efficacy for the
treatment of a metabolic-related disorder selected from the group
consisting of:
[1721] (a) dyslipidemia;
[1722] (b) atherosclerosis;
[1723] (c) coronary heart disease;
[1724] (d) stroke;
[1725] (e) insulin resistance; and
[1726] (f) type 2 diabetes;
comprising the steps of:
[1727] (a') administering or not administering said agonist to the
rat; and
[1728] (b') determining whether the disorder is prevented, delayed,
or made less severe on administering said agonist compared to not
administering said agonist; wherein said determination is
indicative of said agonist having therapeutic efficacy.
[1729] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[1730] In a one hundred seventh aspect, the invention features a
method of preventing or treating a disorder of lipid metabolism in
an individual comprising contacting a therapeutically effective
amount of the modulator of the one hundred fifth aspect with an
antilipolytic GPCR, said receptor comprising the amino acid
sequence of SEQ. ID. NO.:8 (hRUP11); or an allelic variant or
biologically active fragment of said amino acid sequence.
[1731] In some preferred embodiments, said modulator is selective
for the GPCR.
[1732] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[1733] In some preferred embodiments, said modulator is
antilipolytic.
[1734] In some preferred embodiments, said modulator is an
agonist.
[1735] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP11 polypeptide having the amino acid
sequence of SEQ. ID. NO.:8. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[1736] In some preferred embodiments, said contacting comprises
oral administration of said modulator to said individual.
[1737] In preferred embodiment, said modulator is an agonist and
said disorder of lipid metabolism is selected from the group
consisting of:
[1738] (a) elevated level of plasma triglycerides;
[1739] (b) elevated level of plasma free fatty acids;
[1740] (c) elevated level of plasma cholesterol;
[1741] (d) elevated level of LDL-cholesterol;
[1742] (e) reduced level of HDL-cholesterol;
[1743] (f) elevated total cholesterol/HDL-cholesterol ratio;
and
[1744] (g) reduced level of plasma adiponectin.
[1745] In a one hundred eighth aspect, the invention features a
method of preventing or treating a metabolic-related disorder in an
individual comprising contacting a therapeutically effective amount
of the modulator of the one hundred fifth aspect with an
antilipolytic GPCR, said receptor comprising the amino acid
sequence of SEQ. ID. NO.:8 (hRUP11); or an allelic variant or
biologically active fragment of said amino acid sequence.
[1746] In some preferred embodiments, said modulator is selective
for the GPCR.
[1747] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[1748] In some preferred embodiments, said modulator is
antilipolytic.
[1749] In some preferred embodiments, said modulator is an
agonist.
[1750] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP-YS binding assay
carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP111 polypeptide having the amino acid
sequence of SEQ. ID. NO.:8. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[1751] In some preferred embodiments, said contacting comprises
oral administration of said modulator to said individual.
[1752] In preferred embodiment, said modulator is an agonist and
said metabolic-related disorder is selected from the group
consisting of:
[1753] (a) dyslipidemia;
[1754] (b) atherosclerosis;
[1755] (c) coronary heart disease;
[1756] (d) stroke;
[1757] (e) insulin resistance; and
[1758] (f) type 2 diabetes.
[1759] In a one hundred ninth aspect, the invention features a
method of preparing a composition which comprises identifying a
modulator of an antilipolytic GPCR and then admixing a carrier and
the modulator, wherein the modulator is identifiable by the method
of the one hundred fourth aspect.
[1760] In some preferred embodiments, said modulator is selected
from the group consisting of agonist, partial agonist, inverse
agonist and antagonist. More preferably, said modulator is an
agonist. In some embodiments, said modulator is a partial
agonist.
[1761] In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding assay
carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP11 polypeptide having the amino acid
sequence of SEQ. ID. NO.:8. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[1762] In some embodiments, said modulator is selective for the
GPCR.
[1763] In some embodiments, said modulator is antilipolytic.
[1764] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to either intraperitoneal or intravenous
administration. In some preferred embodiments, said oral
bioavailability is at least 1%, at least 5%, at least 10%, or at
least 15% relative to either intraperitoneal or intravenous
administration.
[1765] In an one hundred tenth aspect, the invention features a
pharmaceutical or physiologically acceptable composition
comprising, consisting essentially of, or consisting of the
modulator of the one hundred fifth aspect. In preferred
embodiments, said modulator is an agonist.
[1766] In some preferred embodiments, said modulator is selective
for the GPCR.
[1767] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[1768] In some preferred embodiments, said modulator is
antilipolytic.
[1769] In some preferred embodiments, said modulator is an
agonist.
[1770] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP11 polypeptide having the amino acid
sequence of SEQ. ID. NO.:8. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[1771] In a one hundred eleventh aspect, the invention features a
method of changing lipid metabolism comprising providing or
administering to an individual in need of said change said
pharmaceutical or physiologically acceptable composition of the one
hundred tenth aspect, said needed change in lipid metabolism
selected from the group consisting of:
[1772] (a) a decrease in the level of plasma triglycerides;
[1773] (b) a decrease in the level of plasma free fatty acids;
[1774] (c) a decrease in the level of plasma cholesterol;
[1775] (d) a decrease in the level of LDL-cholesterol;
[1776] (e) an increase in the level of HDL-cholesterol;
[1777] (f) a decrease in the total cholesterol/HDL-cholesterol
ratio; and
[1778] (g) an increase in the level of plasma adiponectin.
[1779] In preferred embodiments, a therapeutically effect amount of
said pharmaceutical or physiologically acceptable composition is
provided or administered to said individual.
[1780] In some preferred embodiments, said providing or
administering of said pharmaceutical or physiologically acceptable
composition is oral.
[1781] In other preferred embodiments, said needed change in lipid
metabolism is a decrease in the postprandial increase in plasma
free fatty acids due to a high fat meal or an inhibition of the
progression from impaired glucose tolerance to insulin
resistance.
[1782] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[1783] In a one hundred twelfth aspect, the invention features a
method of preventing or treating a metabolic-related disorder
comprising providing or administering to an individual in need of
said treatment said pharmaceutical or physiologically acceptable
composition of the one hundred tenth aspect, said metabolic-related
disorder selected from the group consisting of:
[1784] (a) dyslipidemia;
[1785] (b) atherosclerosis;
[1786] (c) coronary heart disease;
[1787] (d) stroke;
[1788] (e) insulin resistance; and
[1789] (f) type 2 diabetes.
[1790] In preferred embodiments, a therapeutically effect amount of
said pharmaceutical or physiologically acceptable composition is
provided or administered to said individual.
[1791] In some preferred embodiments, said providing or
administering of said pharmaceutical or physiologically acceptable
composition is oral.
[1792] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, stroke,
Syndrome X, and heart disease. Heart disease includes, but is not
limited to, cardiac insufficiency, coronary insufficiency, and high
blood pressure. In other preferred embodiments, said
metabolic-related disorder is hyperlipidemia.
[1793] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[1794] In an one hundred thirteenth aspect, the invention features
a method of using the modulator of the one hundred fifth aspect for
the preparation of a medicament for the treatment of a disorder in
lipid metabolism in an individual.
[1795] In some preferred embodiments, said modulator is selective
for the GPCR.
[1796] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[1797] In some preferred embodiments, said modulator is
antilipolytic.
[1798] In some preferred embodiments, said modulator is an
agonist.
[1799] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP11 polypeptide having the amino acid
sequence of SEQ. ID. NO.:8. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[1800] In some preferred embodiments, said treatment comprises oral
administration of said medicament to said individual.
[1801] In preferred embodiments, said modulator is an agonist and
said disorder in lipid metabolism is selected from the group
consisting of:
[1802] (a) elevated level of plasma triglycerides;
[1803] (b) elevated level of plasma free fatty acids;
[1804] (c) elevated level of plasma cholesterol;
[1805] (d) elevated level of LDL-cholesterol;
[1806] (e) reduced level of HDL-cholesterol;
[1807] (f) elevated total cholesterol/HDL-cholesterol ratio;
and
[1808] (g) reduced level of plasma adiponectin.
[1809] In other preferred embodiments, said disorder in lipid
metabolism is an elevated postprandial increase in plasma free
fatty acids due to a high fat meal or a progression from impaired
glucose tolerance to insulin resistance.
[1810] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[1811] In a one hundred fourteenth aspect, the invention features a
method of using the modulator of the one hundred fifth aspect for
the preparation of a medicament for the treatment of a
metabolic-related disorder in an individual.
[1812] In some preferred embodiments, said modulator is selective
for the GPCR.
[1813] In some preferred embodiments, said modulator is orally
bioavailable. In some embodiments, said oral bioavailability is at
least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intraperitoneal administration. In some
embodiments, said oral bioavailability is at least 1%, at least 5%,
at least 10%, or at least 15% relative to intraperitoneal
administration. In some embodiments, said oral bioavailability is
at least 1%, at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, or at least
45% relative to intravenous administration. In some embodiments,
said oral bioavailability is at least 1%, at least 5%, at least
10%, or at least 15% relative to intravenous administration.
[1814] In some preferred embodiments, said modulator is
antilipolytic.
[1815] In some preferred embodiments, said modulator is an
agonist.
[1816] In some preferred embodiments, said modulator is an agonist
with an EC.sub.50 of less than 1000 .mu.M in GTP.gamma.S binding
assay carried out with membrane from stably transfected CHO cells
expressing recombinant hRUP11 polypeptide having the amino acid
sequence of SEQ. ID. NO.:8. In some embodiments, said modulator is
an agonist with an EC.sub.50 of less than 900 .mu.M in said assay.
In some embodiments, said modulator is an agonist with an EC.sub.50
of less than 800 .mu.M in said assay. In some embodiments, said
modulator is an agonist with an EC.sub.50 of less than 700 .mu.M in
said assay. In some embodiments, said modulator is an agonist with
an EC.sub.50 of less than 600 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 550 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 500 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 450 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 400 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 350 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 300 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 250 .mu.M in said assay. In some embodiments, said modulator
is an agonist with an EC.sub.50 of less than 200 .mu.M in said
assay. In some embodiments, said modulator is an agonist with an
EC.sub.50 of less than 150 .mu.M in said assay. In some
embodiments, said modulator is an agonist with an EC.sub.50 of less
than 100 .mu.M in said assay. In some preferred embodiments, said
modulator is an agonist with an EC.sub.50 in said assay of less
than a value selected from the interval of 600 .mu.M to 1000
.mu.M.
[1817] In some preferred embodiments, said treatment comprises oral
administration of said medicament to said individual.
[1818] In preferred embodiments, said modulator is an agonist and
said metabolic-related disorder is selected from the group
consisting of:
[1819] (a) dyslipidemia;
[1820] (b) atherosclerosis;
[1821] (c) coronary heart disease;
[1822] (d) stroke;
[1823] (e) insulin resistance; and
[1824] (f) type 2 diabetes.
[1825] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[1826] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[1827] In a one hundred fifteenth aspect, the invention features a
method of identifying whether a candidate compound binds to an
antilipolytic GPCR, said receptor comprising the amino acid
sequence of SEQ. ID. NO.:8 (hRUP11); or an allelic variant or a
biologically active fragment of said amino acid sequence;
comprising the steps of: [1828] (a) contacting the receptor with a
labeled reference compound known to bind to the GPCR in the
presence or absence of the candidate compound; and [1829] (b)
determining whether the binding of said labeled reference compound
to the receptor is inhibited in the presence of the candidate
compound; wherein said inhibition is indicative of the candidate
compound binding to an antilipolytic GPCR.
[1830] In some embodiments, the antilipolytic acid GPCR comprises a
biologically active fragment of said amino acid sequence.
[1831] In some embodiments, the antilipolytic GPCR is
endogenous.
[1832] In some embodiments, the antilipolytic GPCR is
recombinant.
[1833] In preferred embodiments, said G protein is Gi.
[1834] In some preferred embodiments, said reference compound is
the modulator of the one hundred fifth aspect.
[1835] In other embodiments, said reference compound is an antibody
specific for the GPCR, or a derivative thereof.
[1836] In preferred embodiments, said reference compound comprises
a label selected from the group consisting of:
[1837] (a) radioisotope;
[1838] (b) enzyme; and
[1839] (c) fluorophore.
In some preferred embodiments, said label is .sup.3H.
[1840] In other embodiments, said method further comprises the step
of comparing the level of inhibition of binding of a labeled first
reference compound by the candidate compound to a second level of
inhibition of binding of said labeled first reference compound by a
second reference compound known to bind to the GPCR.
[1841] In a one hundred sixteenth aspect, the invention features a
method of making a transgenic mouse, comprising the step of
engineering said mouse to carry as part of its own genetic material
the gene encoding the human antilipolytic GPCR polypeptide of SEQ.
ID. NO.:8 (hRUP11).
[1842] In some preferred embodiments, expression of said gene is
placed under the control of an essentially adipocyte specific
promoter.
[1843] In a one hundred seventeenth aspect, the invention features
the transgenic mouse according to the method of the one hundred
sixteenth aspect.
[1844] In a one hundred eighteenth aspect, the invention features a
method of using the transgenic mouse of the one hundred seventeenth
aspect to identify whether an agonist of said human receptor has
therapeutic efficacy for the treatment of a disorder of lipid
metabolism selected from the group consisting of:
[1845] (a) elevated level of plasma triglycerides;
[1846] (b) elevated level of plasma free fatty acids;
[1847] (c) elevated level of plasma cholesterol;
[1848] (d) elevated level of LDL-cholesterol;
[1849] (e) reduced level of HDL-cholesterol;
[1850] (f) elevated total cholesterol/HDL-cholesterol ratio;
and
[1851] (g) reduced level of plasma adiponectin;
comprising the steps of:
[1852] (a') administering or not administering the agonist to the
mouse; and
[1853] (b') determining whether on administering the agonist there
is a change selected from the group consisting of: [1854] (i) a
decrease in the level of plasma triglycerides; [1855] (ii) a
decrease in the level of plasma free fatty acids; [1856] (iii) a
decrease in the level of plasma cholesterol; [1857] (iv) a decrease
in the level of LDL-cholesterol; [1858] (v) an increase in the
level of HDL-cholesterol; [1859] (vi) a decrease in the total
cholesterol/HDL-cholesterol ratio; and [1860] (vii) an increase in
the level of plasma adiponectin;
[1861] wherein said change is indicative of the agonist having
therapeutic efficacy.
[1862] In a one hundred nineteenth aspect, the invention features a
method of using the transgenic mouse of the one hundred seventeenth
aspect to identify whether an agonist of said human receptor has
therapeutic efficacy for the treatment of a metabolic-related
disorder selected from the group consisting of:
[1863] (a) dyslipidemia;
[1864] (b) atherosclerosis;
[1865] (c) coronary heart disease;
[1866] (d) stroke;
[1867] (e) insulin resistance; and
[1868] (f) type 2 diabetes;
comprising the steps of:
[1869] (a') administering or not administering the agonist to the
mouse; and
[1870] (b') determining whether on administering the agonist there
is a change selected from the group consisting of: [1871] (i) a
decrease in the level of plasma triglycerides; [1872] (ii) a
decrease in the level of plasma free fatty acids; [1873] (iii) a
decrease in the level of plasma cholesterol; [1874] (iv) a decrease
in the level of LDL-cholesterol; [1875] (v) an increase in the
level of HDL-cholesterol; [1876] (vi) a decrease in the total
cholesterol/HDL-cholesterol ratio; and [1877] (vii) an increase in
the level of plasma adiponectin; wherein said change is indicative
of the agonist having therapeutic efficacy.
[1878] In a one hundred twentieth aspect, the invention features a
method of making a transgenic rat, comprising the step of
engineering said rat to carry as part of its own genetic material
the gene encoding the human antilipolytic GPCR polypeptide of SEQ.
ID. NO.:8 (hRUP11).
[1879] In some preferred embodiments, expression of said gene is
placed under the control of an essentially adipocyte specific
promoter.
[1880] In a one hundred twenty-first aspect, the invention features
the transgenic rat according to the method of the one hundred
twentieth aspect.
[1881] In a one hundred twenty-second aspect, the invention
features a method of using the transgenic rat of the one hundred
twenty-first aspect to identify whether an agonist of said human
receptor has therapeutic efficacy for the treatment of a disorder
of lipid metabolism selected from the group consisting of:
[1882] (a) elevated level of plasma triglycerides;
[1883] (b) elevated level of plasma free fatty acids;
[1884] (c) elevated level of plasma cholesterol;
[1885] (d) elevated level of LDL-cholesterol;
[1886] (e) reduced level of HDL-cholesterol;
[1887] (f) elevated total cholesterol/HDL-cholesterol ratio;
and
[1888] (g) reduced level of plasma adiponectin;
comprising the steps of:
[1889] (a') administering or not administering the agonist to the
rat; and
[1890] (b') determining whether on administering the agonist there
is a change selected from the group consisting of: [1891] (i) a
decrease in the level of plasma triglycerides; [1892] (ii) a
decrease in the level of plasma free fatty acids; [1893] (iii) a
decrease in the level of plasma cholesterol; [1894] (iv) a decrease
in the level of LDL-cholesterol; [1895] (v) an increase in the
level of HDL-cholesterol; [1896] (vi) a decrease in the total
cholesterol/HDL-cholesterol ratio; and [1897] (vii) an increase in
the level of plasma adiponectin; wherein said change is indicative
of the agonist having therapeutic efficacy.
[1898] In a one hundred twenty-third aspect, the invention features
a method of using the transgenic rat of the one hundred
twenty-first aspect to identify whether an agonist of said human
receptor has therapeutic efficacy for the treatment of a
metabolic-related disorder selected from the group consisting
of:
[1899] (a) dyslipidemia;
[1900] (b) atherosclerosis;
[1901] (c) coronary heart disease;
[1902] (d) stroke;
[1903] (e) insulin resistance; and
[1904] (f) type 2 diabetes;
comprising the steps of:
[1905] (a') administering or not administering the agonist to the
rat; and
[1906] (b') determining whether on administering the agonist there
is a change selected from the group consisting of: [1907] (i) a
decrease in the level of plasma triglycerides; [1908] (ii) a
decrease in the level of plasma free fatty acids; [1909] (iii) a
decrease in the level of plasma cholesterol; [1910] (iv) a decrease
in the level of LDL-cholesterol; [1911] (v) an increase in the
level of HDL-cholesterol; [1912] (vi) a decrease in the total
cholesterol/HDL-cholesterol ratio; and [1913] (vii) an increase in
the level of plasma adiponectin; wherein said change is indicative
of the agonist having therapeutic efficacy.
[1914] In a one hundred twenty-fourth aspect, the invention
features an isolated, purified or recombinant RUP11 polynucleotide
selected from the group consisting of:
[1915] (a) a polynucleotide comprising a contiguous span of at
least 75 nucleotides of SEQ.ID. NO.:7, or an allelic variant of
said polynucleotide;
[1916] (b) a polynucleotide comprising a contiguous span of at
least 150 nucleotides of SEQ. ID. NO.:7, or an allelic variant of
said polynucleotide;
[1917] (c) a polynucleotide comprising a contiguous span of at
least 250 nucleotides of SEQ. ID. NO.:7, or an allelic variant of
said polynucleotide;
[1918] (d) a polynucleotide comprising a contiguous span of at
least 350 nucleotides of SEQ. ID. NO.:7, or an allelic variant of
said polynucleotide;
[1919] (e) a polynucleotide comprising a contiguous span of at
least 500 nucleotides of SEQ. ID. NO.:7, or an allelic variant of
said polynucleotide;
[1920] (f) a polynucleotide comprising a contiguous span of at
least 750 nucleotides of SEQ. ID. NOs.:7, or an allelic variant of
said polynucleotide;
[1921] (g) a polynucleotide comprising a contiguous span of at
least 1000 nucleotides of SEQ. ID. NO.:7, or an allelic variant of
said polynucleotide;
[1922] (h) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 20 amino acids of SEQ. ID. NO.:8 or an
allelic variant of said polypeptide;
[1923] (i) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 30 amino acids of SEQ. ID. NO.:8, or an
allelic variant of said polypeptide;
[1924] (j) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 40 amino acids of SEQ. ID. NO.:8, or an
allelic variant of said polypeptide;
[1925] (k) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 50 amino acids of SEQ. ID. NO.:8, or an
allelic variant of said polypeptide;
[1926] (l) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 75 amino acids of SEQ. ID. NO.:8, or an
allelic variant of said polypeptide;
[1927] (m) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 100 amino acids of SEQ. ID. NO.:8, or
an allelic variant of said polypeptide;
[1928] (n) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 150 amino acids of SEQ. ID. NO.:8, or
an allelic variant of said polypeptide;
[1929] (O) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 200 amino acids of SEQ. ID. NO.:8, or
an allelic variant of said polypeptide;
[1930] (p) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 250 amino acids of SEQ. ID. NO.:8, or
an allelic variant of said polypeptide; and
[1931] (q) a polynucleotide encoding a polypeptide comprising a
contiguous span of at least 300 amino acids of SEQ. ID. NO.:8, or
an allelic variant of said polypeptide.
[1932] The invention also relates to an isolated, purified or
recombinant RUP11 polynucleotide wherein said polynucleotide is
selected from the group consisting of:
[1933] (a) a polynucleotide comprising the nucleotide sequence of
SEQ. ID. NO.:7 or an allelic variant of said nucleotide
sequence;
[1934] (b) the polynucleotide of SEQ. ID. NO.:7, or an allelic
variant of said polynucleotide;
[1935] (c) a polynucleotide comprising a nucleotide sequence
encoding a polypeptide having the amino acid sequence of SEQ. ID.
NO.:8 or an allelic variant of said amino acid sequence; and
[1936] (d) a polynucleotide encoding a polypeptide having the amino
acid sequence of SEQ. ID. NO.:8, or an allelic variant of said
polypeptide.
[1937] In preferred embodiments, said isolated, purified or
recombinant polynucleotide comprises at least 8 contiguous
nucleotides of a polynucleotide of the present invention. In other
preferred embodiments, said isolated, purified or recombinant
polynucleotide comprises at least 10, 12, 15, 18, 20, 25, 28, 30,
35, 40, 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or
1000 contiguous nucleotides of a polynucleotide of the present
invention. Preferably said polynucleotide encodes full-length RUP11
polypeptide or a biologically active fragment thereof.
[1938] The polynucleotides of the present invention include genomic
polynucleotides comprising RUP11 polynucleotides of the
invention.
[1939] The present invention also relates to a polynucleotide
encoding a fusion protein, wherein said fusion protein comprises an
RUP11 polypeptide of the invention fused to a heterologous
polypeptide. In a preferred embodiment, said polypeptide of the
invention is constitutively active and said heterologous
polypeptide is a G protein. In other embodiments, said heterologous
polypeptide provides an antigenic epitope. In a preferred
embodiment, said heterologous polypeptide provides a hemaglutinin
(HA) antigenic epitope. Methods relating to a polynucleotide
encoding a fusion protein are well known to those of ordinary skill
in the art.
[1940] The polynucleotides of the present invention also include
variant polynucleotides at least 60%, 70%, 80%, 90%, 95%, 96%, 97%,
98% or 99% identical to an RUP11 polynucleotide of the invention.
In a particularly preferred embodiments, polynucleotide sequence
homologies are evaluated using the Basic Local Alignment Search
Tool ("BLAST"), which is well known in the art [See, e.g., Karlin
and Altschul, Proc Natl Acad Sci USA (1990) 87:2264-8; Altschul et
al., J Mol Biol (1990) 215:403-410; Altschul et al., Nature
Genetics (1993) 3:266-72; and Altschul et al., Nucleic Acids Res
(1997) 25:3389-3402; the disclosures of which are incorporated by
reference in their entirety].
[1941] In further preferred embodiments, the invention features the
complement of said polynucleotide.
[1942] In a one hundred twenty-fifth aspect, the invention features
an isolated, purified or recombinant RUP11 polypeptide selected
from the group consisting of:
[1943] (a) a polypeptide comprising a contiguous span of at least
20 amino acids of SEQ. ID. NO.:8 or an allelic variant of said
contiguous span of amino acids;
[1944] (b) a polypeptide comprising a contiguous span of at least
30 amino acids of SEQ. ID. NO.:8 or an allelic variant of said
contiguous span of amino acids;
[1945] (c) a polypeptide comprising a contiguous span of at least
40 amino acids of SEQ. ID. NO.:8 or an allelic variant of said
contiguous span of amino acids;
[1946] (d) a polypeptide comprising a contiguous span of at least
50 amino acids of SEQ. ID. NO.:8 or an allelic variant of said
contiguous span of amino acids;
[1947] (e) a polypeptide comprising a contiguous span of at least
75 amino acids of SEQ. ID. NO.:8 or an allelic variant of said
contiguous span of amino acids;
[1948] (f) a polypeptide comprising a contiguous span of at least
100 amino acids of SEQ. ID. NO.:8 or an allelic variant of said
contiguous span of amino acids;
[1949] (g) a polypeptide comprising a contiguous span of at least
150 amino acids of SEQ. ID. NO.:8 or an allelic variant of said
contiguous span of amino acids;
[1950] (h) a polypeptide comprising a contiguous span of at least
200 amino acids of SEQ. ID. NO.:8 or an allelic variant of said
contiguous span of amino acids;
[1951] (i) a polypeptide comprising a contiguous span of at least
250 amino acids of SEQ. ID. NO.:8 or an allelic variant of said
contiguous span of amino acids; and
[1952] (j) a polypeptide comprising a contiguous span of at least
300 amino acids of SEQ. ID. NO.:8 or an allelic variant of said
contiguous span of amino acids.
[1953] The invention also relates to an isolated, purified or
recombinant RUP11 polypeptide wherein said polypeptide is selected
from the group consisting of:
[1954] (a) a polypeptide comprising the amino acid sequence of SEQ.
ID. NO.:8 or an allelic variant or a biologically active mutant of
said amino acid sequence; and
[1955] (b) the polypeptide having the amino acid sequence of SEQ.
ID. NO.:8 or an allelic variant or a biologically active mutant of
said amino acid sequence; or a biologically active fragment of said
polypeptide.
[1956] In preferred embodiments, said isolated, purified or
recombinant polypeptide comprises at least 6 contiguous amino acids
of an RUP11 polypeptide of the invention. In further embodiments,
said isolated, purified or recombinant polypeptide comprises at
least 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 125, 150,
175, 200, 225, 250, 275 or 300 contiguous amino acids of a
polypeptide of the present invention. Preferably, said polypeptide
is full-length RUP11 polypeptide or an active fragment thereof.
[1957] The present invention also relates to a fusion protein,
wherein said fusion protein comprises an RUP11 polypeptide of the
invention fused to a heterologous polypeptide. In a preferred
embodiment, said polypeptide of the invention is constitutively
active and said heterologous polypeptide is a G protein. In other
preferred embodiments, said heterologous polypeptide provides an
antigenic epitope. In particularly preferred embodiment, said
heterologous polypeptide provides a hemaglutinin (HA) antigenic
epitope. Methods relating to a fusion protein are well known to
those of ordinary skill in the art.
[1958] The polypeptides of the present invention also include
variant polypeptides at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% identical to an RUP11 polypeptide of the
invention. In a particularly preferred embodiments, polypeptide
sequence homologies are evaluated using the Basic Local Alignment
Search Tool ("BLAST"), which is well known in the art [See, e.g.,
Karlin and Altschul, Proc Natl Acad Sci USA (1990) 87:2264-8;
Altschul et al., J Mol Biol (1990) 215:403-410; Altschul et al.,
Nature Genetics (1993) 3:266-72; and Altschul et al., Nucleic Acids
Res (1997) 25:3389-3402; the disclosures of which are incorporated
by reference in their entirety].
[1959] In an one hundred twenty-sixth aspect, the invention
features a composition comprising, consisting essentially of, or
consisting of the RUP11 polypeptide of the one hundred twenty-fifth
aspect.
[1960] In a one hundred twenty-seventh aspect, the invention
features a recombinant vector, said vector comprising, consisting
essentially of, or consisting of the polynucleotide of the one
hundred twenty-fourth aspect. In some embodiments, said vector is a
targeting vector used in a method of inactivating a gene encoding
an antilipolytic GPCR of the invention. In some preferred
embodiments, said vector is used in a method of transient or stable
transfection. In other preferred embodiments, said vector is used
in a method of transgenic expression.
[1961] In particularly preferred embodiment, said vector is an
expression vector for the expression of a an antilipolytic GPCR in
a recombinant host cell wherein said expression vector comprises,
consists essentially of, or consists of the polynucleotide of the
one hundred twenty-fourth aspect.
[1962] Although a variety of expression vectors are available to
those in the art, for purposes of utilization for both the
endogenous and non-endogenous human, mouse and rat GPCRs, it is
most preferred that the vector utilized be pCMV. In some
alternative embodiments as relates to said human, mouse and rat
antilipolytic GPCRs, it is preferred that the vector utilized be an
adenoviral expression vector.
[1963] In a one hundred twenty-eighth aspect, the invention
features a prokaryotic or eukaryotic host cell comprising,
consisting essentially of, or consisting of the recombinant vector
of the one hundred twenty-seventh aspect. In some embodiments, said
host cell is a eukaryotic embryonic stem cell wherein said vector
of the one hundred twenty-seventh aspect has been used in a method
to inactivate a gene encoding an antilipolytic GPCR of the
invention within said cell. In some embodiments, said host cell is
a eukaryotic embryonic somatic cell wherein said vector of the one
hundred twenty-seventh aspect has been used in a method to
inactivate a gene encoding an antilipolytic GPCR of the invention
within said cell. In some preferred embodiments, said host cell is
derived from a mouse or rat made transgenic for a human RUP11
antilipolytic GPCR of the invention. In other preferred
embodiments, said host cell is prokaryotic and has been transformed
using the vector of the one hundred twenty-seventh aspect. In
further preferred embodiments, said host cell is eukaryotic and has
been transiently transfected using the vector of the one hundred
twenty-seventh aspect. In other further preferred embodiments, said
host cell is eukaryotic and has been stably transfected using the
vector of the one hundred twenty-seventh aspect.
[1964] In particularly preferred embodiment, said host cell
expresses a recombinant antilipolytic GPCR wherein said host cell
comprises, consists essentially of, or consists of the expression
vector of the one hundred twenty-seventh aspect.
[1965] A further embodiment includes a prokaryotic or eukaryotic
host cell recombinant for the polynucleotide of the one hundred
twenty-fourth aspect.
[1966] In some embodiments the host cell is eukaryotic, more
preferably, mammalian, and more preferably selected from the group
consisting of 293, 293T, CHO, and COS-7 cells. In other
embodiments, the host cell is eukaryotic, more preferably
melanophore.
[1967] In a one hundred twenty-ninth aspect, the invention features
a process for the expression of a antilipolytic GPCR in a
recombinant host cell comprising the steps of:
[1968] (a) transfecting the expression vector of the one hundred
twenty-seventh aspect into a suitable host cell; and
[1969] (b) culturing the host cells under conditions which allow
expression of the antilipolytic GPCR protein from the expression
vectors.
[1970] In a one hundred thirtieth aspect, the invention features an
antibody that specifically binds to the polypeptide of the one
hundred twenty-fifth aspect. In some preferred embodiments, the
antibody is monoclonal. In some embodiments, the antibody is
polyclonal.
[1971] In a one hundred thirty-first aspect, the invention features
a method of binding the polypeptide of the one hundred twenty-fifth
aspect to the antibody of the one hundred thirtieth aspect,
comprising contacting said antibody with said polypeptide under
conditions in which said antibody can specifically bind to said
polypeptide.
[1972] In a one hundred thirty-second aspect, the invention
features a method of detecting an antilipolytic GPCR polypeptide in
a biological sample obtained from an individual comprising the
steps of: [1973] (a) obtaining said biological sample from said
individual; [1974] (b) contacting said biological sample with the
antibody of the one hundred thirtieth aspect; and [1975] (c)
detecting the presence or absence of binding of said antibody to
said biological sample; wherein a detection of said binding is
indicative of the receptor polypeptide being expressed in said
biological sample.
[1976] In preferred embodiments, said detecting is through the use
of an enzyme-labeled secondary reagent. In other preferred
embodiments, said detecting is through the use of a
fluorophore-labeled secondary reagent. In other preferred
embodiments, said detecting is through the use of a
radioisotope-labeled secondary reagent. In other embodiments, the
antibody is directly labeled with enzyme, fluorophore or
radioisotope.
[1977] In other preferred embodiments, said biological sample is
taken from adipose, skin or blood.
[1978] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[1979] In further embodiments, said individual has a disorder of
lipid metabolism selected from the group consisting of:
[1980] (a) elevated level of plasma triglycerides;
[1981] (b) elevated level of plasma free fatty acids;
[1982] (c) elevated level of plasma cholesterol;
[1983] (d) elevated level of LDL-cholesterol;
[1984] (e) reduced level of HDL-cholesterol;
[1985] (f) elevated total cholesterol/HDL-cholesterol ratio;
and
[1986] (g) reduced level of plasma adiponectin.
[1987] In other preferred embodiments, said disorder in lipid
metabolism is an elevated postprandial increase in plasma free
fatty acids due to a high fat meal or a progression from impaired
glucose tolerance to insulin resistance.
[1988] In further embodiments, said individual has a
metabolic-related disorder selected from the group consisting
of:
[1989] (a) dyslipidemia;
[1990] (b) atherosclerosis;
[1991] (c) coronary heart disease;
[1992] (d) stroke;
[1993] (e) insulin resistance; and
[1994] (f) type 2 diabetes.
[1995] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[1996] In other embodiments, said method further comprises the step
of comparing the level of detection of said binding for a first
individual to the level of detection of said binding for a second
individual.
[1997] In a one hundred thirty-third aspect, the invention features
a method of detecting expression of a gene encoding an
antilipolytic GPCR in a biological sample obtained from an
individual comprising the steps of: [1998] (a) obtaining said
biological sample from said individual; [1999] (b) contacting said
biological sample with the complementary polynucleotide of the one
hundred twenty-fourth aspect, optionally labeled, under conditions
permissive for hybridization; and [2000] (c) detecting the presence
or absence of said hybridization between said complementary
polynucleotide and an RNA species within said sample;
[2001] wherein a detection of said hybridization is indicative of
expression of said GPCR gene in said biological sample.
[2002] In preferred embodiments, the biological sample is taken
from adipose, skin or blood.
[2003] In preferred embodiments, said individual is a mammal. In
more preferred embodiments, said mammal is a horse, cow, sheep,
pig, cat, dog, rabbit, mouse, rat, non-human primate or human. Yet
more preferred is mouse, rat or human. Most preferred is human.
[2004] In preferred embodiments, said individual has a disorder of
lipid metabolism selected from the group consisting of:
[2005] (a) elevated level of plasma triglycerides;
[2006] (b) elevated level of plasma free fatty acids;
[2007] (c) elevated level of plasma cholesterol;
[2008] (d) elevated level of LDL-cholesterol;
[2009] (e) reduced level of HDL-cholesterol;
[2010] (f) elevated total cholesterol/HDL-cholesterol ratio;
and
[2011] (g) reduced level of plasma adiponectin.
[2012] In other preferred embodiments, said disorder in lipid
metabolism is an elevated postprandial increase in plasma free
fatty acids due to a high fat meal or a progression from impaired
glucose tolerance to insulin resistance.
[2013] In other preferred embodiments, said individual has a
metabolic-related disorder selected from the group consisting
of:
[2014] (a) dyslipidemia;
[2015] (b) atherosclerosis;
[2016] (c) coronary heart disease;
[2017] (d) stroke;
[2018] (e) insulin resistance; and
[2019] (f) type 2 diabetes.
[2020] In other preferred embodiments, said metabolic-related
disorder is selected from the group consisting of obesity, impaired
glucose tolerance, atheromatous disease, hypertension, Syndrome X,
and heart disease. Heart disease includes, but is not limited to,
cardiac insufficiency, coronary insufficiency, and high blood
pressure. In other preferred embodiments, said metabolic-related
disorder is hyperlipidemia.
[2021] In other embodiments, said method further comprises the step
of comparing the level of detection of said hybridization for a
first individual to the level of detection of said hybridization
for a second individual.
[2022] In some preferred embodiments, said complementary
polynucleotide is a primer and said hybridization is detected by
detecting the presence of an amplification product comprising the
sequence of said primer. In more preferred embodiments, said method
is RT-PCR.
[2023] In a one hundred thirty-fourth aspect, the invention
features a GPCR Fusion Protein construct comprising a
constitutively active GPCR and a G protein, said receptor
comprising the amino acid sequence of SEQ. ID. NO.:8 (hRUP11) or an
allelic variant or a biologically active fragment of said amino
acid sequence.
[2024] The invention also relates to a GPCR Fusion Protein
construct wherein the methionine at amino acid position 294 of SEQ.
ID. NO.:8 is substituted by lysine.
[2025] In a one hundred thirty-fifth aspect, the invention features
a method of binding a known ligand of RUP11 antilipolytic GPCR to a
polypeptide selected from the group consisting of:
[2026] (a) a polypeptide comprising a contiguous span of at least 6
amino acids of SEQ. ID. NO.:8;
[2027] (b) a polypeptide comprising a contiguous span of at least
10 amino acids of SEQ. ID. NO.:8;
[2028] (c) a polypeptide comprising a contiguous span of at least
15 amino acids of SEQ. ID. NO.:8;
[2029] (d) a polypeptide comprising a contiguous span of at least
20 amino acids of SEQ. ID. NO.:8;
[2030] (e) a polypeptide comprising a contiguous span of at least
25 amino acids of SEQ. ID. NO.:8;
[2031] (f) a polypeptide comprising a contiguous span of at least
30 amino acids of SEQ. ID. NO.:8;
[2032] (g) a polypeptide comprising a contiguous span of at least
35 amino acids of SEQ. ID. NO.:8;
[2033] (h) a polypeptide comprising a contiguous span of at least
40 amino acids of SEQ. ID. NO.:8;
[2034] (i) a polypeptide comprising a contiguous span of at least
45 amino acids of SEQ. ID. NO.:8; and
[2035] (j) a polypeptide comprising a contiguous span of at least
50 amino acids of SEQ. ID. NO.:8; or an allelic variant of said
polypeptide;
comprising the step of contacting said known ligand with said
polypeptide under conditions which allow said binding to occur.
[2036] In some embodiments, said known ligand is a modulator of the
GPCR. In some embodiments, said known ligand is an agonist of the
GPCR. In some embodiments, said known ligand is the modulator of
the one hundred fifth aspect. In some embodiments, said known
ligand is an antibody specific for the GPCR, or a derivative
thereof.
[2037] In other preferred embodiments, said method is used to
identify whether a candidate compound inhibits said binding of said
known ligand to said polypeptide, comprising the steps of: [2038]
(a) contacting said polypeptide with said known ligand, optionally
labeled, in the presence or absence of said candidate compound;
[2039] (b) detecting the complex between said known ligand and said
polypeptide; and [2040] (c) determining whether less of said
complex is formed in the presence of the [2041] compound than in
the absence of the compound; wherein said determination is
indicative of the candidate compound being an inhibitor of said
binding of said known ligand to said polypeptide.
[2042] In some embodiments, said known ligand is a modulator of the
GPCR. In some embodiments, said known ligand is an agonist of the
GPCR. In some embodiments, said known ligand is the modulator of
the one hundred fifth aspect. In some embodiments, said known
ligand is an antibody specific for the GPCR, or a derivative
thereof.
[2043] In other preferred embodiments, said method is used to
identify whether a candidate compound is an inhibitor of said
binding of said known ligand to said polypeptide, comprising the
steps of:
[2044] (a) contacting said polypeptide with said known ligand,
optionally labeled, in the presence separately of a plurality of
concentrations of said candidate compound for a time sufficient to
allow equilibration of binding;
[2045] (b) measuring unbound ligand and bound ligand; and
[2046] (c) determining K.sub.i for the candidate compound;
[2047] wherein a K.sub.i value of less than 50 uM is indicative of
the candidate compound being an inhibitor of said binding of said
known ligand to said polypeptide. Preferably said K.sub.i value is
less than 25 .mu.M, 10 .mu.M, 5 .mu.M, 1 .mu.M, 750 nM, 500 nM, 400
nM, 300 nM, 250 nM, 200 nM, 150 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60
nM, 50 nM, 40 nM, 30 nM, 20 nM or 10 nM. In preferred embodiments,
K.sub.i determination is made through nonlinear curve fitting with
the program SCTFIT [De Lean et al. (1982) Mol Pharmacol 21:5-16;
cited in Lorenzen et al. (2001) Mol Pharmacol 59:349-357, the
disclosures of which are incorporated by reference herein in their
entireties].
[2048] In some embodiments, said known ligand is a modulator of the
GPCR. In some embodiments, said known ligand is an agonist of the
GPCR. In some embodiments, said known ligand is the modulator of
the one hundred fifth aspect. In some embodiments, said known
ligand is an antibody specific for the GPCR, or a derivative
thereof.
[2049] In a one hundred thirty-six aspect, the invention features a
method of binding an optionally labeled affinity reagent specific
for an antilipolytic GPCR to said receptor in a biological sample,
said receptor comprising the amino acid sequence of SEQ. ID. NO.:8
(hRUP11); or an allelic variant, a biologically active mutant, or a
biologically active fragment of said amino acid sequence,
comprising the steps of:
[2050] (a) obtaining said biological sample;
[2051] (b) contacting the affinity reagent with said receptor in
said biological sample; and
[2052] (c) detecting the complex of said affinity reagent with said
receptor.
[2053] In some embodiments, the antilipolytic GPCR has the amino
acid sequence of SEQ. ID. NO.:8 (hRUP11); or an allelic variant, a
biologically active mutant, or a biologically active fragment of
said amino acid sequence.
[2054] In some embodiments, the antilipolytic GPCR comprises a
biologically active fragment of said amino acid sequence.
[2055] In some embodiments, the antilipolytic GPCR is
endogenous.
[2056] In some embodiments, the antilipolytic GPCR is
recombinant.
[2057] In some embodiments, said biologically active mutant is CART
or EFA. In preferred embodiments, said CART mutant has the amino
acid sequence of SEQ. ID. NO.:8 further substituted at amino acid
position 294 with lysine in place of methionine.
[2058] In preferred embodiments, said G protein is Gi.
[2059] In some embodiments, said affinity reagent is a modulator of
the GPCR. In some embodiments, said affinity reagent is an agonist
of the GPCR. In some embodiments, said affinity reagent is the
modulator of the one hundred fifth aspect. In some embodiments,
said affinity reagent is an antibody specific for the GPCR, or a
derivative thereof.
[2060] In further preferred embodiments, said affinity reagent
comprises a label selected from the group consisting of:
[2061] (a) radioisotope;
[2062] (b) enzyme; and
[2063] (c) fluorophore.
In preferred embodiments, said radioisotope is .sup.3H.
[2064] In a one hundred thirty-seventh aspect, the invention
features the method of the one hundred thirty-sixth aspect further
comprising the step of comparing the level of detection of said
complex in a first biological sample to a second level of detection
of said complex in a second biological sample.
[2065] In a one hundred thirty-eighth aspect, the invention
features the method of the one hundred thirty-seventh aspect
wherein the relationship between said first and second biological
samples is selected from the group consisting of:
[2066] (a) said second biological sample is a replicate of said
first biological sample;
[2067] (b) said first biological sample was obtained prior to an
experimental intervention whereas said second biological sample was
obtained after the experimental intervention, from the same
individual;
[2068] (c) said second biological sample was obtained at a
different time point after an experimental intervention than was
said first biological sample, from the same individual;
[2069] (d) said second biological sample corresponds to a different
subcellular compartment than does said first biological sample;
[2070] (e) said second biological sample represents a different
cell type than does said first biological sample;
[2071] (f) said second biological sample corresponds to a different
tissue than does said first biological sample;
[2072] (g) said second biological sample was obtained from a
different individual than was said first biological sample;
[2073] (h) said second biological sample was obtained at a
different point in time than was said first biological sample, from
the same individual;
[2074] (i) said first biological samples was obtained from a normal
individual, whereas said second biological sample was obtained from
an individual having a metabolic-related disorder;
[2075] (j) said first biological sample was obtained from a normal
individual, whereas said second biological sample was obtained from
an individual having a disorder in lipid metabolism;
[2076] (k) said first biological sample was obtained before a
therapeutic intervention whereas said second biological sample was
obtained after the therapeutic intervention, from the same
individual;
[2077] (l) said second biological sample was obtained at a
different time point after therapeutic intervention than was said
first biological sample, from the same individual; and
[2078] (m) said first biological sample was not exposed to a
compound, whereas said second biological sample was exposed to said
compound.
[2079] In a one hundred thirty-ninth aspect, the invention features
an isolated EFA-hRUP25 polynucleotide selected from the group
consisting of:
[2080] (a) a polynucleotide comprising the nucleotide sequence of
SEQ. ID. NO.:158;
[2081] (b) a polynucleotide having the nucleotide sequence of SEQ.
ID. NO.:158;
[2082] (c) a polynucleotide comprising a polynucleotide encoding
the polypeptide having the amino acid sequence of SEQ. ID. NO.:159
or a biologically active fragment of said polypeptide; and
[2083] (d) a polynucleotide encoding the polypeptide having the
amino acid sequence of SEQ. ID. NO.:159 or a biologically active
fragment of said polypeptide.
[2084] In a one hundred fortieth aspect, the invention features an
isolated EFA-hRUP25 polypeptide selected from the group consisting
of:
[2085] (a) a polypeptide comprising the amino acid sequence of SEQ.
ID. NO.:159, or a biologically active fragment of said polypeptide;
and
[2086] (b) a polypeptide having the amino acid sequence of SEQ. ID.
NO.:159, or a biologically active fragment of said polypeptide.
[2087] In an one hundred forty-first aspect, the invention features
a composition comprising, consisting essentially of, or consisting
of the EFA-hRUP25 polypeptide of the one hundred fortieth
aspect.
[2088] In a one hundred forty-second aspect, the invention features
a recombinant vector comprising the polynucleotide of the one
hundred thirty-ninth aspect. In some preferred embodiments, said
vector is used in a method of transient or stable transfection.
[2089] In particularly preferred embodiment, said vector is an
expression vector for the expression of an EFA-hRUP25 nicotinic
acid GPCR in a recombinant host cell wherein said expression vector
comprises, consists essentially of, or consists of the
polynucleotide of the one hundred thirty-ninth aspect.
[2090] Although a variety of expression vectors are available to
those in the art, it is most preferred that the vector utilized be
pCMV. In some alternative embodiments as relates to EFA-hRUP25
nicotinic acid GPCR, it is preferred that the vector utilized be an
adenoviral expression vector.
[2091] In a one hundred forty-third aspect, the invention features
a prokaryotic or eukaryotic host cell comprising, consisting
essentially of, or consisting of the recombinant vector of the one
hundred forty-second aspect. In some embodiments, said host cell is
prokaryotic and has been transformed using the vector of the one
hundred forty-second aspect. In some embodiments, said host cell is
eukaryotic and has been transiently transfected using the vector of
the one hundred forty-second aspect. In some preferred embodiments,
said host cell is eukaryotic and has been stably transfected using
the vector of the one hundred forty-second aspect.
[2092] In particularly preferred embodiment, said host cell
expresses a recombinant EFA-hRUP25 nicotinic acid GPCR wherein said
host cell comprises, consists essentially of, or consists of the
expression vector of the one hundred forty-second aspect.
[2093] A further embodiment includes a prokaryotic or eukaryotic
host cell recombinant for the polynucleotide of the one hundred
thirty-ninth aspect.
[2094] In some embodiments the host cell is eukaryotic, more
preferably, mammalian, and more preferably selected from the group
consisting of 293, 293T, CHO, and COS-7 cells. In other
embodiments, the Host Cell is eukaryotic, more preferably
melanophore.
[2095] In a one hundred forty-fourth aspect, the invention features
a process for the expression of an EFA-hRUP25 nicotinic acid GPCR
in a recombinant host cell comprising the steps of: [2096] (a)
transfecting the expression vector of the one hundred forty-second
aspect into a suitable host cell; and [2097] (b) culturing the host
cells under conditions which allow expression of the EFA-hRUP25
nicotinic acid GPCR protein from the expression vectors.
[2098] In a one hundred forty-fifth aspect, the invention features
a method of making an EFA mutant of an endogenous GPCR polypeptide
having constitutive activity, comprising the steps of:
[2099] (a) introducing 1, 2, 3, 4, or 5 substitutions, insertions,
or deletions into the amino acid sequence of the endogenous GPCR
polypeptide;
[2100] (b) measuring the activity of the mutant GPCR of (a) in the
absence of agonist and in the presence of a known agonist;
[2101] (c) measuring the activity of the endogenous GPCR in the
absence of agonist and in the presence of said known agonist;
and
[2102] (d) comparing (b) and (c);
wherein a determination that the agonist screening window of (b) is
at least 20% greater than that of (c) identifies the mutant
resulting from (a) to be an EFA mutant of the endogenous GPCR.
[2103] In some embodiments, said number of substitutions,
insertions, or deletions is 1.
[2104] In some embodiments, said number of substitutions,
insertions, or deletions is 1 or 2.
[2105] In some embodiments, said number of substitutions,
insertions, or deletions is 1, 2 or 3.
[2106] In some embodiments, said number of substitutions,
insertions, or deletions is 1, 2, 3 or 4.
[2107] In more preferred embodiments, said number of substitutions,
insertions, or deletions is 1, 2, 3, 4 or 5.
[2108] Applicant reserves the right to exclude any one or more
candidate compounds from any of the embodiments of the invention.
Applicant also reserves the right to exclude any one or more
modulators from any of the embodiments of the invention, including
but not limited to nicotinic acid or any analog or derivative
thereof. Applicant further reserves the right to exclude any
polynucleotide or polypeptide from any of the embodiments of the
invention. Applicant additionally reserves the right to exclude any
metabolic-related disorder or any disorder of lipid metabolism from
any of the embodiments of the invention.
[2109] Throughout this application, various publications, patents
and published patent applications are cited. The disclosures of
these publications, patents and published patent applications
referenced in this application are hereby incorporated by reference
in their entirety into the present disclosure. Citation herein by
Applicant of a publication, patent, or published patent application
is not an admission by Applicant of said publication, patent, or
published patent application as prior art.
[2110] Modifications and extension of the disclosed inventions that
are within the purview of the skilled artisan are encompassed
within the above disclosure and the claims that follow.
BRIEF DESCRIPTION OF THE DRAWINGS
[2111] FIG. 1. FIG. 1 depicts second messenger IP.sub.3 production
from endogenous version hRUP12 ("HRUP12") as compared with the
control ("CMV").
[2112] FIG. 2. FIG. 2 depicts the results of a second messenger
cell-based cyclic AMP assay providing comparative results for
constitutive signaling of endogenous hRUP13 ("hRUP13") and a
control vector ("CMV").
[2113] FIG. 3. FIG. 3 depicts the signal measured comparing CMV,
endogenous hRUP13 ("hRUP13 wt") and non-endogenous, constitutively
activated hRUP13 ("hRUP13(A268K)"), utilizing 8XCRE-Luc reporter
plasmid.
[2114] FIG. 4. FIG. 4 depicts the results of a
[.sup.35S]GTP.gamma.S assay providing comparative results for
constitutive signaling by hRUP13:Gs Fusion Protein ("hRUP13-Gs")
and a control vector ("CMV").
[2115] FIG. 5. FIG. 5 depicts the signal measured comparing CMV,
endogenous hRUP14 ("hRUP14 wt") and non-endogenous, constitutively
activated hRUP13 ("hRUP14(L246K)"), utilizing 8XCRE-Luc reporter
plasmid.
[2116] FIG. 6. FIG. 6 depicts the signal measured comparing CMV,
endogenous hRUP15 ("hRUP15 wt") and non-endogenous, constitutively
activated hRUP15 ("hRUP 15(A398K)"), utilizing 8XCRE-Luc reporter
plasmid.
[2117] FIG. 7. FIG. 7 depicts the results of a second messenger
cell-based cyclic AMP assay providing comparative results for
constitutive signaling of endogenous hRUP15 ("hRUP15 wt"),
non-endogenous, constitutively activated version of hRUP15
("hRUP15(A398K)") and a control vector ("CMV").
[2118] FIG. 8. FIG. 8 depicts the results of a
[.sup.35S]GTP.gamma.S assay providing comparative results for
constitutive signaling by hRUP15:Gs Fusion Protein ("hRUP15-Gs")
and a control vector ("CMV").
[2119] FIG. 9. FIG. 9 depicts second messenger IP.sub.3 production
from endogenous version hRUP17 ("hRUP17") as compared with the
control ("CMV").
[2120] FIG. 10. FIG. 10 depicts messenger IP.sub.3 production from
endogenous version hRUP21 ("hRUP21") as compared with the control
("CMV").
[2121] FIG. 11. FIG. 11 depicts the signal measured comparing CMV,
endogenous hRUP23 ("hRUP23 wt") and non-endogenous, constitutively
activated hRUP23 ("hRUP23(W275K)"), utilizing 8XCRE-Luc reporter
plasmid.
[2122] FIG. 12. FIG. 12 depicts results from a primary screen of
several candidate compounds against hRUP13; results for "Compound
A" are provided in well A2 and "Compound "B" are provided in well
G9.
[2123] FIGS. 13A-C. FIGS. 13A and 13B are histograms representing
relative expression levels of hRUP25 (FIG. 13A) and hRUP38 (FIG.
13B) detected in different human tissues via DNA microarray. The
horizontal axis displays the different tissues, identified in
vertical text above the bar. The vertical axis indicates level of
expression of either hRUP25 (FIG. 13A) or hRUP38 (FIG. 13B). In
FIG. 13A and FIG. 13B, note the high level of expression in primary
adipocytes of hRUP25 and hRUP38, respectively (the signal toward
the left of each of the histograms corresponding to primary
adipocytes is identified by a vertical arrow above the bar, for
ease of reference).
[2124] FIG. 13C is a photograph of an ethidium bromide stained gel
illustrating the relative expression of hRUP25 and hRUP38 as
detected by RT-PCR using cDNA derived from a number of human
tissues as template. Note the controls of the far right three
lanes.
[2125] FIGS. 14A-C. FIGS. 14A, 14B and 14C depict melanophores
transfected with DNA plasmids expressing hRUP25 (FIG. 14A), hRUP38
(FIG. 14B) and hRUP19 (FIG. 14C) without treatment. These cells are
pigment-aggregated because hRUP25 (FIG. 14A), hRUP38 (FIG. 14B) and
hRUP19 (FIG. 14C) are Gi-coupled receptors having a high basal
level of activity, and therefore driving the aggregation to a
measurable level in the absence of a ligand. hRUP11 is also a
Gi-coupled receptor having a high basal level of activity (not
shown).
[2126] FIGS. 15A-B. FIGS. 15A and 15B illustrate the
dose-dependant, nicotinic acid induced aggregation response of
melanophores transfected with increasing amounts of plasmid DNA
encoding hRUP25 (FIG. 15A). Cells transfected with 10 .mu.g of
plasmid DNA encoding hRUP25, respond to nicotinic acid with an
EC.sub.50 of about 54 nM.
[2127] As negative controls, FIG. 15B depicts melanophores
transfected with either salmon sperm DNA (Mock) or plasmid DNA
encoding the .alpha..sub.2AAR. As is evident there is no
aggregation response in these cells upon nicotinic acid treatment
at doses up to 10 .mu.M.
[2128] FIG. 16. FIG. 16 illustrates the nicotinic acid
induced-inositol phosphates (IPs) accumulation in HEK293 cells
co-expressing hRUP25 and the chimeric G.alpha.q-subunit in which
the last five amino acids have been replaced with the corresponding
amino acids of G.alpha.i (Gq.DELTA.Gi). This construct has been
shown to convert the signaling of a Gi-coupled receptor to the Gq
pathway (i.e. accumulation of inositol phosphates) in response to
receptor activation. Cells transfected with Gq.DELTA.Gi plus either
empty plasmid or the constitutively activated .alpha..sub.2AAR
(.alpha..sub.2AK) are non-responsive to nicotinic acid and served
as controls for the IP assay. Cells transfected with Gq.DELTA.Gi
plus either hRUP19 or hRUP38 are also unresponsive to nicotinic
acid, indicating that nicotinic acid is not an agonist for either
hRUP19 or hRUP38.
[2129] FIG. 17. FIG. 17 shows the results from saturation binding
of [.sup.3H]nicotinic acid to membranes from cells expressing
either hRUP25, hRUP38, hRUP19 or vector alone [CHO(-)]. Note that
only hRUP25 binds nicotinic acid in a specific and high-affinity
manner.
[2130] FIGS. 18A-B. FIG. 18A is a set of immunofluorescent
photomicrographs illustrating the expression of hemaglutinin
(HA)-tagged hRUP25 in a stably transfected line of CHO cells (top;
clone #46). No significant labeling is detected in mock
stably-transfected CHO cells (Mock). The lower panels identify the
nuclear (DAPI) staining of cells in the same field.
[2131] FIG. 18B illustrates nicotinic acid and (-)-nicotine
induced-inhibition of forskolin stimulated cAMP accumulation in
hRUP25-CHO cell stable line #46 (described in preceding paragraph).
The EC.sub.50 for nicotinic acid is 23.6 nM and that for
(-)-nicotine is 9.8 .mu.M.
[2132] FIG. 19. FIG. 19 indicates that, in response to nicotinic
acid, both hRUP25 and the mouse ortholog mRUP25 can inhibit TSHR
stimulated cAMP production (in the presence and absence of
TSH).
[2133] FIG. 20. FIG. 20 shows the saturation binding curves of
[.sup.3H]nicotinic acid ([.sup.3H]NA) to membranes prepared from
HEK293 cells transiently expressing either hRUP25 or mRUP25. Note
the significant binding of [.sup.3H]NA relative to either that
found in membranes derived from mock transfected cells or in the
presence of an excess of non-labeled nicotinic acid (200
.mu.M).
[2134] FIG. 21. FIG. 21 is a table comparing the rank order of
potency of various compounds on hRUP25 and the pharmacologically
defined nicotinic acid receptor. The potencies at hRUP25 derived
both by a functional analysis measuring the inhibition of forskolin
induced cAMP production and competitive radioligand binding assays,
closely match the order of potencies of the pharmacologically
defined nicotinic acid receptor.
[2135] FIGS. 22A-B. FIG. 22A depicts nicotinic acid and related
compounds inhibiting isoproterenol induced lipolysis in rat
epididymal fat derived adipocytes at a concentration of 10 .mu.M.
P-3-T represents 3-tetrazole-5-pyridine.
[2136] FIG. 22B illustrates a nicotinic acid dose-dependent
inhibition of isoproterenol induced-lipolysis in rat epididymal fat
derived adipocytes. Note the rightward shift in the dose-response
curves with increasing concentrations of nicotinic acid.
[2137] FIG. 23. FIG. 23 illustrates the ability of both nicotinic
acid and the related compound P-3-T (3-tetrazole-5-pyridine) to
inhibit isoproterenol induced lipolysis in adipocyte primary
cultures derived from human subcutaneous fat in a dose-dependant
manner. The EC.sub.50 value for nicotinic acid and P-3-T were 716
nM and 218 nM respectively.
[2138] FIG. 24. FIG. 24 presents screening data via adenylyl
cyclase assay for hRUP38. Note that nicotinic acid does not
activate inhibition of forskolin stimulated cAMP in
hRUP38-expressing CHO cells whereas
1-Isopropyl-1H-benzotriazole-5-carboxylic acid does.
1-Isopropyl-1H-benzotriazole-5-carboxylic acid has no effect on CHO
cells expressing either hRUP25 or hRUP19. The EC.sub.50 for
nicotinic acid is 25.8 nM and that for
1-Isopropyl-1H-benzotriazole-5-carboxylic acid is 166 nM. NT
indicates not tested. (Also see the legend to FIG. 18A above for
details directed to stable CHO transfectants.) Also see Example 30,
infra.
[2139] FIG. 25. Nicotinic acid and
1-Isopropyl-1H-benzotriazole-5-carboxylic acid were separately
dose-dependently applied to isoproterenol stimulated (100 nM)
primary human adipocytes. FIG. 25 illustrates the ability of
1-Isopropyl-1H-benzotriazole-5-carboxylic acid to inhibit
isoproterenol stimulated lipolysis in adipocyte primary cultures
derived from human subcutaneous fat in a dose-dependant manner
comparable to that of nicotinic acid.
[2140] FIG. 26. FIG. 26 presents screening data via adenylyl
cyclase assay for hRUP38. The horizontal axis indicates the
concentration of 3-(5-Bromo-2-ethoxy-phenyl)-acrylic acid. The
vertical axis indicates "% inhibition of cAMP". Note that a value
of 100% on the vertical axis corresponds to the cAMP level of
forskolin stimulated cells in the absence of
3-(5-Bromo-2-ethoxy-phenyl)-acrylic acid, whereas a value of 200%
on the vertical axis corresponds to the cAMP level of unstimulated
cells in the absence of 3-(5-Bromo-2-ethoxy-phenyl)-acrylic acid.
Note that 3-(5-Bromo-2-ethoxy-phenyl)-acrylic acid activates
inhibition of forskolin stimulated cAMP in hRUP38-expressing CHO
cells but has no effect on CHO cells expressing either hRUP25 or
hRUP19. The EC.sub.50 for 3-(5-Bromo-2-ethoxy-phenyl)-acrylic acid
is 1.17 .mu.M. (Also see the legend to FIG. 18A above for details
directed to stable CHO transfectants.)
[2141] FIG. 27. FIG. 27 presents an RT-PCR analysis of hRUP19
expression using a panel of human tissues. The analysis indicates
that hRUP19 is selectively expressed in fat cells. Low expression
is also evident in testis, placenta, kidney and spleen.
[2142] FIG. 28. FIG. 28 presents a Northern blot analysis of hRUP19
expression using a panel of human tissues. The analysis indicates
that hRUP19 is strongly expressed in mammary gland, probably due to
fat cell-specific expression of hRUP19. Ad, adrenal gland; Bl,
bladder, BM, bone marrow; Br, brain (whole); LN, lymph node; MG,
mammary gland; Pr, prostate; Sp, spinal cord; St, stomach; Thyr,
thyroid; Trch, trachea; Ut, uterus.
[2143] FIG. 29. FIG. 29 presents an analysis of RUP19 expression as
a function of adipocyte differentiation. RT-PCR and Northern blot
analysis of mRUP19 expression by mouse 3T3 pre-adipocytes and
differentiated 3T3 adipocytes was carried out. The analysis
indicates that RUP19 expression is induced during adipocyte
differentiation. Pre-diff 3T3-L1, mouse 3T3 pre-adipocytes;
Post-diff 3T3-L1, differentiated 3T3 adipocytes; .beta.-TC-6, a
mouse insulin-producing cell line; NIT-1, a mouse insulin-producing
cell line.
[2144] FIG. 30. FIG. 30 presents a CART analysis of signal
transduction by hRUP19. The analysis indicates that CART-activated
hRUP19 inhibits cAMP production in membranes of transfected 293
cells.
[2145] FIG. 31. FIG. 31 presents screening data via adenylyl
cyclase assay for hRUP25. The horizontal axis indicates the
concentration of
(5-hydroxy-1-methyl-3-propyl-1H-pyrazol-4-yl)-pyridin-3-yl-methanone.
The vertical axis indicates "% inhibition of cAMP". Note that a
value of 100% on the vertical axis corresponds to the cAMP level of
forskolin stimulated cells in the absence of
(5-hydroxy-1-methyl-3-propyl-1H-pyrazol-4-yl)-pyridin-3-yl-methanone,
whereas a value of 200% on the vertical axis corresponds to the
cAMP level of unstimulated cells in the absence of
(5-hydroxy-1-methyl-3-propyl-1H-pyrazol-4-yl)-pyridin-3-yl-methanone.
Note that
(5-hydroxy-1-methyl-3-propyl-1H-pyrazol-4-yl)-pyridin-3-yl-meth-
anone activates inhibition of forskolin stimulated cAMP in
hRUP25-expressing CHO cells and has an EC.sub.50 of 352 nM.
(5-Hydroxy-1-methyl-3-propyl-1H-pyrazol-4-yl)-pyridin-3-yl-methanone
has no activity on hRUP38-expressing CHO cells up to a
concentration of at least 100 .mu.M (not shown). Also see Example
29, infra.
[2146] FIG. 32. FIG. 32 presents a time-course analysis of plasma
free fatty acids (FFA) concentration in rats administered either
vehicle or niacin [NA] at 15 mg/kg, 30 mg/kg, or 45 mg/kg. Also see
Example 31, infra.
[2147] FIG. 33. FIG. 33 presents an analysis of the agonist
screening window for EFA-hRUP25 GPCR polypeptide of SEQ. ID.
NO.:159 ["hRUP25-S91"] relative to that for endogenous hRUP25 GPCR
polypeptide of SEQ. ID. NO.:36 ["hRUP25 wt"]. Samples were set up
in triplicate. HEK293 cells were transfected with pCMV vector alone
["CMV"], with TSHR alone ["CMV+TSHR"], or were co-transfected with
TSHR and either a2AK (a constitutively activated lysine mutant of
alpha2A adrenergic receptor) ["a2AK"] or endogenous hRUP25 ["hRUP25
wt"] or EFA-hRUP25 ["hRUP25-S91"]. Niacin ["Ni"] was taken as a
known agonist of hRUP25. UK14,304 ["UK"] was taken as a known
agonist of a2AK, a positive control for the assay. The level of
intracellular cAMP was determined for each sample. Also see Example
32, infra, for more details.
DETAILED DESCRIPTION
Definitions
[2148] The scientific literature that has evolved around receptors
has adopted a number of terms to refer to ligands having various
effects on receptors. For clarity and consistency, the following
definitions will be used throughout this patent document. To the
extent that these definitions conflict with other definitions for
these terms, the following definitions shall control:
[2149] ADIPONECTIN. ADIPONECTIN is a plasma protein secreted by
adipocytes and comprised of an N-terminally disposed collagen-like
region and a C-terminal globular region. Reduced levels of plasma
ADIPONECTIN have been associated with a number of metabolic-related
disorders, including atherosclerosis, coronary heart disease,
stroke, insulin resistance and type 2 diabetes. The serum
ADIPONECTIN level for women has been reported to be higher than
that for men, for example 13.5 .mu.g ml.sup.-1 versus 7.2 .mu.g
ml.sup.-1 in one study [Yamamoto Y et al., Clin Sci (Lond) (2002)
103:13742; the disclosure of which is hereby incorporated by
reference in its entirety].
[2150] AFFINITY REAGENTS shall mean compounds that specifically and
measurably bind to a target molecule. Preferably the target
molecule is a GPCR.
[2151] AGONISTS shall mean materials (e.g., ligands, candidate
compounds) that activate an intracellular response when they bind
to the receptor. In some embodiments, AGONISTS are those materials
not previously known to activate the intracellular response when
they bind to the receptor (e.g. to enhance GTP.gamma.S binding to
membranes or to lower intracellular cAMP level). In some
embodiments, AGONISTS are those materials not previously known to
inhibit lipolysis when they bind to the receptor.
[2152] ALLELIC VARIANT. See VARIANT.
[2153] ALLOSTERIC MODULATORS shall mean materials (e.g., ligands,
candidate compounds) that affect the functional activity of the
receptor but which do not inhibit the endogenous ligand from
binding to the receptor. Allosteric modulators include inverse
agonists, partial agonists and agonists.
[2154] AMINO ACID ABBREVIATIONS used herein are set out in Table A:
TABLE-US-00002 TABLE A ALANINE ALA A ARGININE ARG R ASPARAGINE ASN
N ASPARTIC ACID ASP D CYSTEINE CYS C GLUTAMIC ACID GLU E GLUTAMINE
GLN Q GLYCINE GLY G HISTIDINE HIS H ISOLEUCINE ILE I LEUCINE LEU L
LYSINE LYS K METHIONINE MET M PHENYLALANINE PHE F PROLINE PRO P
SERINE SER S THREONINE THR T TRYPTOPHAN TRP W TYROSINE TYR Y VALINE
VAL V
[2155] ANTAGONISTS shall mean materials (e.g., ligands, candidate
compounds) that competitively bind to the receptor at the same site
as the agonists but which do not activate an intracellular
response, and can thereby inhibit the intracellular responses
elicited by agonists. ANTAGONISTS do not diminish the baseline
intracellular response in the absence of an agonist. In some
embodiments, ANTAGONISTS are those materials not previously known
to compete with an agonist to inhibit the cellular response when
they bind to the receptor, e.g. wherein the cellular response is
GTP.gamma.S binding to membranes or to the lowering of
intracellular cAMP level.
[2156] ANTIBODIES are intended herein to encompass monoclonal
antibodies and polyclonal antibodies. ANTIBODIES are further
intended to encompass IgG, IgA, IgD, IgE, and IgM. ANTIBODIES
include whole antibodies, including single-chain whole antibodies,
and antigen binding fragments thereof, including Fab, Fab', F(ab)2
and F(ab')2. ANTIBODIES may be from any animal origin. Preferably,
ANTIBODIES are human, murine, rabbit, goat, guinea pig, hamster,
camel, donkey, sheep, horse or chicken. Preferably ANTIBODIES have
binding affinities with a dissociation constant or Kd value less
than 5.times.10.sup.-6 M, 10.sup.-6 M, 5.times.10.sup.-7 M,
10.sup.-7 M, 5.times.10.sup.-8 M, 10.sup.-8 M, 5.times.10.sup.-9
M,
10.sup.-9 M, 5.times.10.sup.-10 M 10.sup.-10 M, 5.times.10.sup.-11
M, 10.sup.-11 M, 5.times.10.sup.-12 M, 10.sup.-12 M,
5.times.10.sup.-13 M, 10.sup.-13 M,
5.times.10.sup.-14 M 10.sup.-14 M, 5.times.10.sup.-15 M and
10.sup.-15 M. ANTIBODIES of the present invention may be prepared
by any suitable method known in the art.
[2157] ATHEROSCLEROSIS is intended herein to encompass disorders of
large and medium-sized arteries that result in the progressive
accumulation within the intima of smooth muscle cells and lipids.
Atherosclerosis is the primary cause of heart disease and
stroke.
[2158] BIOLOGICALLY ACTIVE FRAGMENT is interchangeable herein with
ACTIVE FRAGMENT and shall mean a fragment of full-length
polypeptide or full-length amino acid sequence retaining part or
all of the functionality of said full-length polypeptide or
full-length amino acid sequence. In particular embodiment, a GPCR
comprising an active fragment of a full-length GPCR polypeptide or
full-length GPCR amino acid sequence retains part or all of the
functionality of said GPCR comprising said full-length polypeptide
or said full-length amino acid sequence. Said GPCR functionality is
understood to include but not intended to be limited to ligand
binding, G protein coupling, and ligand-facilitated coupling to G
protein. By way of illustration and not limitation, BIOLOGICALLY
ACTIVE FRAGMENT is intended herein to encompass full-length GPCR
polypeptide absent the N-terminal methionine.
[2159] 3-(5-BROMO-2-ETHOXY-PHENYL)-ACRYLIC ACID shall be understood
herein to have the formula: ##STR95##
[2160] and to encompass the E isomer, the Z isomer, and mixtures of
E and Z isomers.
[2161] CANDIDATE COMPOUND shall mean a molecule (for example, and
not limitation, a chemical compound) that is amenable to a
screening technique. Preferably, the phrase "candidate compound"
does not include compounds which were publicly known to be
compounds selected from the group consisting of inverse agonist,
agonist or antagonist to a receptor, as previously determined by an
indirect identification process ("indirectly identified compound");
more preferably, not including an indirectly identified compound
which has previously been determined to have therapeutic efficacy
in at least one mammal; and, most preferably, not including an
indirectly identified compound which has previously been determined
to have therapeutic utility in humans.
[2162] CHOLESTEROL. Generally, the total
cholesterol/HDL-cholesterol (i.e., TC/HDL) ratio represents a
useful predictor as to the risk of an individual in developing a
more serious condition, such as an HDL-related condition, such as
but not limited to atherosclerosis and complications therefrom. The
classification of plasma lipid levels is shown in Chart A:
TABLE-US-00003 CHART A CLASSIFICATION OF PLASMA LIPID LEVELS TOTAL
<200 mg/dl Desirable CHOLESTEROL 200-239 mg/dl Borderline High
>240 mg/dl High HDL- <40 mg/dl Low (Men) CHOLESTEROL <50
mg/dl Low (Women) >60 mg/dl High From: 2001 National Cholesterol
Education Program Guidelines
Accordingly, the recommended total cholesterol/HDL-C (i.e., TC/HDL)
ratio indicates that a ratio of less than or equal to 3.5 is ideal
and a ratio of greater than 4.5 is considered an increased "at
risk." The value of determining the TC/HDL ratio is clearly evident
in the circumstance where an individual presents with "normal" LDL
and total cholesterol but possesses low HDL-cholesterol. Based on
LDL and total cholesterol the individual may not qualify for
treatment however, factor in the HDL-cholesterol level then a more
accurate risk assessment may be obtained. Thus, if the individual's
level of HDL-cholesterol is such that the ratio is greater than 4.5
then therapeutic or preventive intervention may be warranted. A
physician or care provider may determine the need of prevention or
treatment based on a TC/HDL ratio; for example, a TC/HDL ratio of
2.5 or greater, 3.0 or greater, 3.5 or greater, 4.0 or greater, 4.5
or greater, 5.0 or greater, or a TC/HDL ratio of 5.5 or
greater.
[2163] CODON shall mean a grouping of three nucleotides (or
equivalents to nucleotides) which generally comprise a nucleoside
[adenosine (A), guanosine (G), cytidine (C), uridine (U) and
thymidine (T)] coupled to a phosphate group and which, when
translated, encodes an amino acid.
[2164] COMPOSITION means a material comprising at least one
component; a "pharmaceutical composition" is an example of a
composition.
[2165] COMPOUND EFFICACY shall mean a measurement of the ability of
a compound to inhibit or stimulate receptor functionality; i.e. the
ability to activate/inhibit a signal transduction pathway, in
contrast to receptor binding affinity. Exemplary means of detecting
compound efficacy are disclosed in the Example section of this
patent document.
[2166] COMPRISING, CONSISTING ESSENTIALLY OF, and CONSISTING OF are
defined herein according to their standard meaning. A defined
meaning set forth in the M.P.E.P. controls over a defined meaning
in the art and a defined meaning set forth in controlling Federal
Circuit case law controls over a meaning set forth in the
M.P.E.P.
[2167] CONSTITUTIVELY ACTIVE RECEPTOR shall mean a receptor
stabilized in an active state by means other than through binding
of the receptor to its ligand or a chemical equivalent thereof. A
CONSTITUTIVELY ACTIVE RECEPTOR may be endogenous or
non-endogenous.
[2168] CONSTITUTIVELY ACTIVATED RECEPTOR shall mean an endogenous
receptor that has been modified so as to be constitutively active.
CART is an acronym for Constitutively Activated Receptor Technology
and when used herein prefixing or suffixing a GPCR, shall be
understood to identify said prefixed or suffixed GPCR as a
CONSTITUTIVELY ACTIVATED RECEPTOR.
[2169] CONSTITUTIVE RECEPTOR ACTIVATION shall mean activation of a
receptor in the absence of binding to its ligand or a chemical
equivalent thereof.
[2170] CONTACT or CONTACTING shall mean bringing at least two
moieties together, whether in an in vitro system or an in vivo
system.
[2171] CORONARY HEART DISEASE is intended herein to encompass
disorders comprising a narrowing of the small blood vessels that
supply blood and oxygen to the heart. CORONARY HEART DISEASE
usually results from the build up of fatty material and plaque. As
the coronary arteries narrow, the flow of blood to the heart can
slow or stop. CORONARY HEART DISEASE can cause chest pain (stable
angina), shortness of breath, heart attack, or other symptoms.
CORONARY HEART DISEASE is intended herein to include coronary
artery disease, the most common type of heart disease. Coronary
artery disease results from atherosclerosis.
[2172] DECREASE is used to refer to a reduction in a measurable
quantity and is used synonymously with the terms "reduce",
"diminish", "lower", and "lessen".
[2173] DIABETES as used herein is intended to encompass the usual
diagnosis of DIABETES made from any of the methods including, but
not limited to, the following list: symptoms of diabetes (e.g.,
polyuria, polydipsia, polyphagia) plus casual plasma glucose levels
of greater than or equal to 200 mg/dl, wherein casual plasma
glucose is defined any time of the day regardless of the timing of
meal or drink consumption; 8 hour fasting plasma glucose levels of
less than or equal to 126 mg/dl; and plasma glucose levels of
greater than or equal to 200 mg/dl 2 hours following oral
administration of 75 g anhydrous glucose dissolved in water.
[2174] DIRECTLY IDENTIFYING or DIRECTLY IDENTIFIED, in relationship
to the phrase "candidate compound", shall mean the screening of a
candidate compound against a constitutively activated receptor,
preferably a constitutively activated orphan receptor, and most
preferably against a constitutively activated G protein-coupled
cell surface orphan receptor, and assessing the compound efficacy
of such compound. This phrase is, under no circumstances, to be
interpreted or understood to be encompassed by or to encompass the
phrase "indirectly identifying" or "indirectly identified."
[2175] DISORDERS OF LIPID METABOLISM are intended herein to
include, but not be limited to, dyslipidemia.
[2176] DYSLIPIDEMIA is intended herein to encompass disorders
comprising any one of elevated level of plasma free fatty acids,
elevated level of plasma cholesterol, elevated level of
LDL-cholesterol, reduced level of HDL-cholesterol, elevated ratio
of total cholesterol to HDL-cholesterol, and elevated level of
plasma triglycerides.
[2177] EFA-GPCR shall mean a mutant GPCR polypeptide that consists
of 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or
insertions relative to the amino acid sequence of an endogenous
GPCR polypeptide having constitutive activity, wherein the agonist
screening window of the mutant GPCR is expanded by greater than
20%, greater than 25%, greater than 30%, greater than 31%, greater
than 32%, greater than 33%, greater than 34%, greater than 35%,
greater than 36%, greater than 37%, greater than 38%, greater than
39%, or greater than 40% relative to that of said endogenous
GPCR.
[2178] ENDOGENOUS shall mean a material that a mammal naturally
produces. ENDOGENOUS in reference to, for example and not
limitation, the term "receptor," shall mean that which is naturally
produced by a mammal (for example, and not limitation, a human) or
a virus. ENDOGENOUS shall be understood to encompass allelic
variants of a gene as well as the allelic polypeptide variants so
encoded. By contrast, the term NON-ENDOGENOUS in this context shall
mean that which is not naturally produced by a mammal (for example,
and not limitation, a human) or a virus. For example, and not
limitation, a receptor which is not constitutively active in its
endogenous form, but when manipulated becomes constitutively
active, is most preferably referred to herein as a "non-endogenous,
constitutively activated receptor." Both terms can be utilized to
describe both "in vivo" and "in vitro" systems. For example, and
not limitation, in a screening approach, the endogenous or
non-endogenous receptor may be in reference to an in vitro
screening system. As a further example and not limitation, where
the genome of a mammal has been manipulated to include a
non-endogenous constitutively activated receptor, screening of a
candidate compound by means of an in vivo system is viable.
[2179] EXPRESSION VECTOR is defined herein as a DNA sequence that
is required for the transcription of cloned DNA and the translation
of the transcribed mRNAs in an appropriate host cell recombinant
for said EXPRESSION VECTOR. An appropriately constructed EXPRESSION
VECTOR should contain an origin of replication for autonomous
replication in host cells, selectable markers, a limited number of
useful restriction enzyme sites, a potential for high copy number,
and active promoters. By way of illustration and not limitation,
pCMV is an expression vector.
[2180] G PROTEIN COUPLED RECEPTOR FUSION PROTEIN and GPCR FUSION
PROTEIN, in the context of the invention disclosed herein, each
mean a non-endogenous protein comprising an endogenous,
constitutively activate GPCR or a non-endogenous, constitutively
activated GPCR fused to at least one G protein, most preferably the
alpha (a) subunit of such G protein (this being the subunit that
binds GTP), with the G protein preferably being of the same type as
the G protein that naturally couples with endogenous orphan GPCR.
For example, and not limitation, in an endogenous state, if the G
protein "G.sub.s.alpha." is the predominate G protein that couples
with the GPCR, a GPCR Fusion Protein based upon the specific GPCR
would be a non-endogenous protein comprising the GPCR fused to
G.sub.s.alpha.; in some circumstances, as will be set forth below,
a non-predominant G protein can be fused to the GPCR. The G protein
can be fused directly to the C-terminus of the constitutively
active GPCR or there may be spacers between the two.
[2181] HOST CELL shall mean a cell capable of having a Plasmid
and/or Vector incorporated therein. In the case of a prokaryotic
Host Cell, a Plasmid is typically replicated as a autonomous
molecule as the Host Cell replicates (generally, the Plasmid is
thereafter isolated for introduction into a eukaryotic Host Cell);
in the case of a eukaryotic Host Cell, a Plasmid may be integrated
into the cellular DNA of the Host Cell such that when the
eukaryotic Host Cell replicates, the Plasmid replicates. In some
embodiments the Host Cell is eukaryotic, more preferably,
mammalian, and more preferably selected from the group consisting
of 293, 293T, CHO, and COS-7 cells. In other embodiments, the Host
Cell is eukaryotic, more preferably melanophore.
[2182]
(5-HYDROXY-1-METHYL-3-PROPYL-1H-PYRAZOL-4-YL)-PYRIDIN-3-YL-METHANO-
NE shall be understood herein to have the formula: ##STR96##
[2183] IN NEED OF PREVENTION OR TREATMENT as used herein refers to
a judgement made by a caregiver (e.g. physician, nurse, nurse
practitioner, etc. in the case of humans; veterinarian in the case
of animals, including non-human mammals) that an individual or
animal requires or will benefit from treatment. This judgement is
made based on a variety of factors that are in the realm of a
caregiver's expertise, but that include the knowledge that the
individual or animal is ill, or will be ill, as the result of a
condition that is treatable by the compounds of the invention.
[2184] INDIRECTLY IDENTIFYING or INDIRECTLY IDENTIFIED means the
traditional approach to the drug discovery process involving
identification of an endogenous ligand specific for an endogenous
receptor, screening of candidate compounds against the receptor for
determination of those which interfere and/or compete with the
ligand-receptor interaction, and assessing the efficacy of the
compound for affecting at least one second messenger pathway
associated with the activated receptor.
[2185] INDIVIDUAL as used herein refers to any animal, including
mammals, preferably mice, rats, other rodents, rabbits, dogs, cats,
swine, cattle, sheep, horses, or primates, and most preferably
humans.
[2186] INHIBIT or INHIBITING, in relationship to the term
"response" shall mean that a response is decreased or prevented in
the presence of a compound as opposed to in the absence of the
compound.
[2187] INSULIN RESISTANCE as used herein is intended to encompass
the usual diagnosis of insulin resistance made by any of a number
of methods, including but not restricted to: the intravenous
glucose tolerance test or measurement of the fasting insulin level.
It is well known that there is an excellent correlation between the
height of the fasting insulin level and the degree of insulin
resistance. Therefore, one could use elevated fasting insulin
levels as a surrogate marker for insulin resistance for the purpose
of identifying which normal glucose tolerance (NGT) individuals
have insulin resistance. A diagnosis of insulin resistance can also
be made using the euglycemic glucose clamp test.
[2188] INVERSE AGONISTS shall mean materials (e.g., ligand,
candidate compound) that bind either to the endogenous form or to
the constitutively activated form of the receptor so as to reduce
the baseline intracellular response of the receptor observed in the
absence of agonists.
[2189] ISOLATED shall mean that the material is removed from its
original environment (e.g., the natural environment if it is
naturally occurring). For example, a naturally occurring
polynucleotide or polypeptide present in a living animal is not
isolated, but the same polynucleotide or DNA or polypeptide,
separated from some or all of the coexisting materials in the
natural system, is isolated. Such a polynucleotide could be part of
a vector and/or such a polynucleotide or polypeptide could be part
of a composition, and still be isolated in that the vector or
composition is not part of its natural environment.
[2190] 1-ISOPROPYL-1H-BENZOTRIAZOLE-5-CARBOXYLIC ACID shall be
understood herein to have the formula: ##STR97##
[2191] KNOCKOUT MOUSE/RAT is intended herein to encompass a mouse
or rat that has been manipulated by recombinant means such that a
single gene of choice has been inactivated or "knocked-out" in a
manner that leaves all other genes unaffected.
[2192] KNOWN RECEPTOR shall mean an endogenous receptor for which
the endogenous ligand specific for that receptor has been
identified.
[2193] LIGAND shall mean a molecule specific for a naturally
occurring receptor.
[2194] METABOLIC-RELATED DISORDERS are intended herein to include,
but not be limited to, dyslipidemia, atherosclerosis, coronary
heart disease, stroke, insulin resistance and type 2 diabetes.
[2195] As used herein, the terms MODULATE or MODIFY are meant to
refer to an increase or decrease in the amount, quality, or effect
of a particular activity, function or molecule.
[2196] MUTANT or MUTATION in reference to an endogenous receptor's
nucleic acid and/or amino acid sequence shall mean a specified
change or changes to such endogenous sequences such that a mutated
form of an endogenous non-constitutively activated receptor
evidences constitutive activation of the receptor. In terms of
equivalents to specific sequences, a subsequent mutated form of a
human receptor is considered to be equivalent to a first mutation
of the human receptor if (a) the level of constitutive activation
of the subsequent mutated form of a human receptor is substantially
the same as that evidenced by the first mutation of the receptor;
and (b) the percent sequence (amino acid and/or nucleic acid)
homology between the subsequent mutated form of the receptor and
the first mutation of the receptor is at least 80%, at least 85%,
at least 90%, at least 92%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, and most preferably at least 99%.
In some embodiments, owing to the fact that some preferred
cassettes disclosed herein for achieving constitutive activation
include a single amino acid and/or codon change between the
endogenous and the non-endogenous forms of the GPCR, it is
preferred that the percent sequence homology should be at least
98%.
[2197] (-)-NICOTINE shall be understood herein to have the formula:
##STR98##
[2198] NICOTINIC ACID shall be understood herein to have the
formula: ##STR99##
[2199] As used herein, the term NICOTINIC ACID ANALOG OR DERIVATIVE
is meant to molecules which bind to nicotinic acid receptors and
have substantially similar effects on the receptor. Such analogs
and derivatives are well-known to those skilled in the art and
include, but are not limited to, Acipimox.TM. and niacinamide.
[2200] NON-ORPHAN RECEPTOR shall mean an endogenous naturally
occurring molecule specific for an identified ligand wherein the
binding of a ligand to a receptor activates an intracellular
signaling pathway.
[2201] ORPHAN RECEPTOR shall mean an endogenous receptor for which
the ligand specific for that receptor has not been identified or is
not known.
[2202] PARTIAL AGONISTS shall mean materials (e.g., ligands,
candidate compounds) that activate the intracellular response when
they bind to the receptor to a lesser degree/extent than do full
agonists.
[2203] PHARMACEUTICAL COMPOSITION shall mean a composition
comprising at least one active ingredient, whereby the composition
is amenable to investigation for a specified, efficacious outcome
in a mammal (for example, and not limitation, a human). Those of
ordinary skill in the art will understand and appreciate the
techniques appropriate for determining whether an active ingredient
has a desired efficacious outcome based upon the needs of the
artisan.
[2204] PLASMID shall mean the combination of a Vector and cDNA.
Generally, a Plasmid is introduced into a Host Cell for the
purposes of replication and/or expression of the cDNA as a
protein.
[2205] POLYNUCLEOTIDES shall mean RNA, DNA, or RNA/DNA hybrid
sequences of more than one nucleotide in either single chain or
duplex form. The polynucleotides of the invention may be prepared
by any known method, including synthetic, recombinant, ex vivo
generation, or a combination thereof, as well as utilizing any
purification methods known in the art.
[2206] POLYPEPTIDE shall refer to a polymer of amino acids without
regard to the length of the polymer. Thus, peptides, oligopeptides,
and proteins are included within the definition of polypeptide.
This term also does not specify or exclude post-expression
modifications of polypeptides. For example, polypeptides that
include the covalent attachment of glycosyl groups, acetyl groups,
phosphate groups, lipid groups and the like are expressly
encompassed by the term POLYPEPTIDE.
[2207] PRIMER is used herein to denote a specific oligonucleotide
sequence which is complementary to a target nucleotide sequence and
used to hybridize to the target nucleotide sequence. A primer
serves as an initiation point for nucleotide polymerization
catalyzed by DNA polymerase, RNA polymerase, or reverse
transcriptase.
[2208] PURIFIED is used herein to describe a polynucleotide or
polynucleotide vector of the invention that has been separated from
other compounds including, but not limited to, other nucleic acids,
carbohydrates, lipids and proteins (such as the enzymes used in the
synthesis of the polynucleotide). A polynucleotide is substantially
pure when at least about 50%, 60%, 75%, or 90% of a sample contains
a single polynucleotide sequence. A substantially pure
polynucleotide typically comprises about 50, 60, 70, 80, 90, 95,
99% weight/weight of a nucleic acid sample. Polynucleotide purity
or homogeneity may be indicated by a number of means well known in
the art, such as agarose or polyacrylamide gel electrophoresis of a
sample, followed by visualizing a single polynucleotide band upon
staining the gel.
[2209] Similarly, the term PURIFIED is used herein to describe a
polypeptide of the invention that has been separated from other
compounds including, but not limited to, nucleic acids, lipids,
carbohydrates and other proteins. In some preferred embodiments, a
polypeptide is substantially pure when at least about 50%, 60%,
75%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the polypeptide
molecules of a sample have a single amino acid sequence. In some
preferred embodiments, a substantially pure polypeptide typically
comprises about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or
99.5% weight/weight of a protein sample. Polypeptide purity or
homogeneity is indicated by a number of methods well known in the
art, such as agarose or polyacrylamide gel electrophoresis of a
sample, followed by visualizing a single polypeptide band upon
staining the gel.
[2210] Further, as used herein, the term PURIFIED does not require
absolute purity; rather, it is intended as a relative definition.
Purification of starting material or natural material to at least
one order of magnitude, preferably two or three orders, and more
preferably four or five orders of magnitude is expressly
contemplated.
[2211] RECEPTOR FUNCTIONALITY shall refer to the normal operation
of a
[2212] receptor to receive a stimulus and moderate an effect in the
cell, including, but not limited to regulating gene transcription,
regulating the influx or efflux of ions, effecting a catalytic
reaction, and/or modulating activity through G-proteins.
[2213] SECOND MESSENGER shall mean an intracellular response
produced as a result of receptor activation. A second messenger can
include, for example, inositol triphosphate (IP.sub.3),
diacylglycerol (DAG), cyclic AMP (cAMP), cyclic GMP (cGMP), and
Ca.sup.2+. Second messenger response can be measured for a
determination of receptor activation. In addition, second messenger
response can be measured for the direct identification of candidate
compounds, including for example, inverse agonists, partial
agonists, agonists, and antagonists.
[2214] SIGNAL TO NOISE RATIO shall mean the signal generated in
response to activation, amplification, or stimulation wherein the
signal is above the background noise or the basal level in response
to non-activation, non-amplification, or non-stimulation.
[2215] SPACER shall mean a translated number of amino acids that
are located after the last codon or last amino acid of a gene, for
example a GPCR of interest, but before the start codon or beginning
regions of the G protein of interest, wherein the translated number
amino acids are placed in-frame with the beginnings regions of the
G protein of interest. The number of translated amino acids can be
one, two, three, four, etc., and up to twelve.
[2216] STIMULATE or STIMULATING, in relationship to the term
"response" shall mean that a response is increased in the presence
of a compound as opposed to in the absence of the compound.
[2217] STROKE is a cardiovascular disease that affects the blood
vessels supplying blood to the brain and is intended herein to
include cerebral thrombosis, the most common type of STROKE.
Cerebral thrombosis occurs when a blood clot (thrombus) forms and
blocks blood flow in an artery bringing blood to part of the brain.
Blood clots usually form in arteries damaged by
atherosclerosis.
[2218] SUBJECT shall mean primates, including but not limited to
humans and baboons, as well as pet animals such as dogs and cats,
laboratory animals such as rats and mice, and farm animals such as
horses, sheep, and cows.
[2219] SUBSTANTIALLY shall refer to a result which is within 40% of
a control result, preferably within 35%, more preferably within
30%, more preferably within 25%, more preferably within 20%, more
preferably within 15%, more preferably within 10%, more preferably
within 5%, more preferably within 2%, and most preferably within 1%
of a control result. For example, in the context of receptor
functionality, a test receptor may exhibit substantially similar
results to a control receptor if the transduced signal, measured
using a method taught herein or similar method known to the
art-skilled, is within 40% of the signal produced by a control
signal.
[2220] TRANSGENIC MOUSE/RAT shall be intended herein to encompass a
mouse or rat that has been engineered through recombinant means to
carry a foreign gene, or transgene, of choice as part of its own
genetic material.
[2221] VARIANT as the term is used herein, is a polynucleotide or
polypeptide that differs from a reference polynucleotide or
polypeptide respectively, but retains essential properties. A
typical variant of a polynucleotide differs in nucleotide sequence
from another, reference polynucleotide. Changes in the nucleotide
sequence of the variant may or may not alter the amino acid
sequence of a polypeptide encoded by the reference polynucleotide.
A typical variant of a polypeptide differs in amino acid sequence
from another, reference polypeptide. A variant and reference
polypeptide may differ in amino acid sequence by one or more
substitutions, additions, deletions in any combination. A variant
of a polynucleotide or polypeptide may be a naturally occurring one
such as an ALLELIC VARIANT, or it may be a variant that is not
known to occur naturally. Non-naturally occurring variants of
polynucleotides and polypeptides may be made by mutagenesis
techniques or by direct synthesis.
[2222] VECTOR in reference to cDNA shall mean a circular DNA
capable of incorporating at least one cDNA and capable of
incorporation into a Host Cell.
[2223] The order of the following sections is set forth for
presentational efficiency and is not intended, nor should be
construed, as a limitation on the disclosure or the claims to
follow.
A. Introduction
[2224] The traditional study of receptors has always proceeded from
the a priori assumption (historically based) that the endogenous
ligand must first be identified before discovery could proceed to
find antagonists and other molecules that could affect the
receptor. Even in cases where an antagonist might have been known
first, the search immediately extended to looking for the
endogenous ligand. This mode of thinking has persisted in receptor
research even after the discovery of constitutively activated
receptors. What has not been heretofore recognized is that it is
the active state of the receptor that is most useful for
discovering agonists, partial agonists, and inverse agonists of the
receptor. For those diseases which result from an overly active
receptor or an under-active receptor, what is desired in a
therapeutic drug is a compound which acts to diminish the active
state of a receptor or enhance the activity of the receptor,
respectively, not necessarily a drug which is an antagonist to the
endogenous ligand. This is because a compound that reduces or
enhances the activity of the active receptor state need not bind at
the same site as the endogenous ligand. Thus, as taught by the
present invention, in some preferred embodiments, a search for
therapeutic compounds should start by screening compounds against
the ligand-independent active state.
B. Identification of Human GPCRs
[2225] The efforts of the Human Genome project has led to the
identification of a plethora of information regarding nucleic acid
sequences located within the human genome; it has been the case in
this endeavor that genetic sequence information has been made
available without an understanding or recognition as to whether or
not any particular genomic sequence does or may contain
open-reading frame information that translate human proteins.
Several methods of identifying nucleic acid sequences within the
human genome are within the purview of those having ordinary skill
in the art. For example, and not limitation, a variety of human
GPCRs, disclosed herein, were discovered by reviewing the
GenBank.TM. database. Table B, below, lists several endogenous
GPCRs that we have discovered, along with other GPCRs that are
homologous to the disclosed GPCR. TABLE-US-00004 TABLE B Disclosed
Open PerCent Human Accession Reading Reference To Homology Orphan
Number Frame Homologous To Designated GPCRs Identified (Base Pairs)
GPCR GPCR hRUP8 AL121755 1,152 bp NPY2R 27% hRUP9 AC0113375 1,260
bp GAL2R 22% hRUP10 AC008745 1,014 bp C5aR 40% hRUP11 AC013396
1,272 bp HM74 36% hRUP12 AP000808 966 bp Mas1 34% hRUP13 AC011780
1,356 bp Fish GPRX- 43% ORYLA hRUP14 AL137118 1,041 bp CysLT1R 35%
hRUP15 AL016468 1,527 bp RE2 30% hRUP16 AL136106 1,068 bp GLR101
37% hRUP17 AC023078 969 bp Mas1 37% hRUP18 AC008547 1,305 bp
Oxytocin 31% hRUP19 AC026331 1,041 bp HM74 52% hRUP20 AL161458
1,011 bp GPR34 25% hRUP21 AC026756 1,014 bp P2Y1R 37% hRUP22
AC027026 993 bp hRUP17 67% Mas1 37% hRUP23 AC007104 1,092 bp Rat
GPR26 31% hRUP24 AL355388 1,125 bp SALPR 44% hRUP25 AC026331 1,092
bp HM74 95% hRUP26 AC023040 1,044 bp Rabbit 5HT1D 27% hRUP27
AC027643 1,020 bp MCH 38% hRUP38 AC026331 1,164 bp HM74 100%
[2226] Such receptors are disclosed, for example, in application
Ser. No. 09/714,008, filed Nov. 16, 2000, which is incorporated by
reference in its entirety.
[2227] Receptor homology is useful in terms of gaining an
appreciation of a role of the receptors within the human body. As
the patent document progresses, techniques for mutating these
receptors to establish non-endogenous, constitutively activated
versions of these receptors will be discussed.
[2228] The techniques disclosed herein have also been applied to
other human, orphan GPCRs known to the art, as will be apparent as
the patent document progresses.
[2229] C. Identification of the Mouse (m) and Rat (r) Orthologs of
Human (h) RUP25 and Identification of the Mouse (m) and Rat (r)
Orthologs of Human (h) RUP19 TABLE-US-00005 TABLE C Reference
Disclosed Open To PerCent Mouse (m) Accession Reading Orthologous
Homology or Rat (r) Number Frame Human To Designated RUP25
Identified (Base Pairs) GPCR GPCR mRUP25 AJ300199 1,083 bp hRUP25
83% rRUP25 None 1,086 bp hRUP25 71% mRUP19 XM_144529 1,032 bp
hRUP19 81% rRUP19 None 1,056 bp hRUP19 83%
D. Receptor Screening
[2230] Screening candidate compounds against a non-endogenous,
constitutively activated version of the GPCRs disclosed herein
allows for the direct identification of candidate compounds which
act at the cell surface receptor, without requiring use, or, in
some embodiments, of the knowledge of the identity of the
receptor's endogenous ligand. Using routine and often commercially
available techniques, one can determine areas within the body where
the endogenous version of human GPCRs disclosed herein is expressed
and/or over-expressed. The expression location of a receptor in a
specific tissue provides a scientist with the ability to assign a
physiological functional role of the receptor. It is also possible
using these techniques to determine related disease/disorder states
which are associated with the expression and/or over-expression of
the receptor; such an approach is disclosed in this patent
document. Furthermore, expression of a receptor in diseased organs
can assist one in determining the magnitude of the clinical
relevance of the receptor.
[2231] Constitutive activation of the GPCRs disclosed herein is
based upon the distance from the proline residue at which is
presumed to be located within TM6 of the GPCR; this algorithmic
technique is disclosed in co-pending and commonly assigned patent
document PCT Application Number PCT/US99/23938, published as WO
00/22129 on Apr. 20, 2000, which, along with the other patent
documents listed herein, is incorporated herein by reference in its
entirety. The algorithmic technique is not predicated upon
traditional sequence "alignment" but rather a specified distance
from the aforementioned TM6 proline residue (or, of course,
endogenous constitutive substitution for such proline residue). By
mutating the amino acid residue located 16 amino acid residues from
this residue (presumably located in the IC3 region of the receptor)
to, preferably, a lysine residue, constitutive activation of the
receptor may be obtained. Other amino acid residues may be useful
in the mutation at this position to achieve this objective and will
be discussed in detail, below.
E. Disease/Disorder Identification and/or Selection
[2232] As will be set forth in greater detail below, inverse
agonists and agonists to the non-endogenous, constitutively
activated GPCR can be identified by the methodologies of this
invention. Such inverse agonists and agonists are good candidates
as lead compounds in drug discovery programs for treating diseases
and/or disorders related to this receptor. Because of the ability
to directly identify inverse agonists and agonists to the GPCR,
thereby allowing for the development of pharmaceutical
compositions, a search for diseases and disorders associated with
the GPCR is relevant. The expression location of a receptor in a
specific tissue provides a scientist with the ability to assign a
physiological function to the receptor. For example, scanning both
diseased and normal tissue samples for the presence of the GPCR now
becomes more than an academic exercise or one which might be
pursued along the path of identifying an endogenous ligand to the
specific GPCR. Tissue scans can be conducted across a broad range
of healthy and diseased tissues. Such tissue scans provide a
potential first step in associating a specific receptor with a
disease and/or disorder. Furthermore, expression of a receptor in
diseased organs can assist one in determining the magnitude of the
clinical relevance of the receptor.
[2233] The DNA sequence of the GPCR can be used to make a
probe/primer. In some preferred embodiments the DNA sequence is
used to make a probe for (a) dot-blot analysis against tissue-mRNA,
and/or (b) RT-PCR identification of the expression of the receptor
in tissue samples. The presence of a receptor in a tissue source,
or a diseased tissue, or the presence of the receptor at elevated
concentrations in diseased tissue compared to a normal tissue, can
be used to correlate location to function and indicate the
receptor's physiological role/function and create a treatment
regimen, including but not limited to, a disease associated with
that function/role. Receptors can also be localized to regions of
organs by this technique. Based on the known or assumed
roles/functions of the specific tissues to which the receptor is
localized, the putative physiological function of the receptor can
be deduced. For example and not limitation, proteins
located/expressed in areas of the thalamus are associated with
sensorimotor processing and arousal (see, Goodman & Gilman's,
The Pharmacological Basis of Therapeutics, 9.sup.th Edition, page
465 (1996)). Proteins expressed in the hippocampus or in Schwann
cells are associated with learning and memory, and myelination of
peripheral nerves, respectively (see, Kandel, E. et al., Essentials
of Neural Science and Behavior pages 657, 680 and 28, respectively
(1995)).
F. Screening of Candidate Compounds
[2234] 1. Generic GPCR Screening Assay Techniques
[2235] When a G protein receptor becomes constitutively active, it
binds to a G protein (e.g., Gq, Gs, Gi, Gz, Go) and stimulates the
binding of GTP to the G protein. The G protein then acts as a
GTPase and slowly hydrolyzes the GTP to GDP, whereby the receptor,
under normal conditions, becomes deactivated. However,
constitutively activated receptors continue to exchange GDP to GTP.
A non-hydrolyzable analog of GTP, [.sup.35S]GTP.gamma.S, can be
used to monitor enhanced binding to membranes which express
constitutively activated receptors. It is reported that
[.sup.35S]GTP.gamma.S can be used to monitor G protein coupling to
membranes in the absence and presence of ligand. An example of this
monitoring, among other examples well-known and available to those
in the art, was reported by Traynor and Nahorski in 1995. The
preferred use of this assay system is for initial screening of
candidate compounds because the system is generically applicable to
all G protein-coupled receptors regardless of the particular G
protein that interacts with the intracellular domain of the
receptor.
[2236] 2. Specific GPCR Screening Assay Techniques
[2237] Once candidate compounds are identified using the "generic"
G protein-coupled receptor assay (i.e., an assay to select
compounds that are agonists or inverse agonists), in some
embodiments further screening to confirm that the compounds have
interacted at the receptor site is preferred. For example, a
compound identified by the "generic" assay may not bind to the
receptor, but may instead merely "uncouple" the G protein from the
intracellular domain.
[2238] a. Gs, Gz and Gi.
[2239] Gs stimulates the enzyme adenylyl cyclase. Gi (and Gz and
Go), on the other hand, inhibit adenylyl cyclase. Adenylyl cyclase
catalyzes the conversion of ATP to cAMP; thus, constitutively
activated GPCRs that couple the Gs protein are associated with
increased cellular levels of cAMP. On the other hand,
constitutively activated GPCRs that couple Gi (or Gz, Go) protein
are associated with decreased cellular levels of cAMP. See,
generally, "Indirect Mechanisms of Synaptic Transmission," Chpt. 8,
From Neuron To Brain (3.sup.rd Ed.) Nichols, J. G. et al eds.
Sinauer Associates, Inc. (1992). Thus, assays that detect cAMP can
be utilized to determine if a candidate compound is, e.g., an
inverse agonist to the receptor (i.e., such a compound would
decrease the levels of cAMP). A variety of approaches known in the
art for measuring cAMP can be utilized; in some embodiments a
preferred approach relies upon the use of anti-cAMP antibodies in
an ELISA-based format. Another type of assay that can be utilized
is a whole cell second messenger reporter system assay. Promoters
on genes drive the expression of the proteins that a particular
gene encodes. Cyclic AMP drives gene expression by promoting the
binding of a cAMP-responsive DNA binding protein or transcription
factor (CREB) that then binds to the promoter at specific sites
called cAMP response elements and drives the expression of the
gene. Reporter systems can be constructed which have a promoter
containing multiple cAMP response elements before the reporter
gene, e.g., .beta.-galactosidase or luciferase. Thus, a
constitutively activated Gs-linked receptor causes the accumulation
of cAMP that then activates the gene and expression of the reporter
protein. The reporter protein such as .beta.-galactosidase or
luciferase can then be detected using standard biochemical assays
(Chen et al. 1995).
[2240] b. Go and Gq.
[2241] Gq and Go are associated with activation of the enzyme
phospholipase C, which in turn hydrolyzes the phospholipid
PIP.sub.2, releasing two intracellular messengers: diacycloglycerol
(DAG) and inistol 1,4,5-triphoisphate (IP.sub.3). Increased
accumulation of IP.sub.3 is associated with activation of Gq- and
Go-associated receptors. See, generally, "Indirect Mechanisms of
Synaptic Transmission," Chpt. 8, From Neuron To Brain (3.sup.rd
Ed.) Nichols, J. G. et al eds. Sinauer Associates, Inc. (1992).
Assays that detect IP.sub.3 accumulation can be utilized to
determine if a candidate compound is, e.g., an inverse agonist to a
Gq- or Go-associated receptor (i.e., such a compound would decrease
the levels of IP.sub.3). Gq-associated receptors can also been
examined using an AP 1 reporter assay in that Gq-dependent
phospholipase C causes activation of genes containing AP1 elements;
thus, activated Gq-associated receptors will evidence an increase
in the expression of such genes, whereby inverse agonists thereto
will evidence a decrease in such expression, and agonists will
evidence an increase in such expression. Commercially available
assays for such detection are available.
[2242] 3. GPCR Fusion Protein
[2243] The use of an endogenous, constitutively activated GPCR or a
non-endogenous, constitutively activated GPCR, for use in screening
of candidate compounds for the direct identification of inverse
agonists or agonists provides an interesting screening challenge in
that, by definition, the receptor is active even in the absence of
an endogenous ligand bound thereto. Thus, in order to differentiate
between, e.g., the non-endogenous receptor in the presence of a
candidate compound and the non-endogenous receptor in the absence
of that compound, with an aim of such a differentiation to allow
for an understanding as to whether such compound may be an inverse
agonist or agonist or have no affect on such a receptor, in some
embodiments it is preferred that an approach be utilized that can
enhance such differentiation. In some embodiments, a preferred
approach is the use of a GPCR Fusion Protein.
[2244] Generally, once it is determined that a non-endogenous GPCR
has been constitutively activated using the assay techniques set
forth above (as well as others known to the art-skilled), it is
possible to determine the predominant G protein that couples with
the endogenous GPCR. Coupling of the G protein to the GPCR provides
a signaling pathway that can be assessed. In some embodiments it is
preferred that screening take place using a mammalian expression
system, such a system will be expected to have endogenous G protein
therein. Thus, by definition, in such a system, the non-endogenous,
constitutively activated GPCR will continuously signal. In some
embodiments it is preferred that this signal be enhanced such that
in the presence of, e.g., an inverse agonist to the receptor, it is
more likely that it will be able to more readily differentiate,
particularly in the context of screening, between the receptor when
it is contacted with the inverse agonist.
[2245] The GPCR Fusion Protein is intended to enhance the efficacy
of G protein coupling with the non-endogenous GPCR. The GPCR Fusion
Protein is preferred for screening with either an endogenous,
constitutively active GPCR or a non-endogenous, constitutively
activated GPCR because such an approach increases the signal that
is generated in such screening techniques. This is important in
facilitating a significant "signal to noise" ratio; such a
significant ratio is preferred for the screening of candidate
compounds as disclosed herein.
[2246] The construction of a construct useful for expression of a
GPCR Fusion Protein is within the purview of those having ordinary
skill in the art. Commercially available expression vectors and
systems offer a variety of approaches that can fit the particular
needs of an investigator. Important criteria in the construction of
such a GPCR Fusion Protein construct include but are not limited
to, that the endogenous GPCR sequence and the G protein sequence
both be in-frame (preferably, the sequence for the endogenous GPCR
is upstream of the G protein sequence), and that the "stop" codon
of the GPCR be deleted or replaced such that upon expression of the
GPCR, the G protein can also be expressed. Other embodiments
include constructs wherein the endogenous GPCR sequence and the G
protein sequence are not in-frame and/or the "stop" codon is not
deleted or replaced. The GPCR can be linked directly to the G
protein, or there can be spacer residues between the two
(preferably, no more than about 12, although this number can be
readily ascertained by one of ordinary skill in the art). Based
upon convenience it is preferred to use a spacer. In some
embodiments it is preferred, that the G protein that couples to the
non-endogenous GPCR will have been identified prior to the creation
of the GPCR Fusion Protein construct. Because there are only a few
G proteins that have been identified, it is preferred that a
construct comprising the sequence of the G protein (i.e., a
universal G protein construct, see Example 5(a) below) be available
for insertion of an endogenous GPCR sequence therein; this provides
for further efficiency in the context of large-scale screening of a
variety of different endogenous GPCRs having different
sequences.
[2247] As noted above, constitutively activated GPCRs that couple
to Gi, Gz and Go are expected to inhibit the formation of cAMP
making assays based upon these types of GPCRs challenging (i.e.,
the cAMP signal decreases upon activation thus making the direct
identification of, e.g., inverse agonists (which would further
decrease this signal), challenging. As will be disclosed herein, it
has been ascertained that for these types of receptors, it is
possible to create a GPCR Fusion Protein that is not based upon the
GPCR's endogenous G protein, in an effort to establish a viable
cyclase-based assay. Thus, for example, an endogenous Gi coupled
receptor can be fused to a Gs protein--such a fusion construct,
upon expression, "drives" or "forces" the endogenous GPCR to couple
with, e.g., Gs rather than the "natural" Gi protein, such that a
cyclase-based assay can be established. Thus, for Gi, Gz and Go
coupled receptors, in some embodiments it is preferred that when a
GPCR Fusion Protein is used and the assay is based upon detection
of adenylyl cyclase activity, that the fusion construct be
established with Gs (or an equivalent G protein that stimulates the
formation of the enzyme adenylyl cyclase). TABLE-US-00006 TABLE D
Effect of Effect of cAMP Effect of IP.sub.3 cAMP Effect on IP.sub.3
Production upon Accumulation Production Accu- Activation of upon
Activation upon mulation GPCR (i.e., of GPCR (i.e., contact upon
contact constitutive constitutive with an with an G activation or
activation or Inverse Inverse protein agonist binding) agonist
binding) Agonist Agonist Gs Increase N/A Decrease N/A Gi Decrease
N/A Increase N/A Gz Decrease N/A Increase N/A Go Decrease Increase
Increase Decrease Gq N/A Increase N/A Decrease
[2248] Equally effective is a G Protein Fusion construct that
utilizes a Gq Protein fused with a Gs, Gi, Gz or Go Protein. In
some embodiments a preferred fusion construct can be accomplished
with a Gq Protein wherein the first six (6) amino acids of the
G-protein .alpha.-subunit ("G.alpha.q") is deleted and the last
five (5) amino acids at the C-terminal end of G.alpha.q is replaced
with the corresponding amino acids of the G.alpha. of the G protein
of interest. For example, a fusion construct can have a Gq (6 amino
acid deletion) fused with a Gi Protein, resulting in a "Gq/Gi
Fusion Construct". This fusion construct will forces the endogenous
Gi coupled receptor to couple to its non-endogenous G protein, Gq,
such that the second messenger, for example, inositol triphosphate
or diacylgycerol, can be measured in lieu of cAMP production.
[2249] 4. Co-transfection of a Target Gi Coupled GPCR with a
Signal-Enhancer Gs Coupled GPCR (cAMP Based Assays)
[2250] A Gi coupled receptor is known to inhibit adenylyl cyclase,
and, therefore, decreases the level of cAMP production, which can
make the assessment of cAMP levels challenging. In some preferred
embodiments, an effective technique in measuring the decrease in
production of cAMP as an indication of constitutive activation of a
receptor that predominantly couples Gi upon activation can be
accomplished by co-transfecting a signal enhancer, e.g., a
non-endogenous, constitutively activated receptor that
predominantly couples with Gs upon activation (e.g., TSHR-A623I,
disclosed below), with the Gi linked GPCR. As is apparent,
constitutive activation of a Gs coupled receptor can be determined
based upon an increase in production of cAMP. Constitutive
activation of a Gi coupled receptor leads to a decrease in
production cAMP. Thus, the co-transfection approach is intended to
advantageously exploit these "opposite" affects. For example,
co-transfection of a non-endogenous, constitutively activated Gs
coupled receptor (the "signal enhancer") with the endogenous Gi
coupled receptor (the "target receptor") provides a baseline cAMP
signal (i.e., although the Gi coupled receptor will decrease cAMP
levels, this "decrease" will be relative to the substantial
increase in cAMP levels established by constitutively activated Gs
coupled signal enhancer). By then co-transfecting the signal
enhancer with a constitutively activated version of the target
receptor, cAMP would be expected to further decrease (relative to
base line) due to the increased functional activity of the Gi
target (i.e., which decreases cAMP).
[2251] Screening of candidate compounds using a cAMP based assay
can then be accomplished, with two `changes` relative to the use of
the endogenous receptor/G-protein fusion: first, relative to the Gi
coupled target receptor, "opposite" effects will result, i.e., an
inverse agonist of the Gi coupled target receptor will increase the
measured cAMP signal, while an agonist of the Gi coupled target
receptor will decrease this signal; second, as would be apparent,
candidate compounds that are directly identified using this
approach should be assessed independently to ensure that these do
not target the signal enhancing receptor (this can be done prior to
or after screening against the co-transfected receptors).
G. Medicinal Chemistry
[2252] Candidate Compounds
[2253] Any molecule known in the art can be tested for its ability
to modulate (increase or decrease) the activity of a GPCR of the
present invention. For identifying a compound that modulates
activity, candidate compounds can be directly provided to a cell
expressing the receptor.
[2254] This embodiment of the invention is well suited to screen
chemical libraries for molecules which modulate, e.g., inhibit,
antagonize, or agonize, the amount of, or activity of, a receptor.
The chemical libraries can be peptide libraries, peptidomimetic
libraries, chemically synthesized libraries, recombinant, e.g.,
phage display libraries, and in vitro translation-based libraries,
other non-peptide synthetic organic libraries, etc. This embodiment
of the invention is also well suited to screen endogenous candidate
compounds comprising biological materials, including but not
limited to plasma and tissue extracts, and to screen libraries of
endogenous compounds known to have biological activity.
[2255] In some embodiments direct identification of candidate
compounds is conducted in conjunction with compounds generated via
combinatorial chemistry techniques, whereby thousands of compounds
are randomly prepared for such analysis. The candidate compound may
be a member of a chemical library. This may comprise any convenient
number of individual members, for example tens to hundreds to
thousand to millions of suitable compounds, for example peptides,
peptoids and other oligomeric compounds (cyclic or linear), and
template-based smaller molecules, for example benzodiazepines,
hydantoins, biaryls, carbocyclic and polycyclic compounds (e.g.,
naphthalenes, phenothiazines, acridines, steroids etc.),
carbohydrate and amino acid derivatives, dihydropyridines,
benzhydryls and heterocycles (e.g., trizines, indoles,
thiazolidines etc.). The numbers quoted and the types of compounds
listed are illustrative, but not limiting. Preferred chemical
libraries comprise chemical compounds of low molecular weight and
potential therapeutic agents.
[2256] Exemplary chemical libraries are commercially available from
several sources (ArQule, Tripos/PanLabs, ChemDesign,
Pharmacopoeia). In some cases, these chemical libraries are
generated using combinatorial strategies that encode the identity
of each member of the library on a substrate to which the member
compound is attached, thus allowing direct and immediate
identification of a molecule that is an effective modulator. Thus,
in many combinatorial approaches, the position on a plate of a
compound specifies that compound's composition. Also, in one
example, a single plate position may have from 1-20 chemicals that
can be screened by administration to a well containing the
interactions of interest. Thus, if modulation is detected, smaller
and smaller pools of interacting pairs can be assayed for the
modulation activity. By such methods, many candidate molecules can
be screened.
[2257] Many diversity libraries suitable for use are known in the
art and can be used to provide compounds to be tested according to
the present invention. Alternatively, libraries can be constructed
using standard methods. Further, more general, structurally
constrained, organic diversity (e.g., nonpeptide) libraries, can
also be used. By way of example, a benzodiazepine library (see
e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:47084712)
may be used.
[2258] In another embodiment of the present invention,
combinatorial chemistry can be used to identify modulators of the
GPCRs of the present invention. Combinatorial chemistry is capable
of creating libraries containing hundreds of thousands of
compounds, many of which may be structurally similar. While high
throughput screening programs are capable of screening these vast
libraries for affinity for known targets, new approaches have been
developed that achieve libraries of smaller dimension but which
provide maximum chemical diversity. (See e.g., Matter, 1997,
Journal of Medicinal Chemistry 40:1219-1229).
[2259] One method of combinatorial chemistry, affinity
fingerprinting, has previously been used to test a discrete library
of small molecules for binding affinities for a defined panel of
proteins. The fingerprints obtained by the screen are used to
predict the affinity of the individual library members for other
proteins or receptors of interest (in the instant invention, the
receptors of the present invention). The fingerprints are compared
with fingerprints obtained from other compounds known to react with
the protein of interest to predict whether the library compound
might similarly react. For example, rather than testing every
ligand in a large library for interaction with a complex or protein
component, only those ligands having a fingerprint similar to other
compounds known to have that activity could be tested. (See, e.g.,
Kauvar et al., 1995, Chemistry and Biology 2:107-118; Kauvar, 1995,
Affinity fingerprinting, Pharmaceutical Manufacturing
International. 8:25-28; and Kauvar, Toxic-Chemical Detection by
Pattern Recognition in New Frontiers in Agrochemical Immunoassay,
D. Kurtz. L. Stanker and J. H. Skerritt. Editors, 1995, AOAC:
Washington, D.C., 305-312).
[2260] In some preferred embodiments, the candidate compound is an
hydroxypyrazole derivative. In some preferred embodiments, the
candidate compound is a benzotriazole carboxylic acid or ester
derivative.
[2261] Candidate Compounds Identified as Modulators
[2262] Generally, the results of such screening will be compounds
having unique core structures; thereafter, these compounds may be
subjected to additional chemical modification around a preferred
core structure(s) to further enhance the medicinal properties
thereof. Such techniques are known to those in the art and will not
be addressed in detail in this patent document.
H. Pharmaceutical Compositions
[2263] The invention provides methods of treatment (and prevention)
by administration to an individual in need of said treatment (or
prevention) a therapeutically effect amount of a modulator of the
invention [also see, e.g., PCT Application Number PCT/IB02/01461
published as WO 02/066505 on 29 Aug. 2002; the disclosure of each
of which is hereby incorporated by reference in its entirety]. In a
preferred aspect, the modulator is substantially purified. The
individual is preferably an animal including, but not limited to
animals such as cows, pigs, horses, chickens, cats, dogs, rabbits,
rats, mice, etc., and is preferably a mammal, and most preferably
human.
[2264] Modulators of the invention can be administered to non-human
animals [see Examples, infra] and/or humans, alone or in
pharmaceutical or physiologically acceptable compositions where
they are mixed with suitable carriers or excipient(s) using
techniques well known to those in the art. Suitable
pharmaceutically-acceptable carriers are available to those in the
art; for example, see Remington's Pharmaceutical Sciences,
16.sup.th Edition, 1980, Mack Publishing Co., (Oslo et al.,
eds.).
[2265] The pharmaceutical or physiologically acceptable composition
is then provided at therapeutically effect dose. A therapeutically
effective dose refers to that amount of a modulator sufficient to
result in prevention or amelioration of symptoms or physiological
status of metabolic-related disorders or disorders of lipid
metabolism as determined illustratively and not by limitation by
the methods described herein.
[2266] It is expressly considered that the modulators of the
invention may be provided alone or in combination with other
pharmaceutically or physiologically acceptable compounds. Other
compounds for the treatment of disorders of the invention are
currently well known in the art. One aspect of the invention
encompasses the use according to embodiments disclosed herein
further comprising one or more agents selected from the group
consisting of .alpha.-glucosidase inhibitor, aldose reductase
inhibitor, biguanide, HMG-CoA reductase inhibitor, squalene
synthesis inhibitor, fibrate, LDL catabolism enhancer, angiotensin
converting enzyme inhibitor, insulin secretion enhancer and
thiazolidinedione. In some embodiments the agent is a
.alpha.-glucosidase inhibitor. In some embodiments the
.alpha.-glucosidase inhibitor is acarbose, voglibose or miglitol.
In some embodiments the .alpha.-glucosidase inhibitor is voglibose.
In some embodiments the agent is an aldose reductase inhibitor. In
some embodiments the aldose reductase inhibitor is tolurestat;
epalrestat; imirestat; zenarestat; zopolrestat; or sorbinil. In
some embodiments the agent is a biguanide. In some embodiments the
biguanide is phenformin, metformin or buformin. In some embodiments
the biguanide is metformin. In some embodiments the agent is a
HMG-CoA reductase inhibitor. In some embodiments the HMG-CoA
reductase inhibitor is rosuvastatin, pravastatin, simvastatin,
lovastatin, atorvastatin, fluvastatin or cerivastatin. In some
embodiments the agent is a fibrate. In some embodiments the fibrate
is bezafibrate, beclobrate, binifibrate, ciplofibrate,
clinofibrate, clofibrate, clofibric acid, etofibrate, fenofibrate,
gemfibrozil, nicofibrate, pirifibrate, ronifibrate, simfibrate, or
theofibrate. In some embodiments the agent is an angiotensin
converting enzyme inhibitor. In some embodiments the angiotensin
converting enzyme inhibitor is captopril, enalapril, alacepril,
delapril; ramipril, lisinopril, imidapril, benazepril, ceronapril,
cilazapril, enalaprilat, fosinopril, moveltopril, perindopril,
quinapril, spirapril, temocapril or trandolapril. In some
embodiments the agent is an insulin secretion enhancer. In some
embodiments the insulin secretion enhancer is tolbutamide;
chlorpropamide; tolazamide; acetohexamide; glycopyramide;
glibenclamide; gliclazide; 1-butyl-3-metanilylurea; carbutamide;
glibonuride; glipizide; gliquidone; glisoxepid; glybuthiazole;
glibuzole; glyhexamide; glymidine; glypinamide; phenbutamide;
tolcyclamide, glimepiride, nateglinide, or mitiglinide. In some
embodiments the agent is a thiazolidinedione. In some embodiments
the thiazolidinedione is rosiglitazone or pioglitazone. In some
embodiments the thiazolidinedione is rosiglitazone. In some
embodiments, the agent is human adiponectin or a fragment thereof
comprising the globular domain.
[2267] In some embodiments the metabolic disorder is selected from
the group consisting of dyslipidemia, atherosclerosis, coronary
heart disease, insulin resistance, obesity, impaired glucose
tolerance, atheromatous disease, hypertension, stroke, Syndrome X,
heart disease and type 2 diabetes. In some embodiments the
metabolic disorder is selected from the group consisting of
dyslipidemia, atherosclerosis, coronary heart disease, stroke,
insulin resistance and type 2 diabetes. In some embodiments, the
disorder of lipid metabolism is selected from the group consisting
of elevated level of plasma triglycerides, elevated level of plasma
free fatty acids, elevated level of plasma cholesterol, elevated
level of LDL-cholesterol, reduced level of HDL-cholesterol,
elevated total cholesterol/HDL-cholesterol ratio, and reduced level
of plasm adiponectin.
[2268] Routes of Administration
[2269] Suitable routes of administration include oral, nasal,
rectal, transmucosal, or intestinal administration, parenteral
delivery, including intramuscular, subcutaneous, intramedullary
injections, as well as intrathecal, direct intraventricular,
intravenous, intraperitoneal, intranasal, intrapulmonary (inhaled)
or intraocular injections using methods known in the art. Other
particularly preferred routes of administration are aerosol and
depot formulation. Sustained release formulations, particularly
depot, of the invented medicaments are expressly contemplated.
[2270] Composition/Formulation
[2271] Pharmaceutical or physiologically acceptable compositions
and medicaments for use in accordance with the present invention
may be formulated in a conventional manner using one or more
physiologically acceptable carriers comprising excipients and
auxiliaries. Proper formulation is dependent upon the route of
administration chosen.
[2272] Certain of the medicaments described herein will include a
pharmaceutically or physiologically acceptable carrier and at least
one modulator of the invention. For injection, the agents of the
invention may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hanks's solution,
Ringer's solution, or physiological saline buffer such as a
phosphate or bicarbonate buffer. For transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
[2273] Pharmaceutical or physiologically acceptable preparations
that can be taken orally include push-fit capsules made of gelatin,
as well as soft, sealed captulse made of gelatin and a plasticizer,
such as glycerol or sorbitol. The push-fit capsules can contain the
active ingredients in admixture with fillers such as lactose,
binders such as starches, and/or lubricants such as talc or
magnesium stearate and, optionally, stabilizers. In soft capsules,
the active compounds may be dissolved or suspended in suitable
liquids, such as fatty oils, liquid paraffin, or liquid
polyethylene glycols. In addition, stabilizers may be added. All
formulations for oral administration should be in dosages suitable
for such administration.
[2274] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[2275] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs
for a nebulizer, with the use of a suitable gaseous propellant,
e.g., carbon dioxide. In the case of a pressurized aerosol the
dosage unit may be determined by providing a valve to deliver a
metered amount. Capsules and cartridges of, e.g., gelatin, for ue
in an inhaler or insufflator, may be formulated containing a powder
mix of the compound and a suitable powder base such as lactose or
starch.
[2276] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage for, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspension, solutions or emulsions in aqueous vehicles, and may
contain formulatory agents such as suspending, stabilizing and/or
dispersing agents.
[2277] Pharmaceutical or physiologically acceptable formulations
for parenteral administration include aqueous solutions of the
active compounds in water-soluble form. Aqueous suspension may
contain substances that increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents that increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[2278] Alternatively, the active ingredient may be in powder or
lyophilized form for constitution with a suitable vehicle, such as
sterile pyrogen-free water, before use.
[2279] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[2280] In a particular embodiment, the compounds can be delivered
via a controlled release system. In one embodiment, a pump may be
used (Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.
14:201-240; Buchwald et al., 1980, Surgery 88:507-516; Saudek et
al., 1989, N. Engl. J. Med. 321:574-579). In another embodiment,
polymeric materials can be used (Medical Applications of Controlled
Release, Langer and Wise, eds., CRC Press, Boca Raton, Fla., 1974;
Controlled Drug Bioavailability, Drug Product Design and
Performance, Smolen and Ball, eds., Wiley, New York, 1984; Ranger
and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; Levy
et al., 1985, Science 228:190-192; During et al., 1989, Ann.
Neurol. 25:351-356; Howard et al., 1989, J. Neurosurg. 71:858-863).
Other controlled release systems are discussed in the review by
Langer (1990, Science 249:1527-1533).
[2281] Additionally, the compounds may be delivered using a
sustained-release system, such as semipermeable matrices of solid
hydrophobic polymers containing the therapeutic agent. Various
sustained release materials have been established and are well
known by those skilled in the art. Sustained-release capsules may,
depending on their chemical nature, release the compounds for a few
weeks up to over 100 days.
[2282] Depending on the chemical nature and the biological
stability of the therapeutic reagent, additional strategies for
modulator stabilization may be employed.
[2283] The pharmaceutical or physiologically acceptable
compositions also may comprise suitable solid or gel phase carriers
or excipients. Examples of such carriers or excipients include but
are not limited to calcium carbonate, calcium phosphate, various
sugars, starches, cellulose derivatives, gelatin, and polymers such
as polyethylene glycols.
[2284] Effective Dosage
[2285] Pharmaceutical or physiologically acceptable compositions
suitable for use in the present invention include compositions
wherein the active ingredients are contained in an effective amount
to achieve their intended purpose. More specifically, a
therapeutically effective amount means an amount effective to
prevent development of or to alleviate the existing symptoms of the
subject being treated. Determination of the effective amounts is
well within the capability of those skilled in the art, especially
in light of the detailed disclosure provided herein.
[2286] For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. For example, a dose can be formulated in animal
models to achieve a circulating concentration range that includes
or encompasses a concentration point or range shown to
antilipolytic in an in vitro system. [See Examples, infra, for in
vitro assays and in vivo animal models.] Such information can be
used to more accurately determine useful doses in humans.
[2287] A therapeutically effective dose refers to that amount of
the compound that results in amelioration of symptoms in a patient.
Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the test population) and the ED.sub.50 (the
dose therapeutically effective in 50% of the test population). The
dose ratio between toxic and therapeutic effects is the therapeutic
index and it can be expressed as the ratio between LD.sub.50 and
ED.sub.50. Compounds that exhibit high therapeutic indices are
preferred.
[2288] The data obtained from these cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50, with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. The exact formulation, route of
administration and dosage can be chosen by the individual physician
in view of the patient's condition. (See, e.g., Fingl et al., 1975,
in "The Pharmacological Basis of Therapeutics", Ch. 1).
[2289] Dosage amount and interval may be adjusted individually to
provide plasma levels of the active compound which are sufficient
to prevent or treat a disorder of the invention, depending on the
particular situation. Dosages necessary to achieve these effects
will depend on individual characteristics and route of
administration.
[2290] Dosage intervals can also be determined using the value for
the minimum effective concentration. Compounds should be
administered using a regimen that maintains plasma levels above the
minimum effective concentration for 10-90% of the time, preferably
between 30-99%, and most preferably between 50-90%. In cases of
local administration or selective uptake, the effective local
concentration of the drug may not be related to plasma
concentration.
[2291] The amount of composition administered will, of course, be
dependent on the subject being treated, on the subject's weight,
the severity of the affliction, the manner of administration, and
the judgement of the prescribing physician.
[2292] A preferred dosage range for the amount of a modulator of
the invention, which can be administered on a daily or regular
basis to achieve desired results, including but not limited to
reduction of the level of plasma triglycerides, reduction of the
level of plasma free fatty acids, elevation of the level of
HDL-cholesterol, reduction of the level of LDL-cholesterol,
reduction of the level of plasma cholesterol, reduction of the
total cholesterol/HDL-cholesterol ratio, or elevation of the level
of plasma adiponectin, is 0.1-100 mg/kg body mass. Other preferred
dosage range is 0.1-30 mg/kg body mass. Other preferred dosage
range is 0.1-10 mg/kg body mass. Other preferred dosage range is
0.1-3.0 mg/kg body mass. Of course, these daily dosages can be
delivered or administered in small amounts periodically during the
course of a day. It is noted that these dosage ranges are only
preferred ranges and are not meant to be limiting to the
invention.
I. Methods of Treatment
[2293] The invention is drawn inter alia to methods of preventing
or treating disorders of lipid metabolism and metabolic-related
disorders comprising providing an individual in need of such
treatment with a modulator of the invention. Preferably the
modulator is provided to the individual in a pharmaceutical
composition that is preferably taken orally. Preferably the
individual is a mammal, and most preferably a human. In preferred
embodiments, the disorder of lipid metabolism is selected from the
group consisting of elevated level of triglycerides, elevated level
of plasma free fatty acids, elevated level of plasma cholesterol,
elevated level of LDL-cholesterol, reduced level of
HDL-cholesterol, elevated total cholesterol/HDL-cholesterol ratio,
and reduced level of plasma adiponectin. In preferred embodiments,
the metabolic-related disorder is selected from the group
consisting of dyslipidemia, atherosclerosis, coronary heart
disease, stroke, insulin resistance, and type 2 diabetes. Other
metabolic-related disorders to be treated by modulators of the
invention include obesity, impaired glucose tolerance, atheromatous
disease, hypertension, Syndrome X, and heart disease. Heart disease
includes, but is not limited to, cardiac insufficiency, coronary
insufficiency, and high blood pressure. Other metabolic-related
disorder to be treated by modulators of the invention is
hyperlipidemia. In other embodiments, the invention provides for a
method of using a modulator of the invention as an inhibitor of the
progression from impaired glucose tolerance to insulin
resistance.
[2294] The invention also features methods of preventing or
treating disorders of lipid metabolism or metabolic-related
disorders comprising providing an individual in need of such
treatment with a modulator identified by assays of the invention.
Preferably, the modulator is provided to the individual in a
pharmaceutical composition that is preferably taken orally.
Preferably the individual is a mammal, and most preferably a human.
In preferred embodiments, the disorder of lipid metabolism is
selected from the group consisting of elevated level of
triglycerides, elevated level of plasma free fatty acids, elevated
level of plasma cholesterol, elevated level of LDL-cholesterol,
reduced level of HDL-cholesterol, elevated total
cholesterol/HDL-cholesterol ratio, and reduced level of plasma
adiponectin. In preferred embodiments, the metabolic-related
disorder is selected from the group consisting of dyslipidemia,
atherosclerosis, coronary heart disease, stroke, insulin
resistance, and type 2 diabetes. Other metabolic-related disorders
to be treated by modulators of the invention include obesity,
impaired glucose tolerance, atheromatous disease, hypertension,
Syndrome X, and heart disease. Heart disease includes, but is not
limited to, cardiac insufficiency, coronary insufficiency, and high
blood pressure. Other metabolic-related disorder to be treated by
modulators of the invention is hyperlipidemia. In other
embodiments, the invention provides for a method of using a
modulator of the invention as an inhibitor of the progression from
impaired glucose tolerance to insulin resistance.
J. Other Utility
[2295] Although a preferred use of the non-endogenous versions of
the GPCRs disclosed herein may be for the direct identification of
candidate compounds as inverse agonists or agonists (preferably for
use as pharmaceutical agents), other uses of these versions of
GPCRs exist. For example, in vitro and in vivo systems
incorporating GPCRs can be utilized to further elucidate and
understand the roles these receptors play in the human condition,
both normal and diseased, as well as understanding the role of
constitutive activation as it applies to understanding the
signaling cascade. In some embodiments it is preferred that the
endogenous receptors be "orphan receptors", i.e., the endogenous
ligand for the receptor has not been identified. In some
embodiments, therefore, the modified, non-endogenous GPCRs can be
used to understand the role of endogenous receptors in the human
body before the endogenous ligand has been identified. Such
receptors can be used to further elucidate known receptors and the
pathways through which they transduce a signal. The present methods
may also be useful in developing treatment regimens for diseases
and disorders associated with the tissues in which the receptors
are localized. Examples of such diseases and disorders and tissues
in which the receptors are localized are set forth supra and
infra.
[2296] Agents that modulate (i.e., increase, decrease, or block)
nicotinic acid receptor functionality may be identified by
contacting a candidate compound with a nicotinic acid receptor and
determining the effect of the candidate compound on nicotinic acid
receptor functionality. The selectivity of a compound that
modulates the functionality of the nicotinic acid receptor can be
evaluated by comparing its effects on the nicotinic acid receptor
to its effects on other receptors. Following identification of
compounds that modulate nicotinic acid receptor functionality, such
candidate compounds may be further tested in other assays
including, but not limited to, in vivo models, in order to confirm
or quantitate their activity. Modulators of nicotinic acid receptor
functionality will be therapeutically useful in treatment of
diseases and physiological conditions in which normal or aberrant
nicotinic acid receptor functionality is involved.
[2297] Agents that modulate (i.e., increase, decrease, or block)
antilipolytic receptor functionality may be identified by
contacting a candidate compound with an antilipolytic receptor and
determining the effect of the candidate compound on antilipolytic
receptor functionality. The selectivity of a compound that
modulates the functionality of an antilipolytic receptor can be
evaluated by comparing its effects on the antilipolytic receptor to
its effects on other receptors. Following identification of
compounds that modulate antilipolytic receptor functionality, such
candidate compounds may be further tested in other assays
including, but not limited to, in vivo models, in order to confirm
or quantitate their activity. Modulators of antilipolytic receptor
functionality will be therapeutically useful in treatment of
diseases and physiological conditions in which normal or aberrant
antilipolytic receptor functionality is involved.
[2298] Other uses of the disclosed receptors and methods will
become apparent to those in the art based upon, inter alia, a
review of this patent document.
EXAMPLES
[2299] The following examples are presented for purposes of
elucidation, and not limitation, of the present invention. While
specific nucleic acid and amino acid sequences are disclosed
herein, those of ordinary skill in the art are credited with the
ability to make minor modifications to these sequences while
achieving the same or substantially similar results reported below.
The traditional approach to application or understanding of
sequence cassettes from one sequence to another (e.g. from rat
receptor to human receptor or from human receptor A to human
receptor B) is generally predicated upon sequence alignment
techniques whereby the sequences are aligned in an effort to
determine areas of commonality. The mutational approach disclosed
herein does not rely upon this approach but is instead based upon
an algorithmic approach and a positional distance from a conserved
proline residue located within the TM6 region of human GPCRs. Once
this approach is secured, those in the art are credited with the
ability to make minor modifications thereto to achieve
substantially the same results (i.e., constitutive activation)
disclosed herein. Such modified approaches are considered within
the purview of this disclosure.
[2300] The following Examples are provided for illustrative
purposes and not as a means of limitation. One of ordinary skill in
the art would be able to design equivalent assays and methods based
on the disclosure herein, all of which form part of the present
invention.
[2301] Although a variety of expression vectors are available to
those in the art, for purposes of utilization for both the
endogenous and non-endogenous human, mouse and rat GPCRs, it is
most preferred that the vector utilized be pCMV. This vector was
deposited with the American Type Culture Collection (ATCC) on Oct.
13, 1998 (10801 University Blvd., Manassas, Va. 20110-2209 USA)
under the provisions of the Budapest Treaty for the International
Recognition of the Deposit of Microorganisms for the Purpose of
Patent Procedure. The DNA was tested by the ATCC and determined to
be viable. The ATCC has assigned the following deposit number to
pCMV: ATCC #203351. In some alternative embodiments as relates to
said human, mouse and rat GPCRs, it is preferred that the vector
utilized be an adenoviral expression vector.
[2302] Recombinant DNA techniques relating to the subject matter of
the present invention and well known to those of ordinary skill in
the art can be found, e.g, in Maniatis T et al., Molecular Cloning:
A Laboratory Manual (1989) Cold Spring Harbor Laboratory; U.S. Pat.
No. 6,399,373; and PCT Application Number PCT/IB02/01461 published
as WO 02/066505 on 29 Aug. 2002; the disclosure of each of which is
hereby incorporated by reference in its entirety.
Example 1
A. Endogenous Human GPCRs
Identification of Human GPCRs
[2303] The disclosed endogenous human GPCRs were identified based
upon a review of the GenBank.TM. database information. While
searching the database, the following cDNA clones were identified
as evidenced below (Table E). TABLE-US-00007 TABLE E Disclosed
Human Accession Complete Open Reading Nucleic Acid Amino Acid
Orphan Number DNA Sequence Frame SEQ.ID. SEQ.ID. GPCRs Identified
(Base Pairs) (Base Pairs) NO. NO. hRUP8 AL121755 147,566 bp 1,152
bp 1 2 hRUP9 AC0113375 143,181 bp 1,260 bp 3 4 hRUP10 AC008745
94,194 bp 1,014 bp 5 6 hRUP11 AC013396 155,086 bp 1,272 bp 7 8
hRUP12 AP000808 177,764 bp 966 bp 9 10 hRUP13 AC011780 167,819 bp
1,356 bp 11 12 hRUP14 AL137118 168,297 bp 1,041 bp 13 14 hRUP15
AL016468 138,828 bp 1,527 bp 15 16 hRUP16 AL136106 208,042 bp 1,068
bp 17 18 hRUP17 AC023078 161,735 bp 969 bp 19 20 hRUP18 AC008547
117,304 bp 1,305 bp 21 22 hRUP19 AC026331 145,183 bp 1,041 bp 23 24
hRUP20 AL161458 163,511 bp 1,011 bp 25 26 hRUP21 AC026756 156,534
bp 1,014 bp 27 28 hRUP22 AC027026 151,811 bp 993 bp 29 30 hRUP23
AC007104 200,000 bp 1,092 bp 31 32 hRUP24 AL355388 190,538 bp 1,125
bp 33 34 hRUP25 AC026331 145,183 bp 1,092 bp 35 36 hRUP26 AC023040
178,508 bp 1,044 bp 37 38 hRUP27 AC027643 158,700 bp 1,020 bp 39 40
hRUP38 AC026331 145,183 bp 1,164 bp 134 135
[2304] 1. Full Length Cloning
[2305] a. hRUP8 (Seq. Id. Nos. 1 & 2)
[2306] The disclosed human HRUP8 was identified based upon the use
of EST database (dbEST) information. While searching the dbEST, a
cDNA clone with accession number AL121755 was identified to encode
a novel GPCR. The following PCR primers were used for RT-PCR with
human testis Marathon-Ready cDNA (Clontech) as templates:
TABLE-US-00008 5'-CTTGCAGACATCACCATGGCAGCC-3' (SEQ.ID.NO.:41;
sense) and 5'-GTGATGCTCTGAGTACTGGACTGG-3'. (SEQ.ID.NO.:42;
antisense)
[2307] PCR was performed using Advantage cDNA polymerase (Clontech;
manufacturing instructions will be followed) in 50 ul reaction by
the following cycles: 94.degree. C. for 30 sec; 94.degree. C. for
10 sec; 65.degree. C. for 20 sec, 72.degree. C. for 1.5 min, and
72.degree. C. for 7 min. Cycles 2 through 4 were repeated 35
times.
[2308] A 1.2 kb PCR fragment was isolated and cloned into the
pCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big Dye
Terminator kit (P.E. Biosystem). See, SEQ.ID.NO.:1. The putative
amino acid sequence for hRUP8 is set forth in SEQ.ID.NO.:2.
[2309] b. hRUP9 (Seq. Id. Nos. 3 & 4)
[2310] The disclosed human hRUP9 was identified based upon the use
of GeneBank database information. While searching the database, a
cDNA clone with Accession Number AC01375 was identified as a human
genomic sequence from chromosome 5. The full length hRUP9 was
cloned by PCR using primers: TABLE-US-00009
5'-GAAGCTGTGAAGAGTGATGC-3', (SEQ.ID.NO.:43; sense)
5'-GTCAGCAATATTGATAAGCAGCAG-3' (SEQ.ID.NO.:44; antisense)
and human genomic DNA (Promega) as a template. Taq Plus Precision
polymerase (Stratagene) was used for the amplification in a 100
.mu.l reaction with 5% DMSO by the following cycle with step 2 to
step 4 repeated 35 times: 94.degree. C. for 1 minute; 94.degree. C.
for 30 seconds; 56.degree. C. for 30 seconds; 72.degree. C. for 2
minutes; 72.degree. C. for 5 minutes.
[2311] A 1.3 Kb PCR fragment was isolated and cloned into the
pCRII-TOPO vector (Invitrogen) from 1% agarose gel and completely
sequenced using the ABI Big Dye Terminator kit (P.E. Biosystems).
See, SEQ.ID.NO.:3. The putative amino acid sequence for hRUP8 is
set forth in SEQ.ID.NO.:4. The sequence of hRUP9 clones isolated
from human genomic DNA matched with the sequence obtained from data
base.
[2312] c. hRUP10 (Seq. Id. Nos. 5 & 6)
[2313] The disclosed human hRUP10 was identified based upon the use
of GenBank database information. While searching the database, a
cDNA clone with accession number AC008754 was identified as a human
genomic sequence from chromosome 19. The full length hRUP10 was
cloned by RT-PCR using primers: TABLE-US-00010
5'-CCATGGGGAACGATTCTGTCAGCTACG-3' (SEQ.ID.NO.:45; sense) and
5'-GCTATGCCTGAAGCCAGTCTTGTG-3' (SEQ.ID.NO.:46; antisense)
and human leukocyte Marathon-Ready cDNA (Clontech) as a template.
Advantage cDNA polymerase (Clontech) was used for the amplification
in a 50 .mu.l reaction by the following cycle with step 2 to step 4
repeated 35 times: 94.degree. C. for 30 seconds; 94.degree. C. for
10 seconds; 62.degree. C. for 20 seconds; 72.degree. C. for 1.5
minutes; 72.degree. C. for 7 minutes. A 1.0 Kb PCR fragment was
isolated and cloned into the pCRII-TOPO vector (Invitrogen) and
completely sequenced using the ABI Big Dye Terminator kit (P.E.
Biosystems). The nucleic acid sequence of the novel human receptor
hRUP10 is set forth in SEQ.ID.NO.:5 and the putative amino acid
sequence thereof is set forth in SEQ.ID.NO.:6.
[2314] d. hRUP11 (Seq. Id. Nos. 7 & 8)
[2315] The disclosed human hRUP11 was identified based upon the use
of GenBank database information. While searching the database, a
cDNA clone with accession number AC013396 was identified as a human
genomic sequence from chromosome 2. The full length hRUP11 was
cloned by PCR using primers: TABLE-US-00011
5'-CCAGGATGTTGTGTCACCGTGGTGGC-3', (SEQ.ID.NO.:47; sense)
5'-CACAGCGCTGCAGCCCTGCAGCTGGC-3' (SEQ.ID.NO.:48; antisense)
and human genomic DNA (Clontech) as a template. TaqPlus Precision
DNA polymerase (Stratagene) was used for the amplification in a 50
.mu.l reaction by the following cycle with step 2 to step 4
repeated 35 times: 94.degree. C. for 3 minutes; 94.degree. C. for
20 seconds; 67.degree. C. for 20 seconds; 72.degree. C. for 1.5
minutes; 72.degree. C. for 7 minutes. A 1.3 Kb PCR fragment was
isolated and cloned into the pCRII-TOPO vector (Invitrogen) and
completely sequenced using the ABI Big Dye Terminator kit (P.E.
Biosystems). The nucleic acid sequence of the novel human receptor
hRUP11 is set forth in SEQ.ID.NO.:7 and the putative amino acid
sequence thereof is set forth in SEQ.ID.NO.:8.
[2316] e. hRUP12 (Seq. Id. Nos. 9 & 10)
[2317] The disclosed human HRUP12 was identified based upon the use
of GenBank database. While searching the database, a cDNA clone
with accession number AP000808 was identified to encode a new GPCR,
having significant homology with rat RTA and human mas1 oncogene
GPCRs. The full length hRUP12 was cloned by PCR using primers:
TABLE-US-00012 5'-CTTCCTCTCGTAGGGATGAACCAGAC-3' (SEQ.ID.NO.:49;
sense) 5'-CTCGCACAGGTGGGAAGCACCTGTGG-3' (SEQ.ID.NO.:50;
antisense)
and human genomic DNA (Clontech) as template. TaqPlus Precision DNA
polymerase (Stratagene) was used for the amplification by the
following cycle with step 2 to step 4 repeated 35 times: 94.degree.
C. for 3 min; 94.degree. C. for 20 sec; 65.degree. C. for 20 sec;
72.degree. C. for 2 min and 72.degree. C. for 7 min. A 1.0 kb PCR
fragment was isolated and cloned into the pCRII-TOPO vector
(Invitrogen) and sequenced using the ABI Big Dye Terminator kit
(P.E. Biosystems) (see, SEQ.ID.NO.:9 for nucleic acid sequence and
SEQ.ID.NO.:10 for deduced amino acid sequence).
[2318] f. hRUP13 (Seq. Id. Nos. 11 & 12)
[2319] The disclosed human HRUP13 was identified based upon the use
of GenBank database. While searching the database, a cDNA clone
with accession number AC011780 was identified to encode a new GPCR,
having significant homology with GPCR fish GPRX-ORYLA. The full
length hRUP13 was cloned by PCR using primers: TABLE-US-00013
5'-GCCTGTGACAGGAGGTACCCTGG-3' (SEQ.ID.NO.:51; sense)
5'-CATATCCCTCCGAGTGTCCAGCGGC-3' (SEQ.ID.NO.:52; antisense)
and human genomic DNA (Clontech) as template. TaqPlus Precision DNA
polymerase (Stratagene) was used for the amplification by the
following cycle with step 2 to step 4 repeated 35 times: 94.degree.
C. for 3 min; 94.degree. C. for 20 sec; 65.degree. C. for 20 sec;
72.degree. C. for 2 min and 72.degree. C. for 7 min. A 1.35 kb PCR
fragment was isolated and cloned into the pCRII-TOPO vector
(Invitrogen) and completely sequenced using the ABI Big Dye
Terminator kit (P.E. Biosystems) (see, SEQ.ID.NO.:11 for nucleic
acid sequence and SEQ.ID.NO.:12 for deduced amino acid
sequence).
[2320] g. hRUP14 (Seq. Id. Nos. 13 & 14)
[2321] The disclosed human hRUP14 was identified based upon the use
of the GenBank database information. While searching the database,
a cDNA clone with Accession Number AL137118 was identified as a
human genomic sequence from chromosome 13. The full length hRUP14
was cloned by PCR using primers: TABLE-US-00014
5'-GCATGGAGAGAAAATTTATGTCCTTGCAACC-3' (SEQ.ID.NO.:53; sense)
5'-CAAGAACAGGTCTCATCTAAGAGCTCC-3' (SEQ.ID.NO.:54; antisense)
and human genomic DNA (Promega) as a template. Taq Plus Precision
polymerase (Stratagene) and 5% DMSO were used for the amplification
by the following cycle with step 2 and step 3 repeated 35 times:
94.degree. C. for 3 minute; 94.degree. C. for 20 seconds;
58.degree. C. for 2 minutes; 72.degree. C. for 10 minutes.
[2322] A 1.1 Kb PCR fragment was isolated and cloned into the
pCRII-TOPO vector (Invitrogen) and completely sequenced using the
ABI Big Dye Terminator kit (P.E. Biosystems) (see, SEQ.ID.NO.:13
for nucleic acid sequence and SEQ.ID.NO.:14 for deduced amino acid
sequence). The sequence of hRUP14 clones isolated from human
genomic DNA matched with the sequence obtained from database.
[2323] h. hRUP15 (Seq. Id. Nos. 15 & 16)
[2324] The disclosed human hRUP15 was identified based upon the use
of the GenBank database information. While searching the database,
a cDNA clone with Accession Number AC016468 was identified as a
human genomic sequence. The full length hRUP15 was cloned by PCR
using primers: TABLE-US-00015 5'-GCTGTTGCCATGACGTCCACCTGCAC-3'
(SEQ.ID.NO.:55; sense) 5'-GGACAGTTCAAGGTTTGCCTTAGAAC-3'
(SEQ.ID.NO.:56; antisense)
and human genomic DNA (Promega) as a template. Taq Plus Precision
polymerase (Stratagene) was used for the amplification by the
following cycle with step 2 to 4 repeated 35 times: 94.degree. C.
for 3 minute; 94.degree. C. for 20 seconds; 65.degree. C. for 20
seconds; 72.degree. C. for 2 minutes and 72.degree. C. for 7
minutes.
[2325] A 1.5 Kb PCR fragment was isolated and cloned into the
pCRII-TOPO vector (Invitrogen) and completely sequenced using the
ABI Big Dye Terminator kit (P.E. Biosystems). See, SEQ.ID.NO.:15
for nucleic acid sequence and SEQ.ID.NO.:16 for deduced amino acid
sequence. The sequence of hRUP15 clones isolated from human genomic
DNA matched with the sequence obtained from database.
[2326] i. hRUP16 (Seq. Id. Nos. 17 & 18)
[2327] The disclosed human hRUP16 was identified based upon the use
of the GenBank database information. While searching the database,
a cDNA clone with Accession Number AL136106 was identified as a
human genomic sequence from chromosome 13. The full length hRUP16
was cloned by PCR using primers: TABLE-US-00016
5'-CTTTCGATACTGCTCCTATGCTC-3', (SEQ.ID.NO.:57; sense, 5' of
initiation codon) 5'-GTAGTCCACTGAAAGTCCAGTGATCC-3' (SEQ.ID.NO.:58;
antisense, 3' of stop codon)
and human skeletal muscle Marathon-Ready cDNA (Clontech) as
template. Advantage cDNA polymerase (Clontech) was used for the
amplification in a 50 ul reaction by the following cycle with step
2 to 4 repeated 35 times: 94.degree. C. for 30 seconds; 94.degree.
C. for 5 seconds; 69.degree. C. for 15 seconds; 72.degree. C. for 1
minute and 72.degree. C. for 5 minutes.
[2328] A 1.1 Kb PCR fragment was isolated and cloned into the
pCRII-TOPO vector (Invitrogen) and completely sequenced using the
T7 Sequenase kit (Amersham). See, SEQ.ID.NO.:17 for nucleic acid
sequence and SEQ.ID.NO.:18 for deduced amino acid sequence. The
sequence of HRUP16 clones matched with four unordered segments of
AL136106, indicating that the hRUP16 cDNA is composed of 4
exons.
[2329] j. hRUP17 (Seq. Id. Nos. 19 & 20)
[2330] The disclosed human hRUP17 was identified based upon the use
of the GenBank database information. While searching the database,
a cDNA clone with Accession Number AC023078 was identified as a
human genomic sequence from chromosome 11. The full length hRUP17
was cloned by PCR using primers: TABLE-US-00017
5'-TTTCTGAGCATGGATCCAACCATCTC-3' (SEQ.ID.NO.:59; sense, containing
initiation codon) 5'-CTGTCTGACAGGGCAGAGGCTCTTC-3' (SEQ.ID.NO.:60;
antisense, 3'of stop codon)
and human genomic DNA (Promega) as template. Advantage cDNA
polymerase mix (Clontech) was used for the amplification in a 100
.mu.l reaction with 5% DMSO by the following cycle with step 2 to 4
repeated 30 times: 94.degree. C. for 1 min; 94.degree. C. for 15
sec; 67.degree. C. for 20 sec; 72.degree. C. for 1 min and 30 sec;
and 72.degree. C. for 5 min.
[2331] A 970 bp PCR fragment was isolated from a 1% agarose gel and
cloned into the pCRII-TOPO vector (Invitrogen) and completely
sequenced using the ABI Big Dye Terminator Kit (P.E. Biosystems).
See, SEQ.ID.NO.:19 for nucleic acid sequence and SEQ.ID.NO.:20 for
deduced amino acid sequence.
[2332] k. hRUP18 (Seq. Id. Nos. 21 & 22)
[2333] The disclosed human hRUP18 was identified based upon the use
of the GenBank database information. While searching the database,
a cDNA clone with Accession Number AC008547 was identified as a
human genomic sequence from chromosome 5. The full length hRUP18
was cloned by PCR using primers: TABLE-US-00018
5'-GGAACTCGTATAGACCCAGCGTCGCTCC-3', (SEQ.ID.NO.:61; sense, 5' of
the initiation codon) 5'-GGAGGTTGCGCCTTAGCGACAGATGACC-3'
(SEQ.ID.NO.:62; antisense, 3' of stop codon)
and human genomic DNA (Promega) as template. TaqPlus precision DNA
polymerase (Stratagene) was used for the amplification in a 100
.mu.l reaction with 5% DMSO by the following cycle with step 2 to 4
repeated 35 times: 95.degree. C. for 5 min; 95.degree. C. for 30
sec; 65.degree. C. for 30 sec; 72.degree. C. for 2 min; and
72.degree. C. for 5 min.
[2334] A 1.3 kb PCR fragment was isolated from a 1% agarose gel and
cloned into the pCRII-TOPO vector (Invitrogen) and completely
sequenced using the ABI Big Dye Terminator Kit (P.E. Biosystems).
See, SEQ.ID.NO.:21 for nucleic acid sequence and SEQ.ID.NO.:22 for
deduced amino acid sequence.
[2335] 1. hRUP19 (Seq. Id. Nos. 23 & 24)
[2336] The disclosed human hRUP19 was identified based upon the use
of the GenBank database information. While searching the database,
a cDNA clone with Accession Number AC026331 was identified as a
human genomic sequence from chromosome 12. The full length hRUP19
was cloned by PCR using primers:
5'-CTGCACCCGGACACTTGCTCTG-3' (SEQ.ID.NO.:63; sense, 5' of
initiation codon),
[2337] 5'-GTCTGCTTGTTCAGTGCCACTCAAC-3' (SEQ.ID.NO.:64; antisense,
containing the stop codon) and human genomic DNA (Promega) as
template. TaqPlus Precision DNA polymerase (Stratagene) was used
for the amplification with 5% DMSO by the following cycle with step
2 to 4 repeated 35 times: 94.degree. C. for 1 min; 94.degree. C.
for 15 sec; 70.degree. C. for 20 sec; 72.degree. C. for 1 min and
30 sec; and 72.degree. C. for 5 min.
[2338] A 1.1 kp PCR fragment was isolated from a 1% agarose gel and
cloned into the pCRII-TOPO vector (Invitrogen) and completely
sequenced using the ABI Big Dye Terminator Kit (P.E. Biosystems).
See, SEQ.ID.NO.:23 for nucleic acid sequence and SEQ.ID.NO.:24 for
deduced amino acid sequence.
[2339] m. hRUP20 (Seq. Id. Nos. 25 & 26)
[2340] The disclosed human hRUP20 was identified based upon the use
of the GenBank database information. While searching the database,
a cDNA clone with Accession Number AL161458 was identified as a
human genomic sequence from chromosome 1. The full length hRUP20
was cloned by PCR using primers:
5'-TATCTGCAATTCTATTCTAGCTCCTG-3' (SEQ.ID.NO.:65; sense, 5' of
initiation codon),
[2341] 5'-TGTCCCTAATAAAGTCACATGAATGC-3' (SEQ.ID.NO.:66; antisense,
3' of stop codon) and human genomic DNA (Promega) as template.
Advantage cDNA polymerase mix (Clontech) was used for the
amplification with 5% DMSO by the following cycle with step 2 to 4
repeated 35 times: 94.degree. C. for 1 min; 94.degree. C. for 15
sec; 60.degree. C. for 20 sec; 72.degree. C. for 1 min and 30 sec;
and 72.degree. C. for 5 min.
[2342] A 1.0 kp PCR fragment was isolated from a 1% agarose gel and
cloned into the pCRII-TOPO vector (Invitrogen) and completely
sequenced using the ABI Big Dye Terminator Kit (P.E. Biosystems).
See, SEQ.ID.NO.:25 for nucleic acid sequence and SEQ.ID.NO.:26 for
deduced amino acid sequence.
[2343] n. hRUP21 (Seq. Id. Nos. 27 & 28)
[2344] The disclosed human hRUP21 was identified based upon the use
of the GenBank database information. While searching the database,
a cDNA clone with Accession Number AC026756 was identified as a
human genomic sequence from chromosome 13. The full length hRUP21
was cloned by PCR using primers: TABLE-US-00019
5'-GGAGACAACCATGAATGAGCCAC-3' (SEQ.ID.NO.:67; sense)
5'-TATTTCAAGGGTTGTTTGAGTAAC-3' (SEQ.ID.NO.:68; antisense)
and human genomic DNA (Promega) as template. Taq Plus Precision
polymerase (Stratagene) was used for the amplification in a 100
.mu.l reaction with 5% DMSO by the following cycle with step 2 to 4
repeated 30 times: 94.degree. C. for 1 min; 94.degree. C. for 15
sec; 55.degree. C. for 20 sec; 72.degree. C. for 1 min and 30 sec;
and 72.degree. C. for 5 min.
[2345] A 1,014 bp PCR fragment was isolated from a 1% agarose gel
and cloned into the pCRII-TOPO vector (Invitrogen) and completely
sequenced using the ABI Big Dye Terminator Kit (P.E. Biosystems).
See, SEQ.ID.NO.:27 for nucleic acid sequence and SEQ.ID.NO.:28 for
deduced amino acid sequence.
[2346] o. hRUP22 (Seq. Id. Nos. 29 & 30)
[2347] The disclosed human hRUP22 was identified based upon the use
of the GenBank database information. While searching the database,
a cDNA clone with Accession Number AC027026 was identified as a
human genomic sequence from chromosome 11. The full length hRUP22
was cloned by PCR using primers:
5'-GGCACCAGTGGAGGTTTTCTGAGCATG-3' (SEQ.ID.NO.:69; sense, containing
initiation codon)
5'-CTGATGGAAGTAGAGGCTGTCCATCTC-3' (SEQ.ID.NO.:70; antisense, 3' of
stop codon)
[2348] and human genomic DNA (Promega) as template. TaqPlus
Precision DNA polymerase (Stratagene) was used for the
amplification in a 100 .mu.l reaction with 5% DMSO by the following
cycle with step 2 to 4 repeated 30 times: 94.degree. C., 1 minutes
94.degree. C., 15 seconds 55.degree. C., 20 seconds 72.degree. C.,
1.5 minute 72.degree. C., 5 minutes.
[2349] A 970 bp PCR fragment was isolated from a 1% agarose gel and
cloned into the pCRII-TOPO vector (Invitrogen) and completely
sequenced using the ABI Big Dye Terminator Kit (P.E. Biosystems).
See, SEQ.ID.NO.:29 for nucleic acid sequence and SEQ.ID.NO.:30 for
deduced amino acid sequence.
[2350] p. hRUP23 (Seq. Id. Nos. 31 & 32)
[2351] The disclosed human hRUP23 was identified based upon the use
of the GenBank database information. While searching the database,
a cDNA clone with Accession Number AC007104 was identified as a
human genomic sequence from chromosome 4. The full length hRUP23
was cloned by PCR using primers:
[2352] 5'-CCTGGCGAGCCGCTAGCGCCATG-3' (SEQ.ID.NO.:71; sense, ATG as
the initiation codon), 5'-ATGAGCCCTGCCAGGCCCTCAGT-3'
(SEQ.ID.NO.:72; antisense, TCA as the stop codon) and human
placenta Marathon-Ready cDNA (Clontech) as template. Advantage cDNA
polymerase (Clontech) was used for the amplification in a 50 ul
reaction by the following cycle with step 2 to 4 repeated 35 times:
95.degree. C. for 30 sec; 95.degree. C. for 15 sec; 66.degree. C.
for 20 sec; 72.degree. C. for 1 min and 20 sec; and 72.degree. C.
for 5 min.
[2353] A 1.0 kb PCR fragment was isolated and cloned into the
pCRII-TOPO vector (Invitrogen) and completely sequenced using the
ABI Big Dye Terminator Kit (P.E. Biosystems). See, SEQ.ID.NO.:31
for nucleic acid sequence and SEQ.ID.NO.:32 for deduced amino acid
sequence.
[2354] q. hRUP24 (Seq. Id. Nos. 33 & 34)
[2355] The disclosed human hRUP24 was identified based upon the use
of the GenBank database information. While searching the database,
a cDNA clone with Accession Number AL355388 was identified as a
human genomic sequence from chromosome 1. The full length hRUP24
was cloned by PCR using primers:
5'-CTGCGATGCCCACACTCAATACTTCTG-3' (SEQ.ID.NO.:73; sense, 5' of
initiation codon),
[2356] 5'-AAGGATCCTACACTTGGTGGATCTCAG-3' (SEQ.ID.NO.:74; antisense,
3' of stop codon) and human genomic DNA (Promega) as template.
Advantage cDNA polymerase mix (Clontech) was used for the
amplification with 5% DMSO by the following cycle with step 2 to 4
repeated 35 times: 94.degree. C. for 1 minute; 94.degree. C. for 15
seconds; 56.degree. C. for 20 seconds 72.degree. C. for 1 minute 30
seconds and 72.degree. C. for 5 minutes.
[2357] A 1.2 kb PCR fragment was isolated from a 1% agarose gel and
cloned into the pCRII-TOPO vector (Invitrogen) and completely
sequenced using the ABI Big Dye Terminator Kit (P.E. Biosystems).
See, SEQ.ID.NO.:33 for nucleic acid sequence and SEQ.ID.NO.:34 for
deduced amino acid sequence.
[2358] r. hRUP25 (Seq. Id. Nos. 35 & 36)
[2359] The disclosed human hRUP25 was identified based upon the use
of the GenBank database information. While searching the database,
a cDNA clone with Accession Number AC026331 was identified as a
human genomic sequence from chromosome 12. The full length hRUP25
was cloned by PCR using primers:
5'-GCTGGAGCATTCACTAGGCGAG-3' (SEQ.ID.NO.:75; sense, 5' of
initiation codon),
[2360] 5'-AGATCCTGGTTCTTGGTGACAATG-3' (SEQ.ID.NO.:76; antisense, 3'
of stop codon) and human genomic DNA (Promega) as template.
Advantage cDNA polymerase mix (Clontech) was used for the
amplification with 5% DMSO by the following cycle with step 2 to 4
repeated 35 times: 94.degree. C. for 1 minute; 94.degree. C. for 15
seconds; 56.degree. C. for 20 seconds 72.degree. C. for 1 minute 30
seconds and 72.degree. C. for 5 minutes.
[2361] A 1.2 kb PCR fragment was isolated from a 1% agarose gel and
cloned into the pCRII-TOPO vector (Invitrogen) and completely
sequenced using the ABI Big Dye Terminator Kit (P.E. Biosystems).
See, SEQ.ID.NO.:35 for nucleic acid sequence and SEQ.ID.NO.:36 for
deduced amino acid sequence.
[2362] s. hRUP26 (Seq. Id. Nos. 37 & 38)
[2363] The disclosed human hRUP26 was identified based upon the use
of GenBank database information. While searching the database, a
cDNA clone with Accession Number AC023040 was identified as a human
genomic sequence from chromosome 2. The full length hRUP26 was
cloned by RT-PCR using hRUP26 specific primers:
5'-AGCCATCCCTGCCAGGAAGCATGG-3' (SEQ.ID.NO.:77; sense, containing
initiation codon)
5'-CCAGACTGTGGACTCAAGAACTCTAGG-3' (SEQ.ID.NO.:78; antisense,
containing stop codon)
[2364] and human pancreas Marathon--Ready cDNA (Clontech) as
template. Advantage cDNA polymerase mix (Clontech) was used for the
amplification in a 10011 reaction with 5% DMSO by the following
cycle with step 2 to 4 repeated 35 times: 94.degree. C. for 5
minute; 95.degree. C. for 30 seconds; 65.degree. C. for 30 seconds
72.degree. C. for 2 minute and 72.degree. C. for 5 minutes.
[2365] A 1.1 kb PCR fragment was isolated from 1% agarose gel and
cloned into the pCRII-TOPO vector (Invitrogen) and completely
sequenced using the ABI Big Dye Terminator Kit (P.E. Biosystems).
See, SEQ.ID.NO.:37 for nucleic acid sequence and SEQ.ID.NO.:38 for
deduced amino acid sequence.
[2366] t. hRUP27 (Seq. Id. Nos. 39 & 40)
[2367] The disclosed human hRUP27 was identified based upon the use
of GenBank database information. While searching the database, a
cDNA clone with Accession Number AC027643 was identified as a human
genomic sequence from chromosome 12. The full length hRUP27 was
cloned by PCR using hRUP27 specific primers:
5'-AGTCCACGAACAATGAATCCATITCATG-3' (SEQ.ID.NO.:79; sense,
containing initiation codon),
5'-ATCATGTCTAGACTCATGGTGATCC-3' (SEQ.ID.NO.:80; antisense, 3' of
stop codon)
[2368] and the human adult brain Marathon-Ready cDNA (Clontech) as
template. Advantage cDNA polymerase mix (Clontech) was used for the
amplification in a 5011 reaction with 5% DMSO by the following
cycle with step 2 to 4 repeated 35 times: 94.degree. C. for 1
minute; 94.degree. C. for 10 seconds; 58.degree. C. for 20 seconds
72.degree. C. for 1 minute 30 seconds and 72.degree. C. for 5
minutes.
[2369] A 1.1 kb PCR fragment was isolated from 1% agarose gel and
cloned into the pCRII-TOPO vector (Invitrogen) and completely
sequenced using the ABI Big Dye Terminator Kit (P.E. Biosystems).
See, SEQ.ID.NO.:35 for nucleic acid sequence and SEQ.ID.NO.:36 for
deduced amino acid sequence. The sequence of hRUP27 cDNA clone
isolated from human brain was determined to match with five
unordered segments of AC027643, indicating that the hRUP27 cDNA is
composed of 5 exons.
[2370] a. hRUP38 (Seq. Id. Nos. 134 & 135)
[2371] The disclosed human hRUP38 was identified based upon the use
of GenBank database information. While searching the database, a
cDNA clone was identified as a human genomic sequence from
chromosome 12. The full length hRUP38 was cloned by PCR using
hRUP38 specific primers:
5'-GCACTCATGAATCGGCACCA-3' (SEQ.ID.NO.:148; sense, containing
initiation codon),
5'-CAGTGACATTACTCGATGCA-3' (SEQ.ID.NO.:149; antisense, 3' of stop
codon)
[2372] and human genomic DNA (Promega) as template. Advantage cDNA
polymerase mix (Clontech) was used for the amplification in a 50
.mu.l reaction with 5% DMSO by the following cycle with step 2 to 4
repeated 35 times: 94.degree. C. for 1 minute; 94.degree. C. for 10
seconds; 60.degree. C. for 20 seconds 72.degree. C. for 1 minute 30
seconds and 72.degree. C. for 5 minutes.
[2373] A 1.2 kb PCR fragment was isolated from 1% agarose gel and
cloned into the pCRII-TOPO vector (Invitrogen) and completely
sequenced using the ABI Big Dye Terminator Kit (P.E. Biosystems).
See, SEQ.ID.NO.:134 for nucleic acid sequence and SEQ.ID.NO.:135
for deduced amino acid sequence. The sequence of hRUP38 DNA clone
isolated from human genomic DNA was determined to match with one
genomic sequence on chromosome 12 and is without introns.
B. Endogenous Mouse and Rat GPCRS
[2374] 1. Identification of Mouse and Rat GPCRs
[2375] The mouse and rat orthologs of hRUP25 and the mouse ortholog
of hRUP19 have been identified and are disclosed below as
determined from genomic sequence. The rat ortholog of hRUP19 has
also been identified (PCT Application Number PCT/US02/04397,
published as WO 02/83736 on Oct. 24, 2002; said disclosure is
hereby incorporated by reference in its entirety) and is provided
below.
[2376] Evidence to date suggests that there is no mouse or rat
ortholog of hRUP38. As the hRUP25 polynucleotide sequence is about
95% identical to hRUP38 polynucleotide sequence, as hRUP25 and
hRUP38 are found on the same arm of chromosome 12, and as an hRUP38
ortholog is absent from rodents, one may hypothesize without
wishing to be bound by theory that hRUP38 was the product of gene
duplication. This event must have happened subsequent to the
divergence of human from rodents. Possibly hRUP38 represents a
novel antilipolytic regulatory pathway for which there is no
counterpart in rodent.
[2377] Evidence to date suggests that there may also be no mouse or
rat ortholog of hRUP11. TABLE-US-00020 TABLE F Disclosed Mouse (m)
Accession Complete Open Reading Nucleic Acid Amino Acid and Rat (r)
Number DNA Sequence Frame SEQ.ID. SEQ.ID. GPCRs Identified (Base
Pairs) (Base Pairs) NO. NO. mRU25 AJ300199 -- 1,083 bp 136 137
rRUP25 None -- 1,086 bp 138 139 mRUP19 XM_144529 -- 1,032 bp 150
151 rRUP19 None -- 1,056 bp 156 157
[2378] 2. Full Length Cloning
[2379] a. mRUP25 (Seq. Id. Nos. 136 & 137)
[2380] In order to clone the open reading frame encoding the mouse
RUP25 receptor we applied a PCR based cloning strategy. Primers
were designed and synthesized based on the start and stop codon
sequence of the mouse PUMA-g sequence, published on Genbank, and
used on mouse genomic DNA (Promega). The PCR primers were as
follows: TABLE-US-00021 5'-ATGAGCAAGTCAGACCATTTTCTAGTGATA-3'
(SEQ.ID.NO.:140; sense) 5'-TTATCTGGCTTCCACATCTCGTTAA-3'
(SEQ.ID.NO.:141; antisense)
Advantage cDNA polymerase mix (Clontech) was used for the
amplification with 5% DMSO by the following cycle with step 2 to 4
repeated 35 times: 94.degree. C. for 1 minute; 94.degree. C. for 15
seconds; 56.degree. C. for 20 seconds 72.degree. C. for 1 minute 30
seconds and 72.degree. C. for 5 minutes.
[2381] A 1.2 kb PCR fragment was isolated from a 1% agarose gel and
cloned into the pCRII-TOPO vector (Invitrogen) and completely
sequenced using the ABI Big Dye Terminator Kit (P.E. Biosystems).
See, SEQ.ID.NO.:35 for nucleic acid sequence and SEQ.ID.NO.:36 for
deduced amino acid sequence.
[2382] b. rRUP25 (Seq. Id. Nos. 138 & 139)
[2383] The rat RUP25 receptor was cloned in an analogous fashion,
however this was done assuming the sequence would be similar to the
mouse sequence because there is no previously published rat
sequence. Again, we applied a PCR based cloning strategy. Primers
were designed and synthesized based on the start and stop codon
sequence of the mouse PUMA-g sequence, published on Genbank, and
used on rat genomic DNA (Promega). The PCR primers were as follows:
TABLE-US-00022 5'-ATGAGCAAGTCAGACCATTTTCTAGTGATA-3'
(SEQ.ID.NO.:142; sense) 5'-TTATCTGGCTTCCACATCTCGTTAA-3'
(SEQ.ID.NO.:143; antisense)
Cloned Pfu polymerase was used for the amplification by the
following cycle with step 2 to 4 repeated 35 times: 94.degree. C.
for 1 minute; 94.degree. C. for 30 sec; 55.degree. C. for 1 min;
72.degree. C. for 2 min; and a final extension at 72.degree. C. for
10 minutes.
[2384] A 1.2 kb PCR fragment was isolated from a 1% agarose gel and
cloned into the pCRII-TOPO vector (Invitrogen) and 12 clones were
completely sequenced using the ABI Big Dye Terminator Kit (P.E.
Biosystems).
Example 2
Preparation of Non-Endogenous, Constitutively Activated GPCRS
[2385] Those skilled in the art are credited with the ability to
select techniques for mutation of a nucleic acid sequence.
Presented below are approaches utilized to create non-endogenous
versions of several of the human GPCRs disclosed above. The
mutations disclosed below are based upon an algorithmic approach
whereby the 16.sup.th amino acid (located in the IC3 region of the
GPCR) from a conserved proline (or an endogenous, conservative
substitution therefor) residue (located in the TM6 region of the
GPCR, near the TM6/IC3 interface) is mutated, preferably to an
alanine, histamine, arginine or lysine amino acid residue, most
preferably to a lysine amino acid residue.
[2386] 1. Transformer Site-Directed.TM. Mutagenesis
[2387] Preparation of non-endogenous human GPCRs may be
accomplished on human GPCRs using, inter alia, Transformer
Site-Directed.TM. Mutagenesis Kit (Clontech) according to the
manufacturer instructions. Two mutagenesis primers are utilized,
most preferably a lysine mutagenesis oligonucleotide that creates
the lysine mutation, and a selection marker oligonucleotide. For
convenience, the codon mutation to be incorporated into the human
GPCR is also noted, in standard form (Table G): TABLE-US-00023
TABLE G Receptor Identifier Codon Mutation hRUP8 V274K hRUP9 T249K
hRUP10 R232K hRUP11 M294K hRUP12 F220K hRUP16 A238K hRUP17 Y215K
hRUP18 L294K hRUP19 T219K hRUP20 K248A K248H K248R hRUP21 R240K
hRUP22 Y222K hRUP24 A245K hRUP25 I230K hRUP26 V285K hRUP27
T248K
[2388] 2. QuikChange.TM. Site-Directed.TM. Mutagenesis
[2389] Preparation of non-endogenous human GPCRs can also be
accomplished by using QuikChange.TM. Site-Directed.TM. Mutagenesis
Kit (Stratagene, according to manufacturer's instructions).
Endogenous GPCR is preferably used as a template and two
mutagenesis primers utilized, as well as, most preferably, a lysine
mutagenesis oligonucleotide and a selection marker oligonucleotide
(included in kit). For convenience, the codon mutation incorporated
into the novel human GPCR and the respective oligonucleotides are
noted, in standard form (Table H): TABLE-US-00024 TABLE H Cycle
5'-3' Conditions orientation Min ('), (sense), 5'-3' Sec ('')
(SEQ.ID.NO.) orientation Cycles 2-4 Receptor Codon mutation
(antisense) repeated Identifier Mutation underlined (SEQ.ID.NO.) 16
times hRUP13 A268K GGGGAGGGAAAGCA CCAGGAGAACCACC 98.degree. for 2'
AAGGTGGTCCTCCT TTTGCTTTCCCTCC 98.degree. for 30'' GG CC 56.degree.
C. for 30'' (81) (82) 72.degree. for 11' 40'' 72.degree. for 5'
hRUP14 L246K CAGGAAGGCAAAGA GATGATGATGGTGG 98.degree. for 2'
CCACCATCATCATC TCTTTGCCTTCCTG 98.degree. for 30'' (85) (86)
55.degree. C. for 30'' 72.degree. for 11' 40'' 72.degree. for 5'
hRUP15 A398K CCAGTGCAAAGCTA GAAGATCACTTTCT 98.degree. for 2'
AGAAAGTGATCTTC TAGCTTTGCACTGG 98.degree. for 30'' (89) (90)
55.degree. C. for 30'' 72.degree. for 11' 40'' 72.degree. for 5'
hRUP23 W275K GCCGCCACCGCGCC GCCAATCTTCCTCT 98.degree. for 2'
AAGAGGAAGATTGG TGGCGCGGTGGCGG 98.degree. for 30'' C C 56.degree. C.
for 30'' (93) (94) 72.degree. for 11' 40'' 72.degree. for 5'
[2390] The non-endogenous human GPCRs were then sequenced and the
derived and verified nucleic acid and amino acid sequences are
listed in the accompanying "Sequence Listing" appendix to this
patent document, as summarized in Table I below: TABLE-US-00025
TABLE I Non Endogenous Nucleic Acid Amino Acid Human GPCR Sequence
Listing Sequence Listing hRUP13 SEQ.ID.NO.:83 SEQ.ID.NO.:84 hRUP14
SEQ.ID.NO.:87 SEQ.ID.NO.:88 hRUP15 SEQ.ID.NO.:91 SEQ.ID.NO.:92
hRUP23 SEQ.ID.NO.:95 SEQ.ID.NO.:96
Example 3
Receptor Expression
[2391] Although a variety of cells are available to the art for the
expression of proteins, it is most preferred that mammalian cells
be utilized. The primary reason for this is predicated upon
practicalities, i.e., utilization of, e.g., yeast cells for the
expression of a GPCR, while possible, introduces into the protocol
a non-mammalian cell which may not (indeed, in the case of yeast,
does not) include the receptor-coupling, genetic-mechanism and
secretary pathways that have evolved for mammalian systems--thus,
results obtained in non-mammalian cells, while of potential use,
are not as preferred as that obtained from mammalian cells. Of the
mammalian cells, COS-7, 293 and 293T cells are particularly
preferred, although the specific mammalian cell utilized can be
predicated upon the particular needs of the artisan.
[2392] a. Transient Transfection
[2393] On day one, 6.times.10.sup.6/10 cm dish of 293 cells well
were plated out. On day two, two reaction tubes were prepared (the
proportions to follow for each tube are per plate): tube A was
prepared by mixing 4 .mu.g DNA (e.g., pCMV vector; pCMV vector with
receptor cDNA, etc.) in 0.5 ml serum free DMEM (Gibco BRL); tube B
was prepared by mixing 2411 lipofectamine (Gibco BRL) in 0.5 ml
serum free DMEM. Tubes A and B were admixed by inversions (several
times), followed by incubation at room temperature for 30-45 min.
The admixture is referred to as the "transfection mixture". Plated
293 cells were washed with 1.times.PBS, followed by addition of 5
ml serum free DMEM. 1 ml of the transfection mixture was added to
the cells, followed by incubation for 4 hrs at 37.degree. C./5%
CO.sub.2. The transfection mixture was removed by aspiration,
followed by the addition of 10 ml of DMEM/10% Fetal Bovine Serum.
Cells were incubated at 37.degree. C./5% CO.sub.2. After 48 hr
incubation, cells were harvested and utilized for analysis.
[2394] b. Stable Cell Lines: Gs Fusion Protein
[2395] Approximately 12.times.10.sup.6 293 cells are plated on a 15
cm tissue culture plate. Grown in DME High Glucose Medium
containing ten percent fetal bovine serum and one percent sodium
pyruvate, L-glutamine, and antibiotics. Twenty-four hours following
plating of 293 cells (or to .about.80% confluency), the cells are
transfected using 12 .mu.g of DNA. The 12 .mu.g of DNA is combined
with 60 .mu.l of lipofectamine and 2 mL of DME High Glucose Medium
without serum. The medium is aspirated from the plates and the
cells are washed once with medium without serum. The DNA,
lipofectamine, and medium mixture are added to the plate along with
10 mL of medium without serum. Following incubation at 37 degrees
Celsius for four to five hours, the medium is aspirated and 25 ml
of medium containing serum is added. Twenty-four hours following
transfection, the medium is aspirated again, and fresh medium with
serum is added. Forty-eight hours following transfection, the
medium is aspirated and medium with serum is added containing
geneticin (G418 drug) at a final concentration of 500 .mu.g/mL. The
transfected cells now undergo selection for positively transfected
cells containing the G418 resistant gene. The medium is replaced
every four to five days as selection occurs. During selection,
cells are grown to create stable pools, or split for stable clonal
selection.
Example 4
Assays for Determination of Constitutive Activity of Non-Endogenous
GPCRs
[2396] A variety of approaches are available for assessment of
constitutive activity of the non-endogenous human GPCRs. The
following are illustrative; those of ordinary skill in the art are
credited with the ability to determine those techniques that are
preferentially beneficial for the needs of the artisan.
[2397] 1. Membrane Binding Assays: [.sup.35S]GTP.gamma.S Assay
[2398] When a G protein-coupled receptor is in its active state,
either as a result of ligand binding or constitutive activation,
the receptor couples to a G protein and stimulates the release of
GDP and subsequent binding of GTP to the G protein. The alpha
subunit of the G protein-receptor complex acts as a GTPase and
slowly hydrolyzes the GTP to GDP, at which point the receptor
normally is deactivated. Constitutively activated receptors
continue to exchange GDP for GTP. The non-hydrolyzable GTP analog,
[.sup.35S]GTP.gamma.S, can be utilized to demonstrate enhanced
binding of [.sup.35S]GTP.gamma.S to membranes expressing
constitutively activated receptors. The advantage of using
[.sup.35S]GTP.gamma.S binding to measure constitutive activation is
that:
[2399] (a) it is generically applicable to all G protein-coupled
receptors; (b) it is proximal at the membrane surface making it
less likely to pick-up molecules which affect the intracellular
cascade.
[2400] The assay utilizes the ability of G protein coupled
receptors to stimulate [.sup.35S]GTP.gamma.S binding to membranes
expressing the relevant receptors. The assay can, therefore, be
used in the direct identification method to screen candidate
compounds to known, orphan and constitutively activated G
protein-coupled receptors. The assay is generic and has application
to drug discovery at all G protein-coupled receptors.
[2401] The [.sup.35]GTP.gamma.S assay was incubated in 20 mM HEPES
and between 1 and about 20 mM MgCl.sub.2 (this amount can be
adjusted for optimization of results, although 20 mM is preferred)
pH 7.4, binding buffer with between about 0.3 and about 1.2 nM
[.sup.35S]GTP.gamma.S (this amount can be adjusted for optimization
of results, although 1.2 is preferred) and 12.5 to 75 .mu.g
membrane protein (e.g, 293 cells expressing the Gs Fusion Protein;
this amount can be adjusted for optimization) and 10 .mu.M GDP
(this amount can be changed for optimization) for 1 hour. Wheatgerm
agglutinin beads (25 .mu.l; Amersham) were then added and the
mixture incubated for another 30 minutes at room temperature. The
tubes were then centrifuged at 1500.times.g for 5 minutes at room
temperature and then counted in a scintillation counter.
[2402] 2. Adenylyl Cyclase
[2403] A Flash Plate.TM. Adenylyl Cyclase kit (New England Nuclear;
Cat. No. SMP004A) designed for cell-based assays can be modified
for use with crude plasma membranes. The Flash Plate wells can
contain a scintillant coating which also contains a specific
antibody recognizing cAMP. The cAMP generated in the wells can be
quantitated by a direct competition for binding of radioactive cAMP
tracer to the cAMP antibody. The following serves as a brief
protocol for the measurement of changes in cAMP levels in whole
cells that express the receptors.
[2404] Transfected cells were harvested approximately twenty four
hours after transient transfection. Media is carefully aspirated
off and discarded. 10 ml of PBS is gently added to each dish of
cells followed by careful aspiration. 1 ml of Sigma cell
dissociation buffer and 3 ml of PBS are added to each plate. Cells
were pipetted off the plate and the cell suspension was collected
into a 50 ml conical centrifuge tube. Cells were then centrifuged
at room temperature at 1,100 rpm for 5 min. The cell pellet was
carefully re-suspended into an appropriate volume of PBS (about 3
ml/plate). The cells were then counted using a hemocytometer and
additional PBS was added to give the appropriate number of cells
(with a final volume of about 50 .mu.l/well).
[2405] cAMP standards and Detection Buffer (comprising 1 .mu.Ci of
tracer [.sup.125I cAMP (50 .mu.l] to 11 ml Detection Buffer) was
prepared and maintained in accordance with the manufacturer's
instructions. Assay Buffer was prepared fresh for screening and
contained 50 .mu.l of Stimulation Buffer, 3 ul of test compound (12
.mu.M final assay concentration) and 50 .mu.l cells, Assay Buffer
was stored on ice until utilized. The assay was initiated by
addition of 50 .mu.l of cAMP standards to appropriate wells
followed by addition of 50 ul of PBSA to wells H-11 and H12. 50
.mu.l of Stimulation Buffer was added to all wells. DMSO (or
selected candidate compounds) was added to appropriate wells using
a pin tool capable of dispensing 3 .mu.l of compound solution, with
a final assay concentration of 12 .mu.M test compound and 100 .mu.l
total assay volume. The cells were then added to the wells and
incubated for 60 min at room temperature. 100 .mu.l of Detection
Mix containing tracer cAMP was then added to the wells. Plates were
then incubated additional 2 hours followed by counting in a Wallac
MicroBeta scintillation counter. Values of cAMP/well were then
extrapolated from a standard cAMP curve which was contained within
each assay plate.
[2406] 3. Cell-Based cAMP for Gi Coupled Target GPCRs
[2407] TSHR is a Gs coupled GPCR that causes the accumulation of
cAMP upon activation. TSHR will be constitutively activated by
mutating amino acid residue 623 (i.e., changing an alanine residue
to an isoleucine residue). A Gi coupled receptor is expected to
inhibit adenylyl cyclase, and, therefore, decrease the level of
cAMP production, which can make assessment of cAMP levels
challenging. An effective technique for measuring the decrease in
production of cAMP as an indication of constitutive activation of a
Gi coupled receptor can be accomplished by co-transfecting, most
preferably, non-endogenous, constitutively activated TSHR
(TSHR-A623ID (or an endogenous, constitutively active Gs coupled
receptor) as a "signal enhancer" with a Gi linked target GPCR to
establish a baseline level of cAMP. Upon creating a non-endogenous
version of the Gi coupled receptor, this non-endogenous version of
the target GPCR is then co-transfected with the signal enhancer,
and it is this material that can be used for screening. We will
utilize such approach to effectively generate a signal when a cAMP
assay is used; this approach is preferably used in the direct
identification of candidate compounds against Gi coupled receptors.
It is noted that for a Gi coupled GPCR, when this approach is used,
an inverse agonist of the target GPCR will increase the cAMP signal
and an agonist will decrease the cAMP signal.
[2408] On day one, 2.times.10.sup.4 293 cells/well will be plated
out. On day two, two reaction tubes will be prepared (the
proportions to follow for each tube are per plate): tube A will be
prepared by mixing 2 .mu.g DNA of each receptor transfected into
the mammalian cells, for a total of 4 .mu.g DNA (e.g., pCMV vector;
pCMV vector with mutated THSR (TSHR-A623ID; TSHR-A623I and GPCR,
etc.) in 1.2 ml serum free DMEM (Irvine Scientific, Irvine,
Calif.); tube B will be prepared by mixing 120 .mu.l lipofectamine
(Gibco BRL) in 1.2 ml serum free DMEM. Tubes A and B will then be
admixed by inversions (several times), followed by incubation at
room temperature for 30-45 min. The admixture is referred to as the
"transfection mixture". Plated 293 cells will be washed with
1.times.PBS, followed by addition of 10 ml serum free DMEM. 2.4 ml
of the transfection mixture will then be added to the cells,
followed by incubation for 4 hrs at 37.degree. C./5% CO.sub.2. The
transfection mixture will then be removed by aspiration, followed
by the addition of 25 ml of DMEM/10% Fetal Bovine Serum. Cells will
then be incubated at 37.degree. C./5% CO.sub.2. After 24 hr
incubation, cells will then be harvested and utilized for
analysis.
[2409] A Flash Plate.TM. Adenylyl Cyclase kit (New England Nuclear;
Cat. No. SMP004A) is designed for cell-based assays, however, can
be modified for use with crude plasma membranes depending on the
need of the skilled artisan. The Flash Plate wells will contain a
scintillant coating which also contains a specific antibody
recognizing cAMP. The cAMP generated in the wells can be
quantitated by a direct competition for binding of radioactive cAMP
tracer to the cAMP antibody. The following serves as a brief
protocol for the measurement of changes in cAMP levels in whole
cells that express the receptors.
[2410] Transfected cells will be harvested approximately twenty
four hours after transient transfection. Media will be carefully
aspirated off and discarded. 10 ml of PBS will be gently added to
each dish of cells followed by careful aspiration. 1 ml of Sigma
cell dissociation buffer and 3 ml of PBS will be added to each
plate. Cells will be pipetted off the plate and the cell suspension
will be collected into a 50 ml conical centrifuge tube. Cells will
then be centrifuged at room temperature at 1,100 rpm for 5 min. The
cell pellet will be carefully re-suspended into an appropriate
volume of PBS (about 3 ml/plate). The cells will then be counted
using a hemocytometer and additional PBS is added to give the
appropriate number of cells (with a final volume of about 50
.mu.l/well).
[2411] cAMP standards and Detection Buffer (comprising 1 .mu.Ci of
tracer [.sup.125I cAMP (50 .mu.l] to 11 ml Detection Buffer) will
be prepared and maintained in accordance with the manufacturer's
instructions. Assay Buffer should be prepared fresh for screening
and contained 50 .mu.l of Stimulation Buffer, 3 .mu.l of test
compound (12 .mu.M final assay concentration) and 50 .mu.l cells,
Assay Buffer can be stored on ice until utilized. The assay can be
initiated by addition of 50 .mu.l of cAMP standards to appropriate
wells followed by addition of 50 .mu.l of PBSA to wells H-11 and
H12. Fifty .mu.l of Stimulation Buffer will be added to all wells.
Selected compounds (e.g., TSH) will be added to appropriate wells
using a pin tool capable of dispensing 3 .mu.l of compound
solution, with a final assay concentration of 12 .mu.M test
compound and 100 .mu.l total assay volume. The cells will then be
added to the wells and incubated for 60 min at room temperature.
100 .mu.l of Detection Mix containing tracer cAMP will then be
added to the wells. Plates were then incubated additional 2 hours
followed by counting in a Wallac MicroBeta scintillation counter.
Values of cAMP/well will then be extrapolated from a standard cAMP
curve which is contained within each assay plate.
[2412] 4. Reporter-Based Assays
[2413] a. CRE-LUC Reporter Assay (Gs-Associated Receptors)
[2414] 293 and 293T cells are plated-out on 96 well plates at a
density of 2.times.10.sup.4 cells per well and were transfected
using Lipofectamine Reagent (BRL) the following day according to
manufacturer instructions. A DNA/lipid mixture is prepared for each
6-well transfection as follows: 260 ng of plasmid DNA in 100 .mu.l
of DMEM were gently mixed with 2 .mu.l of lipid in 100 .mu.l of
DMEM (the 260 ng of plasmid DNA consisted of 200 ng of a 8xCRE-Luc
reporter plasmid, 50 ng of pCMV comprising endogenous receptor or
non-endogenous receptor or pCMV alone, and 10 ng of a GPRS
expression plasmid (GPRS in pcDNA3 (Invitrogen)). The 8XCRE-Luc
reporter plasmid was prepared as follows: vector SRIF-.beta.-gal
was obtained by cloning the rat somatostatin promoter (-71/+51) at
BglV-HindIII site in the p.beta.gal-Basic Vector (Clontech). Eight
(8) copies of cAMP response element were obtained by PCR from an
adenovirus template AdpCF126CCRE8 (see, 7 Human Gene Therapy 1883
(1996)) and cloned into the SRIF-.beta.-gal vector at the Kpn-BglV
site, resulting in the 8xCRE-.beta.-gal reporter vector. The
8xCRE-Luc reporter plasmid was generated by replacing the
beta-galactosidase gene in the 8xCRE-.beta.-gal reporter vector
with the luciferase gene obtained from the pGL3-basic vector
(Promega) at the HindIII-BamHI site. Following 30 min. incubation
at room temperature, the DNA/lipid mixture was diluted with 400
.mu.l of DMEM and 100 .mu.l of the diluted mixture was added to
each well. 100 .mu.l of DMEM with 10% FCS were added to each well
after a 4 hr incubation in a cell culture incubator. The following
day the transfected cells were changed with 200 .mu.l/well of DMEM
with 10% FCS. Eight (8) hours later, the wells were changed to 100
.mu.l/well of DMEM without phenol red, after one wash with PBS.
Luciferase activity were measured the next day using the
LucLite.TM. reporter gene assay kit (Packard) following
manufacturer instructions and read on a 1450 MicroBeta.TM.
scintillation and luminescence counter (Wallac).
[2415] b. AP1 Reporter Assay (Gq-Associated Receptors)
[2416] A method to detect Gq stimulation depends on the known
property of Gq-dependent phospholipase C to cause the activation of
genes containing AP1 elements in their promoter. A Pathdetect.TM.
AP-1 cis-Reporting System (Stratagene, Catalogue # 219073) can be
utilized following the protocol set forth above with respect to the
CREB reporter assay, except that the components of the calcium
phosphate precipitate were 410 ng pAP1-Luc, 80 ng pCMV-receptor
expression plasmid, and 20 ng CMV-SEAP.
[2417] C. SRF-Luc Reporter Assay (Gq-Associated Receptors)
[2418] One method to detect Gq stimulation depends on the known
property of Gq-dependent phospholipase C to cause the activation of
genes containing serum response factors in their promoter. A
Pathdetect.TM. SRF-Luc-Reporting System (Stratagene) can be
utilized to assay for Gq coupled activity in, e.g., COS7 cells.
Cells are transfected with the plasmid components of the system and
the indicated expression plasmid encoding endogenous or
non-endogenous GPCR using a Mammalian Transfection.TM. Kit
(Stratagene, Catalogue #200285) according to the manufacturer's
instructions. Briefly, 410 ng SRF-Luc, 80 ng pCMV-receptor
expression plasmid and 20 ng CMV-SEAP (secreted alkaline
phosphatase expression plasmid; alkaline phosphatase activity is
measured in the media of transfected cells to control for
variations in transfection efficiency between samples) are combined
in a calcium phosphate precipitate as per the manufacturer's
instructions. Half of the precipitate is equally distributed over 3
wells in a 96-well plate, kept on the cells in a serum free media
for 24 hours. The last 5 hours the cells are incubated with 1 .mu.M
Angiotensin, where indicated. Cells are then lysed and assayed for
luciferase activity using a Luclite.TM. Kit (Packard, Cat. #
6016911) and "Trilux 1450 Microbeta" liquid scintillation and
luminescence counter (Wallac) as per the manufacturer's
instructions. The data can be analyzed using GraphPad Prism.TM.
2.0a (GraphPad Software Inc.).
[2419] d. Intracellular IP.sub.3 Accumulation Assay (Gq-Associated
Receptors)
[2420] On day 1, cells comprising the receptors (endogenous and/or
non-endogenous) can be plated onto 24 well plates, usually
1.times.10.sup.5 cells/well (although his umber can be optimized.
On day 2 cells can be transfected by firstly mixing 0.25 .mu.g DNA
in 50 .mu.l serum free DMEM/well and 2 .mu.l lipofectamine in 50
.mu.l serum free DMEM/well. The solutions are gently mixed and
incubated for 15-30 min at room temperature. Cells are washed with
0.5 ml PBS and 400 .mu.l of serum free media is mixed with the
transfection media and added to the cells. The cells are then
incubated for 3-4 hrs at 37.degree. C./5% CO.sub.2 and then the
transfection media is removed and replaced with 1 ml/well of
regular growth media. On day 3 the cells are labeled with
.sup.3H-myo-inositol. Briefly, the media is removed and the cells
are washed with 0.5 ml PBS. Then 0.5 ml inositol-free/serum free
media (GIBCO BRL) is added/well with 0.25 .mu.Ci of
.sup.3H-myo-inositol/well and the cells are incubated for 16-18 hrs
o/n at 37.degree. C./5% CO.sub.2. On Day 4 the cells are washed
with 0.5 ml PBS and 0.45 ml of assay medium is added containing
inositol-free/serum free media 10 .mu.M pargyline 10 mM lithium
chloride or 0.4 ml of assay medium and 50 .mu.l of 10 x ketanserin
(ket) to final concentration of 10 .mu.M. The cells are then
incubated for 30 min at 37.degree. C. The cells are then washed
with 0.5 ml PBS and 200 .mu.l of fresh/ice cold stop solution (1M
KOH; 18 mM Na-borate; 3.8 mM EDTA) is added/well. The solution is
kept on ice for 5-10 min or until cells were lysed and then
neutralized by 200 .mu.l of fresh/ice cold neutralization sol.
(7.5% HCL). The lysate is then transferred into 1.5 ml eppendorf
tubes and 1 ml of chloroform/methanol (1:2) is added/tube. The
solution is vortexed for 15 sec and the upper phase is applied to a
Biorad AG1-X8.TM. anion exchange resin (100-200 mesh). Firstly, the
resin is washed with water at 1:1.25 W/V and 0.9 ml of upper phase
is loaded onto the column. The column is washed with 10 mls of 5 mM
myo-inositol and 10 ml of 5 mM Na-borate/60 mM Na-formate. The
inositol tris phosphates are eluted into scintillation vials
containing 10 ml of scintillation cocktail with 2 ml of 0.1 M
formic acid/1 M ammonium formate. The columns are regenerated by
washing with 10 ml of 0.1 M formic acid/3M ammonium formate and
rinsed twice with dd H.sub.2O and stored at 4.degree. C. in
water.
[2421] Exemplary results are presented below in Table J:
TABLE-US-00026 TABLE J Difference (<) Signal Between Signal
Generated: 1. CMV v. Generated: Non- Wild- Assay Endogenous
Endogenous type Utilized Signal Version Version 2. Wild- (Figure
Generated: (Relative (Relative type Receptor Mutation No.) CMV
Light Units) Light Units) v. Mutant hRUP12 N/A IP.sub.3 317.03
cpm/mg 3463.29 cpm/mg -- 1. 11 Fold (FIG. 1) protein protein hRUP13
N/A cAMP 8.06 pmol/cAMP/ 19.10 pmol/cAMP/ -- 1. 2.4 Fold (FIG. 2)
mg protein mg protein A268K 8XCRE- 3665.43 LPCS 83280.17 LPCS
61713.6 LCPS 1. 23 Fold LUC 2. 26% < (FIG. 3) hRUP14 L246K
8XCRE- 86.07 LCPS 1962.87 LCPS 789.73 LCPS 1. 23 Fold LUC 2. 60%
< (FIG. 5) hRUP15 A398K 8XCRE- 86.07 LCPS 18286.77 LCPS 17034.83
LCPS 1. 212 Fold LUC 2. 1% < (FIG. 6) A398K cAMP 15.00
pmol/cAMP/ 164.4 pmol/cAMP/ 117.5 pmol/cAMP/ 1. 11 Fold (FIG. 7) mg
protein mg protein mg protein 2. 29% < hRUP17 N/A IP.sub.3
317.03 cpm/mg 741.07 cpm/ -- 1. 2.3 Fold (FIG. 9) protein mg
protein hRUP21 N/A IP.sub.3 730.5 cpm/mg 1421.9 cpm/mg -- 1. 2 Fold
(FIG. 10) protein protein hRUP23 W275K 8XCRE- 311.73 pmol/cAMP/
13756.00 pmol/cAMP/ 9756.87 pmol/cAMP/ 1. 44 Fold LUC mg protein
protein mg protein 2. 30% < (FIG. 11) N/A = not applied
[2422] Exemplary results of GTP.gamma.S assay for detecting
constitutive activation, as disclosed in Example 4(1) above, was
accomplished utilizing G.sub.s:Fusion Protein Constructs on human
hRUP13 and hRUP15. Table K below lists the signals generated from
this assay and the difference in signals as indicated:
TABLE-US-00027 TABLE K Difference Between: 1. CMV v. Fusion Signal
Protein Signal Signal Generated: 2. CMV + GDP Generated: Generated:
Fusion vs. Signal Fusion CMV + 10 .mu.M Protein + 10 .mu.M Fusion +
GDP Generated: Protein GDP GDP 3. Fusion vs. Receptor: CMV (cpm
(cpm (cpm Fusion + GDP Gs Fusion Assay (cpm bound bound bound bound
(cpm bound Protein Utilized GTP) GTP) GTP) GTP) GTP) hRUP13-
GTP.gamma.S 32494.0 49351.30 11148.30 28834.67 1. 1.5 Fold Gs (FIG.
2. 2.6 Fold 4) 3. 42% < hRUP15- GTP.gamma.S 30131.67 32493.67
7697.00 14157.33 1. 1.1 Fold Gs (FIG. 2. 1.8 Fold 8) 3. 56%
<
Example 5
Fusion Protein Preparation
[2423] a. GPCR:Gs Fusion Constuct
[2424] The design of the constitutively activated GPCR-G protein
fusion construct was accomplished as follows: both the 5' and 3'
ends of the rat G protein Gs.alpha. (long form; Itoh, H. et al., 83
PNAS 3776 (1986)) were engineered to include a HindIII
(5'-AAGCTT-3') sequence thereon. Following confirmation of the
correct sequence (including the flanking HindIII sequences), the
entire sequence was shuttled into pcDNA3.1(-) (Invitrogen, cat. no.
V795-20) by subcloning using the HindIII restriction site of that
vector. The correct orientation for the G.sub.s.alpha. sequence was
determined after subcloning into pcDNA3.1(-). The modified
pcDNA3.1(-) containing the rat G.sub.s.alpha. gene at HindIII
sequence was then verified; this vector was now available as a
"universal" G.sub.s.alpha. (protein vector. The pcDNA3.1 (-) vector
contains a variety of well-known restriction sites upstream of the
HindIII site, thus beneficially providing the ability to insert,
upstream of the Gs protein, the coding sequence of an endogenous,
constitutively active GPCR. This same approach can be utilized to
create other "universal" G protein vectors, and, of course, other
commercially available or proprietary vectors known to the artisan
can be utilized--the important criteria is that the sequence for
the GPCR be upstream and in-frame with that of the G protein.
[2425] hRUP13 couples via Gs. For the following exemplary GPCR
Fusion Proteins, fusion to Gs.alpha. was accomplished.
[2426] A hRUP13Gs.alpha. Fusion Protein construct was made as
follows: primers were designed as follows: TABLE-US-00028
5'-gatc[TGTAGAAT]GGAGTCCTCACCCATCCCCCAG-3' (SEQ ID.NO.:97; sense)
5'-gatc[GATATC]CGTGACTCCAGCCGGGGTGAGGCGGC-3'. (SEQ.ID.NO.:98;
antisense)
[2427] Nucleotides in lower caps are included as spacers in the
restriction sites (designated in brackets) between the G protein
and hRUP13. The sense and anti-sense primers included the
restriction sites for XbaI and EcoRV, respectively, such that
spacers (attributed to the restriction sites) exist between the G
protein and hRUP15.
[2428] PCR was then utilized to secure the respective receptor
sequences for fusion within the Gs.alpha. universal vector
disclosed above, using the following protocol for each: 100 ng cDNA
for hRUP15 was added to separate tubes containing 2 .mu.l of each
primer (sense and anti-sense), 3 .mu.L of 10 mM dNTPs, 10 .mu.L of
10XTaqPlus.TM. Precision buffer, 1 .mu.L of TaqPlus.TM. Precision
polymerase (Stratagene: #600211), and 80 .mu.L of water. Reaction
temperatures and cycle times for hRUP15 were as follows with cycle
steps 2 through 4 were repeated 35 times: 94.degree. C. for 1 min;
94.degree. C. for 30 seconds; 62.degree. C. for 20 sec; 72.degree.
C. 1 min 40 sec; and 72.degree. C. 5 min. PCR product for was run
on a 1% agarose gel and then purified (data not shown). The
purified product was digested with XbaI and EcoRV and the desired
inserts purified and ligated into the Gs universal vector at the
respective restriction site. The positive clones was isolated
following transformation and determined by restriction enzyme
digest; expression using 293 cells was accomplished following the
protocol set forth infra. Each positive clone for hRUP15-Gs Fusion
Protein was sequenced to verify correctness. (See, SEQ.ID.NO.:99
for nucleic acid sequence and SEQ.ID.NO.:100 for amino acid
sequence).
[2429] hRUP15 couples via Gs. For the following exemplary GPCR
Fusion Proteins, fusion to Gs.alpha. was accomplished.
[2430] A hRUP15-Gs.alpha. Fusion Protein construct was made as
follows: primers were designed as follows: TABLE-US-00029
5'-TCTAGAATGACGTCCACCTGCACCAACAGC-3' (SEQ.ID.NO.:101; sense)
5'-gatatcGCAGGAAAAGTAGCAGAATCGTAGGAAG-3'. (SEQ.ID.NO.:102;
antisense)
[2431] Nucleotides in lower caps are included as spacers in the
restriction sites between the G protein and hRUP15. The sense and
anti-sense primers included the restriction sites for EcoRV and
Xba1, respectively, such that spacers (attributed to the
restriction sites) exists between the G protein and hRUP15.
[2432] PCR was then utilized to secure the respective receptor
sequences for fusion within the Gs.alpha. universal vector
disclosed above, using the following protocol for each: 100 ng cDNA
for hRUP15 was added to separate tubes containing 2 .mu.l of each
primer (sense and anti-sense), 3 .mu.L of 10 mM dNTPs, 10 .mu.L of
10XTaqPlus.TM. Precision buffer, 1 uL of TaqPlus.TM. Precision
polymerase (Stratagene: #600211), and 80 .mu.L of water. Reaction
temperatures and cycle times for hRUP15 were as follows with cycle
steps 2 through 4 were repeated 35 times: 94.degree. C. for 1 min;
94.degree. C. for 30 seconds; 62.degree. C. for 20 sec; 72.degree.
C. for 1 min 40 sec; and 72.degree. C. for 5 min. The PCR product
for was run on a 1% agarose gel and then purified (data not shown).
The purified product was digested. The purified product was
digested with EcoRV and Xba1 and the desired inserts purified and
ligated into the Gs universal vector at the respective restriction
site. The positive clones were isolated following transformation
and determined by restriction enzyme digest; expression using 293
cells was accomplished following the protocol set forth infra. Each
positive clone for hRUP15-Gs Fusion Protein was sequenced to verify
correctness. (See, SEQ.ID.NO.:103 for nucleic acid sequence and
SEQ.ID.NO.:104 for amino acid sequence).
[2433] b. Gq(6 Amino Acid Deletion)/Gi Fusion Construct
[2434] The design of a Gq (del)/Gi fusion construct can be
accomplished as follows: the N-terminal six (6) amino acids (amino
acids 2 through 7, having the sequence of TLESIM (SEQ.ID.NO.:129)
G.sub..alpha.q-subunit will be deleted and the C-terminal five (5)
amino acids, having the sequence EYNLV (SEQ.ID.NO.:130) will be
replaced with the corresponding amino acids of the G.sub..alpha.i
Protein, having the sequence DCGLF (SEQ.ID.NO.:131). This fusion
construct will be obtained by PCR using the following primers:
TABLE-US-00030 (SEQ.ID.NO.:132)
5'-gatcaagcttcCATGGCGTGCTGCCTGAGCGAGGAG-3' and (SEQ.ID.NO.:133)
5'-gatcggatccTTAGAACAGGCCGCAGTCCTTCAGGTTCAGCTGCAGG ATGGTG-3'
and Plasmid 63313 which contains the mouse G.sub..alpha.q-wild type
version with a hemagglutinin tag as template. Nucleotides in lower
caps are included as spacers.
[2435] TaqPlus Precision DNA polymerase (Stratagene) will be
utilized for the amplification by the following cycles, with steps
2 through 4 repeated 35 times: 95.degree. C. for 2 min; 95.degree.
C. for 20 sec; 56.degree. C. for 20 sec; 72.degree. C. for 2 min;
and 72.degree. C. for 7 min. The PCR product will be cloned into a
pCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big Dye
Terminator kit (P.E. Biosystems). Inserts from a TOPO clone
containing the sequence of the fusion construct will be shuttled
into the expression vector pcDNA3.1(+) at the HindIII/BamHI site by
a 2 step cloning process.
Example 6
Tissue Distribution of the Disclosed Human GPCRS
[2436] A. RT-PCR
[2437] RT-PCR was applied to confirm the expression and to
determine the tissue distribution of several novel human GPCRs.
Oligonucleotides utilized were GPCR-specific and the human multiple
tissue cDNA panels (MTC, Clontech) as templates. Taq DNA polymerase
(Stratagene) were utilized for the amplification in a 40 .mu.l
reaction according to the manufacturer's instructions. 20 .mu.l of
the reaction will be loaded on a 1.5% agarose gel to analyze the
RT-PCR products. Table L below lists the receptors, the cycle
conditions and the primers utilized.
[2438] By way of illustration, RT-PCR results for hRUP25 and hRUP38
are shown in FIG. 13C. RT-PCR results for hRUP19 are shown in FIG.
27, and RT-PCR results for mRUP19 are shown in FIG. 29. Applicant
discloses herein that hRUP25, hRUP38 and hRUP19 are expressed by
primary adipocytes and, in the case of hRUP38 and hRUP19, have
limited tissue distribution beyond adipose. That hRUP19 has limited
tissue distribution is further apparent by Northern blot analysis
(FIG. 28). TABLE-US-00031 TABLE L Cycle Conditions Min ('), Sec
('') Cycles 2-4 Receptor repeated 5' Primer 3' Primer DNA Tissue
Identifier 30 times (SEQ.ID.NO.) (SEQ.ID.NO.) Fragment Expression
hRUP10 94.degree. for 30'' CATGTATG GCTATGCCT 730 bp Kidney,
94.degree. for 10'' CCAGCGTC GAAGCCAG leukocyte, 62.degree. C. for
20'' CTGCTCC TCTTGTG liver, 72.degree. for 1' (105) (106) placenta
72.degree. for 7' and spleen *cycles 2-4 repeated 35 times hRUP11
94.degree. for 2' GCACCTGC CACAGCGC 630 bp Liver, kidney,
94.degree. for 15'' TCCTGAGC TGCAGCCCT pancreas, 67.degree. C. for
15'' ACCTTCTCC GCAGCTGG colon, small 72.degree. for 45'' (107) C
intestinal, 72.degree. for 5' (108) spleen and prostate hRUP12
94.degree. for 2' CCAGTGAT CAGACACTT 490 bp Brain, colon,
94.degree. for 15'' GACTCTGT GGCAGGGA heart, kidney, 66.degree. C.
for 15'' CCAGCCTG CGAGGTG leukocyte, 72.degree. for 45'' (109)
(110) pancreas, 72.degree. for 5' prostate, small intestinal,
spleen, testis, and thymus hRUP13 94.degree. for 1' CTTGTGGTC
CATATGCCT 700 bp Placenta and 94.degree. for 15'' TACTGCAG
CCGAGTGTC lung 68.degree. C. for 20'' CATGTTCCG CAGCGGC 72.degree.
for 1' 45'' (111) (112) 72.degree. for 5' hRUP14 94.degree. for 1'
ATGGATCC CAAGAACAG 700 bp Not yet 94.degree. for 15'' TTATCATG
GTCTCATCT determined 68.degree. C. for 20'' GCTTCCTC AAGAGCTCC
72.degree. for 1' 45'' (113) (114) 72.degree. for 5' hRUP16
94.degree. for 30'' CTCTGATG GTAGTCCAC 370 bp Fetal brain,
94.degree. for 5'' CCATCTGCT TGAAAGTC fetal kidney 69.degree. C.
for 15'' GGATTCCTG CAGTGATCC and fetal 72.degree. for 30'' (115)
(116) Skeletal 72.degree. for 5' muscle hRUP18 94.degree. for 2'
TGGTGGCG GTTGCGCCT 330 bp Pancreas 94.degree. for 15'' ATGGCCAA
TAGCGACA 60.degree. C. for 20'' CAGCGCTC GATGACC 72.degree. for 1'
(117) (118) 72.degree. for 5' hRUP19 95.degree. for 4' GGCCGTGG
AACCGGGT 492 bp Adipose, 95.degree. for 1' CTGATTTCC CGCCTTCTT
adipocyte 60.5.degree. C. for 30'' TCCTTAT GATCC 72.degree. for 1'
(152) (153) 72.degree. for 7' *cycles 2-4 repeated 35 times hRUP21
94.degree. for 1' TCAACCTG AAGGAGTA Kidney, lung 94.degree. for
15'' TATAGCAG GCAGAATG and testis 56.degree. C. for 20'' CATCCTC
GTTAGCC 72.degree. for 40'' (119) (120) *cycles 2-3 repeated 30
times hRUP22 94.degree. for 30'' GACACCTG CTGATGGAA Testis, thymus
94.degree. for 15'' TCAGCGGT GTAGAGGCT and spleen 69.degree. C. for
20'' CGTGTGTG GTCCATCTC 72.degree. for 40'' (121) (122) *cycles 2-3
repeated 30 times hRUP23 94.degree. for 2' GCGCTGAG CACGGTGA 520 bp
Placenta 94.degree. for 15'' CGCAGACC CGAAGGGC 60.degree. C. for
20'' AGTGGCTG ACGAGCTC 72.degree. for 1' (123) (124) 72.degree. for
5' hRUP25 96.degree. for 2' CTGATGGA GCTGAAGCT 297 bp Adipocyte,
96.degree. for 30'' CAACTATG GCTGCACAA spleen, 55.degree. C. for 1'
TGAGGCGT ATTTGCACC leukocyte, 72.degree. for 2' TGG (145) kidney,
lung, 72.degree. for 10' (144) testis hRuP26 94.degree. for 2'
AGCCATCC CCAGGTAGG 470 bp Pancreas 94.degree. for 15'' CTGCCAGG
TGTGCAGCA 65.degree. C. for 20'' AAGCATGG CAATGGC 72.degree. for 1'
(125) (126) 72.degree. for 5' hRUP27 94.degree. for 30'' CTGTTCAAC
ATCATGTCT 890 bp Brain 94.degree. for 10'' AGGGCTGGT AGACTCAT
55.degree. C. for 20'' TGGCAAC GGTGATCC 72.degree. for 1' (127)
(128) 72.degree. for 3' *cycles 2-4 repeated 35 times hRUP38
96.degree. for 2' CTACTATGTG CCCTTCTTGG 852 bp Adipocyte,
96.degree. for 30'' CGGCGTTCA AATGGTTATTT spleen, lung 55.degree.
C. for 1' (146) (147) 72.degree. for 2' 72.degree. for 10'
[2439] B. Affymetrix GeneChip.RTM. Technology
[2440] Amino acid sequences were submitted to Affymetrix for the
designing and manufacturing of microarray containing
oligonucleotides to monitor the expression levels of G
protein-coupled receptors (GPCRs) using their GeneChip.RTM.
Technology. Also present on the microarray were probes for
characterized human brain tissues from Harvard Brain Band or
obtained from commercially available sources. RNA samples were
amplified, labeled, hybridized to the microarray, and data analyzed
according to manufacturer's instructions.
[2441] Adipose tissues were monitored for the level of gene
expression of each of the GPCRs represented on the microarray.
GPCRs were determined to be expressed if the expression index was
greater than 100 (based upon and according to manufacturer's
instructions). The data was analyzed and had indicated that
classification of GPCRs with an expression index greater than 100
was reasonable because a number of known GPCRs had previously been
reported to be expressed in neuronal tissues with an expression
index greater than 100.
[2442] Using the GeneChip, Applicant has discovered hRUP25 and
hRUP38 to have high levels of expression in adipocytes, consistent
with hRUP25 and hRUP38 playing a role in lipolysis (see, Goodman
& Gilman's, The Pharmacological Basis of Therapeutics, 9.sup.th
Edition, page 235 (1996). See FIGS. 13A and 13B. FIG. 13A is a plot
representing the expression level of hRUP25 in various tissues.
hRUP25 is highly expressed by primary adipocytes. FIG. 13B is a
plot representing the expression level of hRUP38 in various
tissues. hRUP38 is highly expressed by primary adipocytes.
[2443] This patent document discloses the identification of
nicotinic acid as a ligand and agonist of human, mouse and rat
RUP25. See, Examples infra. The patent document further discloses
that nicotinic acid is not an agonist of hRUP38 or hRUP19. In the
case of hRUP38, this was an unexpected result, as hRUP25 and hRUP38
are about 95% identical (Table B), although it is not a result
without precedent [see, e.g., Yan M et al. Science (2000)
290:523-7; the disclosure of which is hereby incorporated by
reference in its entirety].
Example 7
Protocol: Direct Identification of Inverse Agonists and
Agonists
[2444] A. [.sup.35S]GTP.gamma.S Assay
[2445] Although we have utilized endogenous, constitutively active
GPCRs for the direct identification of candidate compounds as,
e.g., inverse agonists, for reasons that are not altogether
understood, intra-assay variation can become exacerbated. In some
embodiments, a GPCR Fusion Protein, as disclosed above, is also
utilized with a non-endogenous, constitutively activated GPCR. When
such a protein is used, intra-assay variation appears to be
substantially stabilized, whereby an effective signal-to-noise
ratio is obtained. This has the beneficial result of allowing for a
more robust identification of candidate compounds. Thus, in some
embodiments it is preferred that for direct identification, a GPCR
Fusion Protein be used and that when utilized, the following assay
protocols be utilized.
[2446] 1. Membrane Preparation
[2447] In some embodiments membranes comprising the constitutively
active orphan GPCR/Fusion Protein of interest and for use in the
direct identification of candidate compounds as inverse agonists or
agonists are preferably prepared as follows:
[2448] a. Materials
[2449] "Membrane Scrape Buffer" is comprised of 20 mM HEPES and 10
mM EDTA, pH 7.4; "Membrane Wash Buffer" is comprised of 20 mM HEPES
and 0.1 mM EDTA, pH 7.4; "Binding Buffer" is comprised of 20 mM
HEPES, 100 mM NaCl, and 10 mM MgCl.sub.2, pH 7.4
[2450] b. Procedure
[2451] All materials will be kept on ice throughout the procedure.
Firstly, the media will be aspirated from a confluent monolayer of
cells, followed by rinse with 10 ml cold PBS, followed by
aspiration. Thereafter, 5 ml of Membrane Scrape Buffer will be
added to scrape cells; this will be followed by transfer of
cellular extract into 50 ml centrifuge tubes (centrifuged at 20,000
rpm for 17 minutes at 4.degree. C.). Thereafter, the supernatant
will be aspirated and the pellet will be resuspended in 30 ml
Membrane Wash Buffer followed by centrifuge at 20,000 rpm for 17
minutes at 4.degree. C. The supernatant will then be aspirated and
the pellet resuspended in Binding Buffer. This will then be
homogenized using a Brinkman Polytron.TM. homogenizer (15-20 second
bursts until the all material is in suspension). This is referred
to herein as "Membrane Protein".
[2452] 2. Bradford Protein Assay
[2453] Following the homogenization, protein concentration of the
membranes will be determined using the Bradford Protein Assay
(protein can be diluted to about 1.5 mg/ml, aliquoted and frozen
(-80.degree. C.) for later use; when frozen, protocol for use will
be as follows: on the day of the assay, frozen Membrane Protein is
thawed at room temperature, followed by vortex and then homogenized
with a Polytron at about 12.times.1,000 rpm for about 5-10 seconds;
it was noted that for multiple preparations, the homogenizor should
be thoroughly cleaned between homogenization of different
preparations).
[2454] a. Materials
[2455] Binding Buffer (as per above); Bradford Dye Reagent;
Bradford Protein Standard will be utilized, following manufacturer
instructions (Biorad, cat. no. 500-0006).
[2456] b. Procedure
[2457] Duplicate tubes will be prepared, one including the
membrane, and one as a control "blank". Each contained 800 .mu.l
Binding Buffer. Thereafter, 10 .mu.l of Bradford Protein Standard
(1 mg/ml) will be added to each tube, and 10 .mu.g of membrane
Protein will then be added to just one tube (not the blank).
Thereafter, 200 .mu.l of Bradford Dye Reagent will be added to each
tube, followed by vortex of each. After five (5) minutes, the tubes
will be re-vortexed and the material therein will be transferred to
cuvettes. The cuvettes will then be read using a CECIL 3041
spectrophotometer, at wavelength 595.
[2458] 3. Direct Identification Assay
[2459] a. Materials
[2460] GDP Buffer consisted of 37.5 ml Binding Buffer and 2 mg GDP
(Sigma, cat. no. G-7127), followed by a series of dilutions in
Binding Buffer to obtain 0.2 .mu.M GDP (final concentration of GDP
in each well was 0.1 .mu.M GDP); each well comprising a candidate
compound, has a final volume of 200 .mu.l consisting of 100 .mu.l
GDP Buffer (final concentration, 0.1 .mu.M GDP), 50 .mu.l Membrane
Protein in Binding Buffer, and 50 .mu.l [.sup.35S]GTP.gamma.S (0.6
nM) in Binding Buffer (2.5 .mu.l [.sup.35S]GTP.gamma.S per 10 ml
Binding Buffer).
[2461] b. Procedure
[2462] Candidate compounds will be preferably screened using a
96-well plate format (these can be frozen at -80.degree. C.).
Membrane Protein (or membranes with expression vector excluding the
GPCR Fusion Protein, as control), will be homogenized briefly until
in suspension. Protein concentration will then be determined using
the Bradford Protein Assay set forth above. Membrane Protein (and
control) will then be diluted to 0.25 mg/ml in Binding Buffer
(final assay concentration, 12.5 .mu.g/well). Thereafter, 100 .mu.l
GDP Buffer was added to each well of a Wallac Scintistrip.TM.
(Wallac). A 5 ul pin-tool will then be used to transfer 5 .mu.l of
a candidate compound into such well (i.e., 5 .mu.l in total assay
volume of 200 .mu.l is a 1:40 ratio such that the final screening
concentration of the candidate compound is 10 .mu.M). Again, to
avoid contamination, after each transfer step the pin tool should
be rinsed in three reservoirs comprising water (1X), ethanol (1X)
and water (2X)--excess liquid should be shaken from the tool after
each rinse and dried with paper and kimwipes. Thereafter, 50 .mu.l
of Membrane Protein will be added to each well (a control well
comprising membranes without the GPCR Fusion Protein was also
utilized), and pre-incubated for 5-10 minutes at room temperature.
Thereafter, 50 .mu.l of [.sup.35S]GTP.gamma.S (0.6 nM) in Binding
Buffer will be added to each well, followed by incubation on a
shaker for 60 minutes at room temperature (again, in this example,
plates were covered with foil). The assay will then be stopped by
spinning of the plates at 4000 RPM for 15 minutes at 22.degree. C.
The plates will then be aspirated with an 8 channel manifold and
sealed with plate covers. The plates will then be read on a Wallac
1450 using setting "Prot. #37" (as per manufacturer
instructions).
[2463] B. Cyclic AMP Assay
[2464] Another assay approach to directly identified candidate
compound was accomplished by utilizing a cyclase-based assay. In
addition to direct identification, this assay approach can be
utilized as an independent approach to provide confirmation of the
results from the [.sup.35S]GTP.gamma.S approach as set forth
above.
[2465] A modified Flash Plate.TM. Adenylyl Cyclase kit (New England
Nuclear; Cat. No. SMP004A) was preferably utilized for direct
identification of candidate compounds as inverse agonists and
agonists to constitutively activated orphan GPCRs in accordance
with the following protocol.
[2466] Transfected cells were harvested approximately three days
after transfection. Membranes were prepared by homogenization of
suspended cells in buffer containing 20 mM HEPES, pH 7.4 and 10 mM
MgCl.sub.2. Homogenization was performed on ice using a Brinkman
Polytron.TM. for approximately 10 seconds. The resulting homogenate
is centrifuged at 49,000.times.g for 15 minutes at 4.degree. C. The
resulting pellet was then resuspended in buffer containing 20 mM
HEPES, pH 7.4 and 0.1 mM EDTA, homogenized for 10 seconds, followed
by centrifugation at 49,000.times.g for 15 minutes at 4.degree. C.
The resulting pellet was then stored at -80.degree. C. until
utilized. On the day of direct identification screening, the
membrane pellet was slowly thawed at room temperature, resuspended
in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCl.sub.2, to
yield a final protein concentration of 0.60 mg/ml (the resuspended
membranes are placed on ice until use).
[2467] cAMP standards and Detection Buffer (comprising 2 .mu.Ci of
tracer [.sup.125I cAMP (100 .mu.l] to 11 ml Detection Buffer) were
prepared and maintained in accordance with the manufacturer's
instructions. Assay Buffer was prepared fresh for screening and
contained 20 mM HEPES, pH 7.4, 10 mM MgCl.sub.2, 20 mM
phospocreatine (Sigma), 0.1 units/ml creatine phosphokinase
(Sigma), 50 .mu.M GTP (Sigma), and 0.2 mM ATP (Sigma); Assay Buffer
was then stored on ice until utilized.
[2468] Candidate compounds identified as per above (if frozen,
thawed at room temperature) were added, preferably, to 96-well
plate wells (3 .mu.l/well; 12 .mu.M final assay concentration),
together with 40 .mu.l Membrane Protein (30 .mu.g/well) and 50
.mu.l of Assay Buffer. This admixture was then incubated for 30
minutes at room temperature, with gentle shaking.
[2469] Following the incubation, 100 .mu.l of Detection Buffer was
added to each well, followed by incubation for 2-24 hours. Plates
were then counted in a Wallac MicroBeta.TM. plate reader using
"Prot. #31" (as per manufacturer instructions).
[2470] A representative screening assay plate (96 well format)
result is presented in FIG. 12. Each bar represents the results for
a different compound in each well, plus hRUP13-Gs.alpha. Fusion
Protein construct, as prepared in Example 5(a) above. The
representative results presented in FIG. 12 also provide standard
deviations based upon the mean results of each plate ("m") and the
mean plus two arbitrary preference for selection of inverse
agonists as "leads" from the primary screen involves selection of
candidate compounds that that reduce the percent response by at
least the mean plate response, minus two standard deviations.
Conversely, an arbitrary preference for selection of agonists as
"leads" from the primary screen involves selection of candidate
compounds that increase the percent response by at least the mean
plate response, plus the two standard deviations. Based upon these
selection processes, the candidate compounds in the following wells
were directly identified as putative inverse agonist (Compound A)
and agonist (Compound B) to hRUP13 in wells A2 and G9,
respectively. See, FIG. 12. It is noted for clarity: these
compounds have been directly identified without any knowledge of
the endogenous ligand for this GPCR. By focusing on assay
techniques that are based upon receptor function, and not compound
binding affinity, we are able to ascertain compounds that are able
to reduce the functional activity of this receptor (Compound A) as
well as increase the functional activity of the receptor (Compound
B). Based upon the location of these receptors in, for example,
lung tissue (see, for example, hRUP13 and hRUP21 in Example 6),
pharmaceutical agents can be developed for potential therapeutic
treatment of lung cancer.
Example 8
Melanophore Technology
[2471] Melanophores are skin cells found in lower vertebrates. They
contain pigmented organelles termed melanosomes. Melanophores are
able to redistribute these melanosomes along a microtubule network
upon G-protein coupled receptor (GPCR) activation. The result of
this pigment movement is an apparent lightening or darkening of the
cells. In melanophores, the decreased levels of intracellular cAMP
that result from activation of a Gi-coupled receptor cause
melanosomes to migrate to the center of the cell, resulting in a
dramatic lightening in color. If cAMP levels are then raised,
following activation of a Gs-coupled receptor, the melanosomes are
re-dispersed and the cells appear dark again. The increased levels
of diacylglycerol that result from activation of Gq-coupled
receptors can also induce this re-dispersion. In addition, the
technology is also suited to the study of certain receptor tyrosine
kinases. The response of the melanophores takes place within
minutes of receptor activation and results in a simple, robust
color change. The response can be easily detected using a
conventional absorbance microplate reader or a modest video imaging
system. Unlike other skin cells, the melanophores derive from the
neural crest and appear to express a full complement of signaling
proteins. In particular, the cells express an extremely wide range
of G-proteins and so are able to functionally express almost all
GPCRs.
[2472] Melanophores can be utilized to identify compounds,
including natural ligands, against GPCRs. This method can be
conducted by introducing test cells of a pigment cell line capable
of dispersing or aggregating their pigment in response to a
specific stimulus and expressing an exogenous clone coding for the
GCPR. A stimulant, e.g., melatonin, sets an initial state of
pigment disposition wherein the pigment is aggregated within the
test cells if activation of the GPCR induces pigment dispersion.
However, stimulating the cell with a stimulant to set an initial
state of pigment disposition wherein the pigment is dispersed if
activation of the GPCR induces pigment aggregation. The test cells
are then contacted with chemical compounds, and it is determined
whether the pigment disposition in the cells changed from the
initial state of pigment disposition. Dispersion of pigments cells
due to the candidate compound, including but not limited to a
ligand, coupling to the GPCR will appear dark on a petri dish,
while aggregation of pigments cells will appear light.
[2473] Materials and methods will be followed according to the
disclosure of U.S. Pat. No. 5,462,856 and U.S. Pat. No. 6,051,386.
These patent disclosures are hereby incorporated by reference in
their entirety.
[2474] Melanophores were transfected by electroporation with
plasmids coding for the GPCRs, for example hRUP25, hRUP38, hRUP11
and hRUP19. Pre-screening of the GPCRs in melanophores was
performed in the absence of nicotinic acid following the protocol
below to determine the G protein coupling. This pre-screen
evidenced that hRUP25 (FIG. 14A), hRUP38 (FIG. 14B) and hRUP19
(FIG. 14C) are strongly Gi-coupled. hRUP11 is also strongly
Gi-coupled (not shown). Consistent with hRUP19 being Gi-coupled,
CART-activated hRUP19 inhibits cAMP production in membranes of
transfected 293 cells (FIG. 30).
[2475] The cells were plated in 96-well plates (one receptor per
plate). 48 hours post-transfection, half of the cells on each plate
were treated with 10 nM melatonin. Melatonin activates an
endogenous Gi-coupled receptor in the melanophores and causes them
to aggregate their pigment. The remaining half of the cells were
transferred to serum-free medium 0.7X L-15 (Gibco). After one hour,
the cells in serum-free media remained in a pigment-dispersed state
while the melatonin-treated cells were in a pigment-aggregated
state. At this point, the cells were treated with a dose response
of nicotinic acid (Sigma). If the plated GPCRs bound to nicotinic
acid, the melanophores would be expected to undergo a color change
in response to the compound. If the receptor were either a Gs or Gq
coupled receptor, then the melatonin-aggregated melanophores would
undergo pigment dispersion. In contrast, if the receptor was a
Gi-coupled receptor, then the pigment-dispersed cells would be
expected to undergo a dose-dependent pigment aggregation.
[2476] Melanophores transfected with hRUP25 were treated with
nicotinic acid. Upon this treatment, the cells underwent pigment
aggregation in a dose-dependent manner. hRUP25-expressing cells
that were pre-aggregated with melatonin did not disperse upon
nicotinic acid treatment, which is consistent with the receptor
being a Gi-coupled receptor. See, FIG. 15 and infra.
[2477] To confirm and extend these results, melanophores were
transfected with a range of hRUP25 DNA from 0 to 10% g. As
controls, melanophores were also transfected with 10 .mu.g of
.alpha..sub.2A Adrenergic receptor (a known Gi-coupled receptor)
and salmon sperm DNA (Gibco), as a mock transfection. On day 3, the
cells were again incubated for 1 hour in serum-free L-15 medium
(Gibco) and remained in a pigment-dispersed state. The cells were
then treated with a dose response of nicotinic acid. See, FIG. 15A.
FIG. 15A depicts the aggregation response of nicotinic acid at
melanophores transfected with various ranges of hRUP25. At 10 .mu.g
of hRUP25, the EC.sub.50 for nicotinic acid is about 54 nM. Stated
differently, at very low concentrations, nicotinic acid evidences
binding to hRUP25.
[2478] Reference is now made to FIG. 15B. In FIG. 15B, both the
mock transfected and .alpha..sub.2A transfected cells did not
respond to nicotinic acid. This data evidences that nicotinic acid
binds specifically to the Gi-coupled receptor hRUP25.
[2479] The data show that the greater the amount of hRUP25 plasmid
DNA transfected, the greater the magnitude of the observed
aggregation response. Collectively these data indicate that hRUP25:
1) is a Gi-coupled receptor that 2) binds to nicotinic acid.
[2480] As set forth herein, nicotinic acid is a ligand for, and
agonist of, human, mouse and rat RUP25. It is further shown that
hRUP38, hRUP11, hRUP19, and human, mouse and rat RUP25 are
Gi-coupled. Additionally, hRUP38, human and mouse RUP19, hRUP11,
and human, mouse, and rat RUP25 may be used in methods described
herein to identify antagonists, agonists, inverse agonists, partial
agonists, allosteric enhancers, and negative allosteric modulators.
As discussed supra, methods of modifying nicotinic acid receptor
activity in adipocytes using a modulator of the receptor are set
forth. Preferably, the modulator is an agonist.
Example 9
Nicotinic Acid Induced-Inositol Phosphates Accumulation in 293
Cells Co-Expressing hRUP AND Gq.DELTA.Gi
[2481] FIG. 16 illustrates the nicotinic acid induced-inositol
phosphates (IPs) accumulation in HEK293 cells co-expressing hRUP25
and the chimeric G.alpha.q-subunit in which the last five amino
acids have been replaced with the corresponding amino acids of
G.alpha.i (Gq.DELTA.Gi). This construct has been shown to convert
the signaling of a Gi-coupled receptor to the Gq pathway (i.e.
accumulation of inositol phosphates) in response to receptor
activation. Cells transfected with Gq.DELTA.Gi plus either empty
plasmid or the constitutively activated .alpha..sub.2AAR
(.alpha..sub.2AK) are non-responsive to nicotinic acid and served
as controls for the IP assay. Cells transfected with Gq.DELTA.Gi
plus either hRUP19 or hRUP38 are also unresponsive to nicotinic
acid, indicating that nicotinic acid is not an agonist for either
hRUP19 or hRUP38.
Example 10
Saturation Binding of [.sup.3H] Nicotinic Acid to Membranes from
Cells Expressing Either hRUP25, hRUP38, hRUP190R Vector Alone
[2482] FIG. 17 shows the results from saturation binding of [3H]
nicotinic acid to membranes from cells expressing either hRUP25,
hRUP38, hRUP19 or vector alone [CHO(-)]. Only hRUP25 was found to
bind nicotinic acid in a specific and high-affinity manner.
Example 11
Nicotinic Acid and (-)-Nicotine Induced-Inhibition of Forskolin
Stimulated cAMP Accumulation in hRUP25-CHO Cell Stable Line #46
[2483] FIG. 18A is a set of immunofluorescent photomicrographs
illustrating the expression of hemaglutinin(HA)-tagged hRUP25 in a
stably transfected line of CHO cells (top; clone #46). No
significant labeling is detected in mock stably-transfected CHO
cells (Mock). The lower panels identify the nuclear (DAPI) staining
of cells in the same field.
[2484] FIG. 18B illustrates nicotinic acid and (-)-nicotine
induced-inhibition of forskolin stimulated cAMP accumulation in
hRUP25-CHO cell stable line #46 (described in preceding paragraph).
The EC.sub.50 for nicotinic acid is 23.6 nM and that for
(-)-nicotine is 9.8 .mu.M.
Example 12
hRUP25 AND mRUP25 Inhibit TSHR Induced-Camp Accumulation Following
Activation by Nicotinic Acid
[2485] FIG. 19 indicates that, in response to nicotinic acid, both
hRUP25 and the mouse ortholog mRUP25 can inhibit TSHR stimulated
cAMP production (in the presence and absence of TSH).
Example 13
hRUP25 AND mRUP25 Bind to Nicotinic Acid Specifically and with High
Affinity
[2486] FIG. 20 shows the saturation binding curves of
[.sup.3H]nicotinic acid ([.sup.3H]NA) to membranes prepared from
HEK293 cells transiently expressing either hRUP25 or mRUP25. Note
the significant binding of [.sup.3H]NA relative to either that
found in membranes derived from mock transfected cells or in the
presence of an excess of non-labeled nicotinic acid (200
.mu.M).
[2487] Radioligand binding was done as follows. Media was removed
from cells grown in culture [either stably or transiently
transfected with negative control (empty plasmid) or with the
individual receptors hRUP25, mRUP25, rRUP25, hRUP38, hRUP11 or
hRUP19] and cells were scraped and homogenized in buffer containing
15 mM HEPES, 5 mM EDTA, 5 mM EGTA, plus protease inhibitors
(leupeptin, PMSF and pepstatin). Membranes were harvested following
centrifugation at 30,000.times.g, 4.degree. C. for 30 min.
Membranes were then resuspended and re-homogenized in CHAPS binding
buffer (50 mM Tris-HCl and 0.02% CHAPS, pH 7.4). Aliquots were
taken for protein analysis via the Bradford protein assay and
normalized such that each binding reaction contained the same
amount of membrane protein (25-50% g). 50 nM [.sup.3H]nicotinic
acid was added to each sample and either buffer (for total samples)
or a desired amount of non-labeled compound (at the same volumes
and in the same diluent) was added and the reactions were left at
room temperature gently shaking for 1 hr. Free ligand was separated
from bound ligand via rapid filtration onto a filter. Appropriate
scintilant was added to each sample and counted in an appropriate
scintillation counter. Data was analyzed using Excel and
PrismGraph. In some cases radioligand binding was performed using a
scintillation proximity assay (SPA) in which case the samples did
not require filtration or the addition of scintilant.
Example 14
The Rank Order of Potency of Compounds on HRUP25 Closely Matches
that of the Pharmacologically Defined Nicotinic Acid Receptor
[2488] FIG. 21 is a table comparing the rank order of potency of
various compounds on hRUP25 and the pharmacologically defined
nicotinic acid receptor. The potencies at hRUP25 derived both by a
functional analysis measuring the inhibition of forskolin induced
cAMP production and competitive radioligand binding assays, closely
match the order of potencies of the pharmacologically defined
nicotinic acid receptor.
Example 15
Nicotinic Acid and Related Compounds Inhibit Isoproterenol Induced
Lipolysis in Rat Epididymal Fat Derived Adipocytes
[2489] FIG. 22A depicts nicotinic acid and related compounds
inhibiting isoproterenol induced lipolysis in rat epididymal fat
derived adipocytes at a concentration of 10 .mu.M. P-3-T represents
3-tetrazole-5-pyridine.
[2490] FIG. 22B illustrates a nicotinic acid dose-dependent
inhibition of isoproterenol induced-lipolysis in rat epididymal fat
derived adipocytes. Note the rightward shift in the dose-response
curves with increasing concentrations of nicotinic acid.
[2491] Lipolysis assays were done following the isolation of
adipocytes from rat or human. The source of fat from rats was the
epididymal fat and from humans was either subcutaneous or omental.
Cells were isolated following collagenase digestion and floatation.
An elevation of intracellular cAMP levels and concomitant
activation of lipolysis via hormone sensitive lipase was
accomplished using isoproterenol, forskolin,
3-isobutyl-1-methyl-xanthine (IBMX) or a combination thereof at
concentrations and times determined empirically and depending on
the source of tissue. Lipolysis was allowed to continue for the
desired time in the presence or absence of drug (e.g. nicotinic
acid, P-3-T, etc). Data was analyzed using Excel and
PrismGraph.
[2492] To show that a modulator of hRUP19 behaves similarly, an
analogous assay is set up using said modulator of hRUP19. Preferred
said modulator is an agonist.
[2493] To show that a modulator of hRUP38 behaves similarly, an
analogous assay is set up using said modulator of hRUP38, wherein
the rat is transgenic for hRUP38. Preferred said modulator is an
agonist.
[2494] To show that a modulator of hRUP11 behaves similarly, an
analogous assay is set up using said modulator of hRUP11, wherein
the rat is transgenic for hRUP11. Preferred said modulator is an
agonist.
Example 16
Dose-Dependent Inhibition of Isoproterenol Induced-Lipolysis in
Human, Subcutaneous-Derived, Primary Adipocytes Via Nicotinic Acid
and P-3-T
[2495] FIG. 23 illustrates the ability of both nicotinic acid and
the related compound P-3-T (3-tetrazole-5-pyridine) to inhibit
isoproterenol induced lipolysis in adipocyte primary cultures
derived from human subcutaneous fat in a dose-dependant manner. The
EC.sub.50 value for nicotinic acid and P-3-T were 716 nM and 218 nM
respectively. (Also see Example 15, supra.)
Example 17
Screening Data for Nicotinic Acid and
1-Isopropyl-1H-Benzotriazole-5-Carboxylic Acid in cAMP Assays
[2496] FIG. 24 presents screening data via adenylyl cyclase assay
for hRUP38. Note that nicotinic acid does not activate inhibition
of forskolin stimulated cAMP hRUP38-expressing CHO cells whereas
1-Isopropyl-1H-benzotriazole-5-carboxylic acid does.
1-Isopropyl-1H-benzotriazole-5-carboxylic acid has no effect on CHO
cells expressing either hRUP25 or hRUP19.
Example 18
Inhibition of Isoproterenol Stimulated Lipolysis 1N Human
Subcutaneous Adipocytes
[2497] Nicotinic acid (an agonist of hRUP25) and
1-Isopropyl-1H-benzotriazole-5-carboxylic acid (an agonist of
hRUP38; see Example 17, supra) were separately dose-dependently
applied to isoproterenol (100 nM) stimulated primary human
adipocytes. FIG. 25 illustrates the ability of
1-Isopropyl-1H-benzotriazole-5-carboxylic acid to inhibit
isoproterenol stimulated lipolysis in adipocyte primary cultures
derived from human subcutaneous fat in a dose-dependent manner
comparable to that of nicotinic acid.
[2498] To show that a modulator of hRUP19 behaves similarly, an
analogous assay is set up using said modulator of hRUP19. Preferred
said modulator is an agonist.
[2499] To show that a modulator of hRUP11 behaves similarly, an
analogous assay is set up using said modulator of hRUP11. Preferred
said modulator is an agonist.
Example 19
Inhibition of Forskolin Stimulated cAMP Accumulation in hRUP38-CHO
Stable Cell Line by 3-(Bromo-2-Ethoxy-Phenyl)-Acrylic Acid
[2500] FIG. 26 presents screening data via adenylyl cyclase assay
for hRUP38. Note that 3-(5-Bromo-2-ethoxy-phenyl)-acrylic acid
activates inhibition of forskolin stimulated cAMP in
hRUP38-expressing CHO cells but has no effect on CHO cells
expressing either hRUP25 or hRUP19. The EC.sub.50 for
3-(5-Bromo-2-ethoxy-phenyl)-acrylic acid is 1.17 .mu.M. (Also see
the legend to Example 11, supra, for details directed to stable CHO
transfectants.)
Example 20
RT-PCR Indicates that hRUP19 is Selectively Expressed 1N Human Fat
Cells
[2501] FIG. 27 presents an RT-PCR analysis of hRUP19 expression
using a panel of human tissues. The analysis indicates that hRUP19
is selectively expressed in fat cells. Low expression is also
evident in testis, placenta, kidney and spleen.
[2502] Oligonucleotides used for PCR had the following sequences:
5'-GGCCGTGGCTGATTTCCTCCTTAT-3' (SEQ. ID. NO.:152; forward primer)
and 5'-AACCGGGTCGCCTTCTTCATCC-3' (SEQ. ID. NO.:153; reverse
primer). Commercially available human multiple tissue cDNA panels
were used as templates [Clontech, MTC panels Human I (#K1420-1) and
Human II (K-1421-1), and human fat cell cDNA (#7128-1)]. 1 ng cDNA
was used per PCR amplification. PCR was performed using Platinum
PCR SuperMix (Life Technologies, Inc.); according to manufacturer
instructions. The following cycles were used: 95.degree. C. for 4
min; 95.degree. C. for 1 min; 60.5.degree. C. for 30 sec,
72.degree. C. for 1 min, and 72.degree. C. for 7 min; cycles 2
through 4 were repeated 35 times. The resulting PCR reactions (15
.mu.l) were loaded on a 1.5% agarose gel to analyze the RT-PCR
products, and a specific 492 base-pair DNA fragment representing
hRUP19 was specifically amplified from cDNA of fat cell origin. Low
expression was also evident in testis, placenta, kidney, and
spleen.
Example 21
Northern Blot Analysis of hRUP19 Expression 1N Selected Tissues
[2503] FIG. 28 presents a Northern blot analysis of hRUP19
expression using a panel of human tissues. The analysis indicates
that hRUP19 is strongly expressed in mammary gland, probably due to
fat cell-specific expression of hRUP19. Ad, adrenal gland; Bl,
bladder, BM, bone marrow; Br, brain (whole); LN, lymph node; MG,
mammary gland; Pr, prostate; Sp, spinal cord; St, stomach; Thyr,
thyroid; Trch, trachea; Ut, uterus.
[2504] A pre-made blot containing Poly A+ RNA from 12 human tissues
was purchased from Clontech (Human 12-Lane II, Cat. # 7784-1).
hRUP19 cDNA probe encompassing the coding region was generated by
PCR using a plasmid containing hRUP19 cDNA as template. The blot
was prehybridized with 20 ml Clontech ExpressHyb solution (Cat. #
8015-2) for 30 minutes in a hybridization oven at 65.degree. C.,
according to the manufacturer's directions. 25 ng of a random
primer-labeled, hRUP19 cDNA probe was added to the prehybridization
solution, and the incubation was continued for an additional 2 hr.
The membrane was then washed according to the manufacturer's
directions (Cat. # 8015-2) and exposed to film for autoradiography
over a period of two days. Ad, adrenal gland; BI, bladder, BM, bone
marrow; Br, brain (whole); LN, lymph node; MG, mammary gland; Pr,
prostate; Sp, spinal cord; St, stomach; Thyr, thyroid; Trch,
trachea; Ut, uterus.
Example 22
RUP19 Expression is Induced During Adipocyte Differentiation:
Characterization of mRUP19 Expression in Mouse 3T3 Pre-Adipocytes
and Adipocytes
[2505] FIG. 29 presents an analysis of RUP19 mRNA expression as a
function of adipocyte differentiation. The analysis indicates that
RUP19 mRNA expression is induced during adipocyte
differentiation.
[2506] 3T3 pre-adipocytes were cultured in DMEM containing 10%
bovine calf serum. These cells were induced to differentiate into
an adipocyte phenotype using a standard protocol [Haraguchi K et al
(1996) Biochem Biophys Res Comm 223:193-198; the disclosure of
which is hereby incorporated by reference in its entirety].
Briefly, 1 day after confluence, cells were treated with DMEM
containing 10% FBS, 10 .mu.g/ml insulin, 0.2 .mu.g/ml
dexamethasone, and 0.5 mM isobutylmethylxanthine. After 3 days, the
cells were shifted to media supplemented with 10 .mu.g/ml insulin
and 10% FBS, and 2 days later, the cells were shifted to media
containing 10% FBS alone. After an additional 48 hrs, total RNA was
isolated from undifferentiated or differentiated cells using RNAzol
according to the manufacturer's directions. Separate populations of
undifferentiated and differentiated cells were subjected to
staining with Oil Red, to confirm the induction of an adipocyte
phenotype with this protocol.
[2507] Northern blot analysis. 10 ug of total RNA from 293 cells,
3T3 preadipocytes and 3T3 adipocytes was subjected to
electrophoresis on a 1% agarose/formaldehyde gel and transferred to
a nylon membrane using standard protocols. The blot was hybridized
to a 361 bp mRUP19 cDNA probe and exposed to film as described in
FIG. 28. The mRUP19 cDNA probe corresponds to nucleotides 5-365 of
SEQ. ID. NO.:150.
[2508] RT-PCR analysis. To detect mRUP19 mRNA by RT-PCR, the
following primers were used: 5'-ACTGTGGTGGCTGTGGATAGGTA-3' (SEQ.
ID. NO.:154; forward primer) and 5'-GCAGATTGTGAGCTTGGCGTAGAA-3'
(SEQ. ID. NO.:155; reverse primer). These are predicted to generate
an mRUP19 product of 567 bp. The cDNA templates were prepared using
a RETROscript.TM. First Strand Synthesis Kit for RT-PCR (Ambion,
Inc., Cat. # 1710), according to the manufacturer's directions,
except that duplicate reactions were done for each input RNA, and
in one of these, reverse transcriptase was excluded from the
reaction. 3 ul of each reaction was used for PCR. The reaction
conditions for the PCR were as follows: 1 cycle @ 94.degree. C./5
min., 25 cycles @ 94.degree. C./30 sec, 59.degree. C./30 sec,
72.degree. C./1 min, and 1 cycle @ 72.degree. C./5 min. The
reactions were then analyzed on a 1% agarose gel. Pre-diff 3T3-L1,
mouse 3T3 pre-adipocytes; Post-diff 3T3-L1, differentiated 3T3
adipocytes; .beta.-TC-6, a mouse insulin-producing cell line;
NIT-1, a mouse insulin-producing cell line.
Example 23
Cart-Activated hRUP19 Inhibits Camp Production in Membranes of
Transfected 293 Cells
[2509] FIG. 30 presents a CART analysis of signal transduction by
hRUP19. The analysis indicates that CART-activated hRUP19 inhibits
cAMP production in membranes of transfected 293 cells.
[2510] Membranes were prepared as follows. 15 ug of the following
expression plasmids were each introduced into 293 cells (one 15 cm
dish per transfection) using Lipofectamine Reagent (Invitrogen,
#18324-020) according to the manufacturer's instructions: pCMV-MCS
(empty CMV expression plasmid), pCMV-hRUP19, pCMV-hRUP19-CART (same
as pCMV-hRUP19, except that codon 219 has been converted from
threonine to lysine). After 48 hours, a crude membrane preparation
was prepared using standard protocols. Briefly, cells were washed
with ice cold PBS, removed from the plate by scraping in the
presence of a hypotonic Tris/EDTA buffer, fragmented using a
pre-chilled dounce homogenizer, spun at low speed to pellet nuclei
and intact cells, and finally, the supernatant is subjected to
centrifugation at 20,000 rpm in a Beckman Avanti J-20 centrifuge.
The membrane pellet is then resuspended at a protein concentration
of 1 mg/ml for use in a membrane cyclase assay. The membrane
cyclase assay was carried out as per the manufacturer's
recommendation using an Adenylyl Cyclase Activation FlashPlate
Assay Kit (Perkin Elmer Life Sciences, Inc., #SMP004B).
Example 24
Summary: hRUP25, mRUP25, rRUP25, hRUP38, hRUP19, mRUP19, rRUP19,
AND hRUP11
[2511] TABLE-US-00032 TABLE M Disclosed Gi-Coupled Nicotinic Acid
(Lowers the Receptor Expression by Level of Shown to Inhibit
Sub-Family Adipocytes or Intracellular Intracellular GPCRs Adipose
cAMP) Lipolysis Agonist hRUP25 yes yes yes nicotinic acid;
(-)-nicotine; see FIG. 21; (5-hydroxy-1- methyl-3-propyl-1H-
pyrazol-4-yl)-pyridin- 3-yl-methanone mRUP25 yes yes n.d. nicotinic
acid rRUP25 yes yes yes nicotinic acid hRUP38 yes yes yes
1-Isopropyl-1H- benzotriazole-5- carboxylic acid;
3-(5-Bromo-2-ethoxy- phenyl)-acrylic acid hRUP11 n.d. yes n.d. n.d.
hRUP19 yes yes n.d. n.d. mRUP19 yes n.d. n.d. n.d. rRUP19 n.d. n.d.
n.d. n.d. n.d.: not displayed
Example 25
Rodent Diabetes Models
[2512] Rodent models of type 2 diabetes associated with obesity and
insulin resistance have been developed. Genetic models such as
db/db and ob/ob [see Diabetes (1982) 31:1-6] in mice and fa/fa in
zucker rats have been developed for understanding the
pathophysiology of disease and for testing candidate therapeutic
compounds [Diabetes (1983) 32:830-838; Annu Rep Sankyo Res Lab
(1994) 46:1-57]. The homozygous animals, C57 BL/KsJ-db/db mice
developed by Jackson Laboratory are obese, hyperglycemic,
hyperinsulinemic and insulin resistant [J Clin Invest (1990)
85:962-967], whereas heterozygotes are lean and normoglycemic. In
the db/db model, mice progressively develop insulinopenia with age,
a feature commonly observed in late stages of human type 2 diabetes
when sugar levels are insufficiently controlled. Since this model
resembles that of human type 2 diabetes, the compounds of the
present invention are tested for activities including, but not
limited to, lowering of plasma glucose and triglycerides. Zucker
(fa/fa) rats are severely obese, hyperinsulinemic, and insulin
resistant {Coleman, Diabetes (1982) 31:1; E Shafrir in Diabetes
Mellitus, H Rifkin and D Porte, Jr, Eds [Elsevier Science
Publishing Co, New York, ed. 4, (1990), pp. 299-340]}, and the
fa/fa mutation may be the rat equivalent of the murine db mutation
[Friedman et al, Cell (1992) 69:217-220; Truett et al, Proc Natl
Acad Sci USA (1991) 88:7806]. Tubby (tub/tub) mice are
characterized by obesity, moderate insulin resistance and
hyperinsulinemia without significant hyperglycemia [Coleman et al,
Heredity (1990) 81:424].
[2513] The present invention encompasses the use of compounds of
the invention for reducing the insulin resistance and hyperglycemia
in any or all of the above rodent diabetes models, in humans with
type 2 diabetes or other preferred metabolic-related disorders or
disorders of lipid metabolism described previously, or in models
based on other mammals. Plasma glucose and insulin levels will be
tested, as well as other factors including, but not limited to,
plasma free fatty acids and triglycerides.
In Vivo Assay for Anti-Hyperglycemic Activity of Compounds of the
Invention
[2514] Genetically altered obese diabetic mice (db/db) (male, 7-9
weeks old) are housed (7-9 mice/cage) under standard laboratory
conditions at 22.degree. C. and 50% relative humidity, and
maintained on a diet of Purina rodent chow and water ad libitum.
Prior to treatment, blood is collected from the tail vein of each
animal and blood glucose concentrations are determined using One
Touch Basic Glucose Monitor System (Lifescan). Mice that have
plasma glucose levels between 250 to 500 mg/dl are used. Each
treatment group consists of seven mice that are distributed so that
the mean glucose levels are equivalent in each group at the start
of the study. db/db mice are dosed by micro-osmotic pumps, inserted
using isoflurane anesthesia, to provide compounds of the invention,
saline, or an irrelevant compound to the mice subcutaneously
(s.c.). Blood is sampled from the tail vein at intervals thereafter
and analyzed for blood glucose concentrations. Significant
differences between groups (comparing compounds of the invention to
saline-treated) are evaluated using Student t-test.
[2515] The foregoing is provided by way of illustration and not
limitation. Other illustrative rodent models for type 2 diabetes
have been described [Moller D E, Nature (2001) 414:821-7 and
references therein; and Reed M J et al., Diabetes, Obesity and
Metabolism (1999) 1:75-86 and reference therein; the disclosure of
each of which is hereby incorporated by reference in its
entirety].
Example 26
Mouse Atherosclerosis Model
[2516] Adiponectin-deficient mice generated through knocking out
the adiponectin gene have been shown to be predisposed to
atherosclerosis and to be insulin resistant. The mice are also a
suitable model for ischemic heart disease [Matsuda, M et al. J Biol
Chem (2002) July, and references cited therein, the disclosures of
which are incorporated herein by reference in their entirety].
[2517] Adiponectin knockout mice are housed (7-9 mice/cage) under
standard laboratory conditions at 22.degree. C. and 50% relative
humidity. The mice are dosed by micro-osmotic pumps, inserted using
isoflurane anesthesia, to provide compounds of the invention,
saline, or an irrelevant compound to the mice subcutaneously
(s.c.). Neointimal thickening and ischemic heart disease are
determined for different groups of mice sacrificed at different
time intervals. Significant differences between groups (comparing
compounds of the invention to saline-treated) are evaluated using
Student t-test.
[2518] The foregoing mouse model of atherosclerosis is provided by
way of illustration and not limitation. By way of further example,
Apolipoprotein E-deficient mice have also been shown to be
predisposed to atherosclerosis [Plump A S et al., Cell (1992)
71:343-353; the disclosure of which is hereby incorporated by
reference in its entirety].
[2519] A preferred model is that of diet-induced atherosclerosis in
C57BL/6J mice, an inbred strain known to be susceptible to
diet-induced atherosclerotic lesion formation. This model is well
known to those persons of ordinary skill in the art [Kamada N et
al., J Atheroscler Thromb (2001) 8:1-6; Garber D W et al., J Lipid
Res (2001) 42:545-52; Smith J D et al., J Intern Med (1997)
242:99-109; the disclosure of each of which is hereby incorporated
by reference in its entirety].
Example 27
Transgenic Mouse/Rat
hRUP38
[2520] The present invention also provides methods and compositions
for the generation of mice and rats that express hRUP38 recombinant
human antilipolytic GPCR polyeptide of the invention.
[2521] Methods of making transgenic animals such as mice and rats
are well known to those of ordinary skill in the art, and any such
method can be used in the present invention. Briefly, transgenic
mammals can be produced, e.g., by transfecting a pluripotential
stem cell such as an ES cell with a polynucleotide encoding hRUP38
polypeptide of the invention. Successfully transformed ES cells can
then be introduced into an early stage embryo that is then
implanted into the uterus of a mammal of the same species. In
certain cases, the transformed ("transgenic") cells will comprise
part of the germ line of the resulting animal and adult animals
comprising the transgenic cells in the germ line can then be mated
to other animals, thereby eventually producing a population of
transgenic animals that have the transgene in each of their cells
and that can stably transmit the transgene to each of their
offspring. Other methods of introducing the polynucleotide can be
used, for example introducing the polynucleotide encoding hRUP38
polypeptide of the invention into a fertilized egg or early stage
embryo via microinjection. Alternatively, the transgene may be
introduced into an animal by infection of zygotes with a retrovirus
containing the transgene [Jaenisch, R, Proc Natl Acad Sci USA
(1976) 73:1260-4]. Methods of making transgenic mammals are
described, e.g., in Wall et al., J Cell Biochem (1992) 49:113-20;
Hogan et al., in Manipulating the Mouse Embryo. A Laboratory
Manual. (1986) Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.; in WO 91/08216; or in U.S. Pat. No. 4,736,866; all of
which disclosures are hereby incorporated by reference in their
entirety.
[2522] In preferred embodiments, expression of said gene is placed
under the control of an essentially adipocyte specific promoter. In
further preferred embodiments, said essentially adipocyte specific
promoter is adiponectin gene promoter [Das, K et al., Biochem
Biophys Res Commun (2001) 280:1120-9; Barth, N et al., Diabetologia
(2002) 45:1425-1433; the disclosures of which are hereby
incorporated by reference in its entirety]. In other further
preferred embodiments, said essentially adipocyte specific promoter
is resistin gene promoter [Hartman, H B et al. J Biol Chem (2002)
277:19754-61, which disclosure is hereby incorporated by reference
in its entirety]. In other preferred embodiments, said essentially
adipocyte specific promoter is aP2 [Felmer, R et al., J Endocrinol
(2002) 175:487-498; the disclosure of which is hereby incorporated
by reference in its entirety]. In other further preferred
embodiments, expression of said gene is kept under the control of
its endogenous promoter.
hRUP11
[2523] The present invention also provides methods and compositions
for the generation of mice and rats that express hRUP11 recombinant
human antilipolytic GPCR polyeptide of the invention.
[2524] Methods of making transgenic animals such as mice and rats
are well known to those of ordinary skill in the art, and any such
method can be used in the present invention. Briefly, transgenic
mammals can be produced, e.g., by transfecting a pluripotential
stem cell such as an ES cell with a polynucleotide encoding hRUP11
polypeptide of the invention. Successfully transformed ES cells can
then be introduced into an early stage embryo that is then
implanted into the uterus of a mammal of the same species. In
certain cases, the transformed ("transgenic") cells will comprise
part of the germ line of the resulting animal and adult animals
comprising the transgenic cells in the germ line can then be mated
to other animals, thereby eventually producing a population of
transgenic animals that have the transgene in each of their cells
and that can stably transmit the transgene to each of their
offspring. Other methods of introducing the polynucleotide can be
used, for example introducing the polynucleotide encoding hRUP11
polypeptide of the invention into a fertilized egg or early stage
embryo via microinjection. Alternatively, the transgene may be
introduced into an animal by infection of zygotes with a retrovirus
containing the transgene [Jaenisch, R, Proc Natl Acad Sci USA
(1976) 73:1260-4]. Methods of making transgenic mammals are
described, e.g., in Wall et al., J Cell Biochem (1992) 49:113-20;
Hogan et al., in Manipulating the Mouse Embryo. A Laboratory
Manual. (1986) Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.; in WO 91/08216; or in U.S. Pat. No. 4,736,866; all of
which disclosures are hereby incorporated by reference in their
entirety.
[2525] In preferred embodiments, expression of said gene is placed
under the control of an adipocyte specific promoter. In further
preferred embodiments, said adipocyte specific promoter is
adiponectin gene promoter [Das, K et al., Biochem Biophys Res
Commun (2001) 280:1120-9; Barth, N et al., Diabetologia (2002)
45:1425-1433; the disclosures of which are hereby incorporated by
reference in its entirety]. In other further preferred embodiments,
said adipocyte specific promoter is resistin gene promoter
[Hartman, H B et al. J Biol Chem (2002) 277:19754-61, which
disclosure is hereby incorporated by reference in its entirety]. In
other preferred embodiments, said adipocyte specific promoter is
aP2 [Felmer, R et al., J Endocrinol (2002) 175:487-498; the
disclosure of which is hereby incorporated by reference in its
entirety]. In other further preferred embodiments, expression of
said gene is kept under the control of its endogenous promoter.
Example 28
Knockout Mouse/Rat
[2526] Mouse
RUP25
[2527] A preferred DNA construct will comprise, from 5'-end to
3'-end: (a) a first nucleotide sequence that is comprised in the
mRUP25 genomic sequence; (b) a nucleotide sequence comprising a
positive selection marker, such as the marker for neomycin
resistance (neo); and (c) a second nucleotide sequence that is
comprised in the mRUP25 genomic sequence and is located on the
genome downstream of the first mRUP25 nucleotide sequence (a).
mRUP25 genomic sequence will be isolated using methods well known
to those of ordinary skill in the art (Maniatis T et al., Molecular
Cloning: A Laboratory Manual (1989) Cold Spring Harbor Laboratory;
the disclosure of which is hereby incorporated by reference in its
entirety).
[2528] In preferred embodiments, this DNA construct also comprises
a negative selection marker located upstream the nucleotide
sequence (a) or downstream the nucleotide sequence (c). Preferably,
the negative selection marker comprises the thymidine kinase (tk)
gene [Thomas et al., Cell (1986) 44:419-28], the hygromycin beta
gene [Te Riele et al., Nature (1990) 348:649-51], the hprt gene
[Van der Lugt et al., Gene (1991) 105:263-7; Reid et al., Proc Natl
Acad Sci USA (1990) 87:4299-4303] or the Diptheria toxin A fragment
(Dt-A) gene [Nada et al., Cell (1993) 73:1125-35; Yagi et al., Proc
Natl Acad Sci USA (1990) 87:9918-9922], which disclosures are
hereby incorporated by reference in their entireties. Preferably,
the positive selection marker is located within an mRUP25 exon
sequence so as to interrupt the sequence encoding an mRUP25
polypeptide. These replacement vectors are described, for example,
by Thomas et al., Cell (1986) 44:419-28; Thomas et al., Cell (1987)
51:503-12; Mansour et al., Nature (1988) 336:348-52; Koller et al.,
Annu Rev Immunol (1992) 10:705-30; and U.S. Pat. No. 5,631,153;
which disclosures are hereby incorporated by reference in their
entireties.
[2529] The first and second nucleotide sequences (a) and (c) may be
indifferently located within an mRUP25 regulatory sequence, an
intronic sequence, an exon sequence or a sequence containing both
regulatory and/or intronic and/or exon sequences. The size of the
nucleotide sequences (a) and (c) ranges from 1 to 50 kb, preferably
from 1 to 10 kb, more preferably from 2 to 6 kb, and most
preferably from 2 to 4 kb.
[2530] Methods of making a knockout mouse are well known to those
of ordinary skill in the art and have been used to successfully
inactivate a wide range of genes.
RUP19
[2531] A preferred DNA construct will comprise, from 5'-end to
3'-end: (a) a first nucleotide sequence that is comprised in the
mRUP19 genomic sequence; (b) a nucleotide sequence comprising a
positive selection marker, such as the marker for neomycin
resistance (neo); and (c) a second nucleotide sequence that is
comprised in the mRUP19 genomic sequence and is located on the
genome downstream of the first mRUP19 nucleotide sequence (a).
mRUP19 genomic sequence will be isolated using methods well known
to those of ordinary skill in the art (Maniatis T et al., Molecular
Cloning: A Laboratory Manual (1989) Cold Spring Harbor Laboratory;
the disclosure of which is hereby incorporated by reference in its
entirety).
[2532] In preferred embodiments, this DNA construct also comprises
a negative selection marker located upstream the nucleotide
sequence (a) or downstream the nucleotide sequence (c). Preferably,
the negative selection marker comprises the thymidine kinase (tk)
gene [Thomas et al., Cell (1986) 44:419-28], the hygromycin beta
gene [Te Riele et al., Nature (1990) 348:649-51], the hprt gene
[Van der Lugt et al., Gene (1991) 105:263-7; Reid et al., Proc Natl
Acad Sci USA (1990) 87:42994303] or the Diptheria toxin A fragment
(Dt-A) gene [Nada et al., Cell (1993) 73:1125-35; Yagi et al., Proc
Natl Acad Sci USA (1990) 87:9918-9922], which disclosures are
hereby incorporated by reference in their entireties. Preferably,
the positive selection marker is located within an mRUP19 exon
sequence so as to interrupt the sequence encoding an mRUP19
polypeptide. These replacement vectors are described, for example,
by Thomas et al., Cell (1986) 44:419-28; Thomas et al., Cell (1987)
51:503-12; Mansour et al., Nature (1988) 336:348-52; Koller et al.,
Annu Rev Immunol (1992) 10:705-30; and U.S. Pat. No. 5,631,153;
which disclosures are hereby incorporated by reference in their
entireties.
[2533] The first and second nucleotide sequences (a) and (c) may be
indifferently located within an mRUP19 regulatory sequence, an
intronic sequence, an exon sequence or a sequence containing both
regulatory and/or intronic and/or exon sequences. The size of the
nucleotide sequences (a) and (c) ranges from 1 to 50 kb, preferably
from 1 to 10 kb, more preferably from 2 to 6 kb, and most
preferably from 2 to 4 kb.
[2534] Methods of making a knockout mouse are well known to those
of ordinary skill in the art and have been used to successfully
inactivate a wide range of genes.
Rat
RUP25
[2535] Gene targeting technology for the rat is less robust than
that for the mouse and is an area of active interest. One approach
will be to inactivate rRUP25 gene in rat embryonic stem cell
(ESC)-like cells and then inject cells with inactivated rRUP25 gene
into rat blastocysts generated after fusion of two-cell embryos
[Krivokharchenko et al., Mol Reprod Dev (2002) 61:460-5].
[2536] An alternative approach will be to inactivate rRUP25 gene in
rat ESC-like cells and then transfer the nucleus of the rat
ESC-like cells having inactivated rRUP25 gene into enucleated
oocytes [Sato K et al., Hum Cell (2001) 14:301-4; Wakayama and
Yanagimachi, Semin Cell Dev Biol (1999) 10:253-8; Hochedlinger and
Jaenisch, Nature (2002) 415:1035-8; Yanagimachi, Mol Cell
Endocrinol (2002) 187:241-8; the disclosures of which are
incorporated herein by reference in their entireties].
[2537] rRUP25 genomic sequence can be isolated using methods well
known to those of ordinary skill in the art (Maniatis T et al.,
Molecular Cloning: A Laboratory Manual (1989) Cold Spring Harbor
Laboratory; the disclosure of which is hereby incorporated by
reference in its entirety).
RUP19
[2538] Gene targeting technology for the rat is less robust than
that for the mouse and is an area of active interest. One approach
will be to inactivate rRUP19 gene in rat embryonic stem cell
(ESC)-like cells and then inject cells with inactivated rRUP19 gene
into rat blastocysts generated after fusion of two-cell embryos
[Krivokharchenko et al., Mol Reprod Dev (2002) 61:460-5].
[2539] An alternative approach will be to inactivate rRUP19 gene in
rat ESC-like cells and then transfer the nucleus of the rat
ESC-like cells having inactivated rRUP25 gene into enucleated
oocytes [Sato K et al., Hum Cell (2001) 14:3014; Wakayama and
Yanagimachi, Semin Cell Dev Biol (1999) 10:253-8; Hochedlinger and
Jaenisch, Nature (2002) 415:1035-8; Yanagimachi, Mol Cell
Endocrinol (2002) 187:241-8; the disclosures of which are
incorporated herein by reference in their entireties].
[2540] rRUP19 genomic sequence can be isolated using methods well
known to those of ordinary skill in the art (Maniatis T et al.,
Molecular Cloning: A Laboratory Manual (1989) Cold Spring Harbor
Laboratory; the disclosure of which is hereby incorporated by
reference in its entirety).
CRE-LOXP System
[2541] These new DNA constructs make use of the site specific
recombination system of the P1 phage. The P1 phage possesses a
recombinase called Cre that interacts with a 34 base pair loxP
site. The loxP site is composed of two palindormic sequences of 13
bp separated by an 8 bp conserved sequence [Hoess R H et al,
Nucleic Acids Res (1986) 14:2287-300; which disclosure is hereby
incorporated by reference in its entirety]. The recombination by
the Cre enzyme between two loxP sites having an identical
orientation leads to the deletion of the DNA fragment.
[2542] The Cre-loxP system used in combination with a homologous
recombination technique has been first described by Gu et al. [Gu H
et al., Cell (1993) 73:1155-64; Gu H et al., Science (1994)
265:103-6; which disclosures are hereby incorporated by reference
in their entirety]. Briefly, a nucleotide sequence of interest to
be inserted in a targeted location of the genome harbors at least
two loxP sites in the same orientation and located at the
respective ends of a nucleotide sequence to be excised from the
recombinant genome. The excision event requires the presence of the
recombinase (Cre) enzyme within the nucleus of the recombinant cell
host. The recombinase enzyme may be brought at the desired time
either by (a) incubating the recombinant cell hosts in a culture
medium containing this enzyme, by injecting the Cre enzyme directly
into the desired cell, such as by lipofection of the enzyme into
the cells, such as described by Baubonis et al. [Baubonis W and
Sauer B, Nucleic Acids Res (1993) 21:2025-9; which disclosure is
hereby incorporated by reference in its entirety]; (b) transfecting
the cell host with a vector comprising the Cre Coding sequence
operably linked to a promoter functional in the recombinant cell
host, which promoter being optionally inducible, said vector being
introduced in the recombinant cell host, such as described by Gu et
al. [Gu H et al., Cell (1993) 73:1155-64; which disclosure is
hereby incorporated by reference in its entirety] and Sauer et al.
[Sauer B and Henderson N, Proc Natl Acad Sci USA (1988) 85:5166-70;
which disclosure is hereby incorporated by reference in its
entirety]; (c) introducing into the genome of the cell host a
polynucleotide comprising the Cre coding sequence operably linked
to a promoter functional in the recombinant cell host, which
promoter is optionally inducible, and said polynucleotide being
inserted in the genome of the cell host either by a random
insertion event or an homologous recombination event, such as
described by Gu et al. [Gu H et al., Science (1994) 265:103-6;
which disclosure is hereby incorporated by reference in its
entirety].
[2543] Vectors and methods using the Cre-loxP system are describe
by Zou et al. (1994), which disclosure is hereby incorporated by
reference in its entirety.
[2544] In preferred embodiments of the invention, Cre is introduced
into the genome of the cell host by strategy (c) above, wherein
said promoter is essentially adipocyte specific and leads to
essentially adipocyte specific knockout of (loxP-flanked) mRUP25 or
mRUP19 in the mouse or rRUP25 or rRUP19 in the rat. In some
embodiments, said essentially adipocyte specific promoter is
adiponectin gene promoter [Das, K et al., Biochem Biophys Res
Commun (2001) 280:1120-9; Barth, N et al., Diabetologia (2002)
45:1425-1433; the disclosures of which are hereby incorporated by
reference in its entirety]. In some embodiments, said essentially
adipocyte specific promoter is resistin gene promoter [Hartman, H B
et al. J Biol Chem (2002) 277:19754-61, which disclosure is hereby
incorporated by reference in its entirety]. In some embodiments,
said essentially adipocyte specific promoter is aP2 [Felmer, R et
al., J Endocrinol (2002) 175:487-498; the disclosure of which is
hereby incorporated by reference in its entirety]. Methods of
constructing a lineage-specific knockout are well known to persons
of ordinary skill in the art, as illustrated but not intended to be
limited by: Kuhn R and Torres R M, Methods Mol Biol (2002)
180:175-204; Sauer B, Methods (1998) 14:381-92; Gutstein D E et
al., Circulation Research (2001) 88:333; Minamino T et al.,
Circulation Research (2001) 88:587; and Bex A et al., J Urol (2002)
168:2641-2644; the disclosure of each of which is hereby
incorporated by reference in its entirety.
Example 29
Fluorometric Imaging Plate Reader (FLIPR) Assay for the Measurement
of Intracellular Calcium Concentration
[2545] Target Receptor (experimental) and pCMV (negative control)
stably transfected cells from respective clonal lines are seeded
into poly-D-lysine pretreated 96-well plates (Becton-Dickinson,
#356640) at 5.5.times.10.sup.4 cells/well with complete culture
medium (DMEM with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate)
for assay the next day. To prepare Fluo-4-AM (Molecular Probe,
#F14202) incubation buffer stock, 1 mg Fluo4-AM is dissolved in 467
.mu.l DMSO and 467 .mu.l Pluoronic acid (Molecular Probe, #P3000)
to give a 1 mM stock solution that can be stored at -20.degree. C.
for a month. Fluo4-AM is a fluorescent calcium indicator dye.
[2546] Candidate compounds are prepared in wash buffer
(1.times.HBSS/2.5 mM Probenicid/20 mM HEPES at pH 7.4).
[2547] At the time of assay, culture medium is removed from the
wells and the cells are loaded with 100 .mu.l of 4 .mu.M
Fluo4-AM/2.5 mM Probenicid (Sigma, #P8761)/20 mM HEPES/complete
medium at pH 7.4. Incubation at 37.degree. C./5% CO.sub.2 is
allowed to proceed for 60 min.
[2548] After the 1 hr incubation, the Fluo4-AM incubation buffer is
removed and the cells are washed 2.times. with 100 .mu.L wash
buffer. In each well is left 100 .mu.l wash buffer. The plate is
returned to the incubator at 37.degree. C./5% CO.sub.2 for 60
min.
[2549] FLIPR (Fluorometric Imaging Plate Reader; Molecular Device)
is programmed to add 50 .mu.l candidate compound on the 30.sup.th
second and to record transient changes in intracellular calcium
concentration ([Ca.sup.2+]) evoked by the candidate compound for
another 150 seconds. Total fluorescence change counts are used to
determine agonist activity using the FLIPR software. The instrument
software normalizes the fluorescent reading to give equivalent
initial readings at zero.
[2550] In some embodiments, the cells comprising Target Receptor
further comprise promiscuous G alpha 15/16 or the chimeric Gq/Gi
alpha unit.
[2551] Although the foregoing provides a FLIPR assay for agonist
activity using stably transfected cells, a person of ordinary skill
in the art would readily be able to modify the assay in order to
characterize antagonist activity. Said person of ordinary skill in
the art would also readily appreciate that, alternatively,
transiently transfected cells could be used.
Example 30
IN VIVO Pig Model of HDL-Cholesterol and Atherosclerosis
[2552] The utility of a modulator of the present invention as a
medical agent in the prevention or treatment of a high total
cholesterol/HDL-cholesterol ratio and conditions relating thereto
is demonstrated, without limitation, by the activity of the
modulator in lowering the ratio of total cholesterol to
HDL-cholesterol, in elevating HDL-cholesterol, or in protection
from atherosclerosis in an in vivo pig model. Pigs are used as an
animal model because they reflect human physiology, especially
lipid metabolism, more closely than most other animal models. An
illustrative in vivo pig model not intended to be limiting is
presented here.
[2553] Yorkshire albino pigs (body weight 25.5.+-.4 kg) are fed a
saturated fatty acid rich and cholesterol rich (SFA-CHO) diet
during 50 days (1 kg chow 35 kg.sup.-1 pig weight), composed of
standard chow supplemented with 2% cholesterol and 20% beef tallow
[Royo T et al., European Journal of Clinical Investigation (2000)
30:843-52; which disclosure is hereby incorporated by reference in
its entirety]. Saturated to unsaturated fatty acid ratio is
modified from 0.6 in normal pig chow to 1.12 in the SFA-CHO diet.
Animals are divided into two groups, one group (n=8) fed with the
SFA-CHO diet and treated with placebo and one group (n=8) fed with
the SFA-CHO diet and treated with the modulator (3.0 mg kg.sup.-1).
Control animals are fed a standard chow for a period of 50 days.
Blood samples are collected at baseline (2 days after the reception
of the animals), and 50 days after the initiation of the diet.
Blood lipids are analyzed. The animals are sacrificed and
necropsied.
[2554] Alternatively, the foregoing analysis comprises a plurality
of groups each treated with a different dose of the modulator.
Preferred said doses are selected from the group consisting of: 0.1
mg kg.sup.-1, 0.3 mg kg.sup.-1, 1.0 mg kg.sup.-1, 3.0 mg kg.sup.-1,
10 mg kg.sup.-1, 30 mg kg.sup.-1 and 100 mg kg.sup.-1.
Alternatively, the foregoing analysis is carried out at a plurality
of timepoints. Preferred said timepoints are selected from the
group consisting of 10 weeks, 20 weeks, 30 weeks, 40 weeks, and 50
weeks.
HDL-Cholesterol
[2555] Blood is collected in trisodium citrate (3.8%, 1:10). Plasma
is obtained after centrifugation (1200 g 15 min) and immediately
processed. Total cholesterol, HDL-cholesterol, and LDL-cholesterol
are measured using the automatic analyzer Kodak Ektachem DT System
(Eastman Kodak Company, Rochester, N.Y., USA). Samples with value
parameters above the range are diluted with the solution supplied
by the manufacturer and then re-analyzed. The total
cholesterol/HDL-cholesterol ratio is determined. Comparison is made
of the level of HDL-cholesterol between groups. Comparison is made
of the total cholesterol/HDL-cholesterol ratio between groups.
[2556] Elevation of HDL-cholesterol or reduction of the total
cholesterol/HDL-cholesterol ratio on administration of the
modulator is taken as indicative of the modulator having the
aforesaid utility.
Atherosclerosis
[2557] The thoracic and abdominal aortas are removed intact, opened
longitudinally along the ventral surface, and fixed in
neutral-buffered formalin after excision of samples from standard
sites in the thoracic and abdominal aorta for histological
examination and lipid composition and synthesis studies. After
fixation, the whole aortas are stained with Sudan IV and pinned out
flat, and digital images are obtained with a TV camera connected to
a computerized image analysis system (Image Pro Plus; Media
Cybernetics, Silver Spring, Md.) to determine the percentage of
aortic surface involved with atherosclerotic lesions [Gerrity R G
et al, Diabetes (2001) 50:1654-65; Cornhill J F et al,
Arteriosclerosis, Thrombosis, and Vascular Biology (1985) 5:415-26;
which disclosures are hereby incorporated by reference in their
entirety]. Comparison is made between groups of the percentage of
aortic surface involved with atherosclerotic lesions.
[2558] Reduction of the percentage of aortic surface involved with
atherosclerotic lesions on administration of the modulator is taken
as indicative of the modulator having the aforesaid utility.
Plasma Free Fatty Acids
[2559] It would be readily apparent to anyone of ordinary skill in
the art that the foregoing in vivo pig model is easily modified in
order to address, without limitation, the activity of the modulator
in lowering plasma free fatty acids.
Example 31
Measurement of Plasma Free Fatty Acids (FFA) in Rats Administered
Niacin
[2560] Catheters were surgically implanted into the jugular veins
of male Sprague Dawley rats. The following day rats were deprived
of food and approximately 16 hours later were given interperitoneal
(IP) injections of either vehicle, or niacin [NA] at 15 mg/kg, 30
mg/kg or 45 mg/kg body weight. Blood was drawn (.about.200 ml) at
the indicated time points and plasma was isolated following
centrifugation. Plasma FFA were then measured via the NEFA C kit
according to manufacturer specifications (Wako Chemicals USA, Inc).
All three concentrations of niacin significantly decrease plasma
FFA levels. [See, FIG. 32.] * Indicates a statistical increase in
plasma FFA versus vehicle control indicating a rebound effect in
the 45 mg/kg niacin treated rats at 3 hr (i.e., plasma FFA levels
go beyond basal levels following inhibition).
[2561] By way of illustration and not limitation, said rat model
has utility as an in vivo animal model for modulators of RUP25
provided by the invention. By way of illustration and not
limitation, said rat model also has utility as an in vivo animal
model for modulators of RUP19 provided by the invention. By way of
illustration and not limitation, hRUP38 and hRUP11 transgenic rats
provided by the invention have utility as in vivo animal models for
modulators of hRUP38 and hRUP11 provided by the invention.
[2562] To show that a modulator of hRUP25 other than niacin behaves
similarly, an analogous assay is set up using said modulator of
hRUP25. Preferred said modulator is an agonist.
[2563] To show that a modulator of hRUP38 behaves similarly, an
analogous assay is set up using said modulator of hRUP38, wherein
the rat is transgenic for hRUP38. Preferred said modulator is an
agonist.
[2564] To show that a modulator of hRUP19 behaves similarly, an
analogous assay is set up using said modulator of hRUP19. Preferred
said modulator is an agonist.
[2565] To show that a modulator of hRUP11 behaves similarly, an
analogous assay is set up using said modulator of hRUP11, wherein
the rat is transgenic for hRUP11. Preferred said modulator is an
agonist.
Example 32
Preparation of Non-Endogenous, Enhanced-for-Agonist [EFA-]
GPCRS
[2566] Constitutive activity of a GPCR reduces the available window
for identification of an agonist of the GPCR, where said window is
taken here to be the difference in assay readout between the GPCR
in the absence of agonist and the GPCR in the presence of a known
agonist. A mutant of a constitutively active GPCR that is less
constitutively active but comparably or more responsive to said
known agonist [Enhanced-for-Agonist GPCR; EFA-GPCR] would have
novel utility for the identification of modulators of said GPCR,
particularly agonists.
[2567] EFA-GPCR is disclosed herewith as a mutant GPCR polypeptide
that consists of 1, 2, 3, 4, or 5 amino acid substitutions,
deletions, or insertions relative to the amino acid sequence of an
endogenous GPCR polypeptide having constitutive activity, wherein
the agonist screening window of the mutant GPCR is expanded by
greater than 20%, greater than 25%, greater than 30%, greater than
31%, greater than 32%, greater than 33%, greater than 34%, greater
than 35%, greater than 36%, greater than 37%, greater than 38%,
greater than 39%, or greater than 40% relative to that of said
endogenous GPCR.
[2568] As the GPCRs of the invention are constitutively active to a
significant degree, an EFA version of said GPCRs would have novel
utility in screening for modulators of said GPCRs, particularly
agonists.
[2569] Herewith Applicant discloses EFA-hRUP25 "hRUP25-S91"
polynucleotide of SEQ. ID. NO:158 and the encoded polypeptide of
SEQ. ID. NO.:159. EFA-hRUP25 polypeptide "hRUP25-S91" differs from
endogenous hRUP25 polypeptide of SEQ. ID. NO.:36 through
substitution of the tryptophan at amino acid position 91 with
serine. Mutagenesis was performed using QuickChange Site-Directed
Mutagenesis Kit (Stratagene) according to the manufacturer's
instructions. The mutagenesis primers had the following
sequences:
[2570] Sense primer: CTATGTGAGGCGTTCAGACTGGAAGTTG (SEQ. ID.
NO.:160;
[2571] Antisense primer: CAAACTTCCAGTCTGAACGCCTCACATAG (SEQ. ID.
NO.:161.
[2572] Activity of EFA-hRUP25 "hRUP25-S91" polypeptide is presented
in FIG. 33. The window for agonist screening is 44% larger for
EFA-hRUP25 (433-210 pmol cAMP/mg protein) relative to that of
endogenous hRUP25 (329-174 pmol cAMP/mg protein).
{[(433-210=223)/(329-174=155)]=1.44.ident.144%.}
[2573] The invention relates in part to an isolated EFA-hRUP25
polypeptide comprising the amino acid sequence of SEQ. ID. NO.:159
and to isolated polynucleotide encoding said EFA-hRUP25
polypeptide. A preferred EFA-hRUP25 polynucleotide has the
nucleotide sequence of SEQ. ID. NO.:158.
[2574] The invention further relates in part to isolated EFA-hRUP25
polypeptide comprising an amino acid sequence consisting of 1, 2,
3, or 4 amino acid substitutions, deletions, or insertions relative
to the amino acid sequence of SEQ. ID. NO.:36 in addition to the
substitution of serine for tryptophan at amino acid position 91, as
well as to isolated polynucleotide encoding said EFA-hRUP25
polypeptide.
[2575] The invention also relates in part to a method of using a
polypeptide comprising an EFA-hRUP25 amino acid sequence to
identify a modulator of EFA-hRUP25. The invention also relates to a
method of using a polypeptide comprising an EFA-hRUP25 amino acid
sequence to identify a modulator of lipolysis. Preferred said
modulator of EFA-hRUP25 is an agonist.
[2576] Other embodiments encompass EFA-mRUP25 and EFA-rRUP25
polypeptide and polynucleotide. Also preferred is a method of using
EFA-mRUP25 or EFA-rRUP25 to identify a modulator of EFA-mRUP25 or
EFA-rRUP25. Also preferred is a method of using EFA-mRUP25 or
EFA-rRUP25 to identify a modulator of lipolysis. Preferred said
modulator of EFA-mRUP25 or EFA-rRUP25 is an agonist.
[2577] Other embodiments encompass EFA-RUP38 polypeptide and
polynucleotide. Also preferred is a method of using EFA-RUP38 to
identify a modulator of EFA-RUP38. Also preferred is a method of
using EFA-RUP38 to identify a modulator of lipolysis. Preferred
said modulator of EFA-RUP38 is an agonist.
[2578] Other embodiments encompass EFA-hRUP19 polypeptide and
polynucleotide. Also preferred is a method of using EFA-hRUP19 to
identify a modulator of EFA-hRUP19. Also preferred is a method of
using EFA-hRUP19 to identify a modulator of lipolysis. Preferred
said modulator of EFA-hRUP19 is an agonist.
[2579] Other embodiments encompass EFA-mRUP19 and EFA-rRUP19
polypeptide and polynucleotide. Also preferred is a method of using
EFA-mRUP19 or EFA-rRUP19 to identify a modulator of EFA-mRUP19 or
EFA-rRUP19. Also preferred is a method of using EFA-mRUP19 or
EFA-rRUP19 to identify a modulator of lipolysis. Preferred said
modulator of EFA-mRUP19 or EFA-rRUP19 is an agonist.
[2580] Other embodiments encompass EFA-RUP11 polypeptide and
polynucleotide. Also preferred is a method of using EFA-RUP11 to
identify a modulator of EFA-RUP11. Also preferred is a method of
using EFA-RUP11 to identify a modulator of lipolysis. Preferred
said modulator of RUP11 is an agonist.
[2581] The invention also provides for a method of making an EFA
mutant of an endogenous GPCR polypeptide having constitutive
activity, comprising the steps of:
[2582] (a) introducing 1, 2, 3, 4, or 5 substitutions, insertions,
or deletions into the amino acid sequence of the endogenous GPCR
polypeptide;
[2583] (b) measuring the activity of the mutant GPCR of (a) in the
absence of agonist and in the presence of a known agonist;
[2584] (c) measuring the activity of the endogenous GPCR in the
absence of agonist and in the presence of said known agonist;
and
[2585] (d) comparing (b) and (c);
[2586] wherein a determination that the agonist screening window of
(b) is greater than 20%, greater than 25%, greater than 30%,
greater than 31%, greater than 32%, greater than 33%, greater than
34%, greater than 35%, greater than 36%, greater than 37%, greater
than 38%, greater than 39%, or greater than 40% than that of (c)
identifies the mutant resulting from (a) to be an EFA mutant of the
endogenous GPCR.
[2587] In some embodiments, the agonist screening window of (b) is
greater than 20% than that of (c).
[2588] In some preferred embodiments, said known agonist is
nicotinic acid.
[2589] Methods of carrying out site-specific mutagenesis are well
known to those of ordinary skill in the art. [See, e.g, in Maniatis
T et al., Molecular Cloning: A Laboratory Manual (1989) Cold Spring
Harbor Laboratory; the disclosure of which is hereby incorporated
by reference in its entirety]. Many commercial kits for carrying
out site-specific mutagenesis are well known to those of ordinary
skill in the art and are readily available. Those skilled in the
art are credited with the ability to select techniques for mutation
of a nucleic acid sequence.
Example 33
Oral Bioavailability
[2590] Based upon the in vivo data developed, as for example by way
of illustration and not limitation data through the rat model of
Example 31 supra, oral bioavailability of a modulator of the
invention is determined. The modulator is administered by oral
gavage at doses ranging from 0.1 mg kg.sup.-1 to 100 mg kg.sup.-.
Oral administration of the modulator is shown to reduce the level
of plasma free fatty acids. The effect of the modulator is shown to
be dose-dependent and comparable to the effect after
intraperitoneal administration. The dose of modulator required to
achieve half-maximal reduction of plasma free fatty acids through
oral administration is compared to the dose of modulator required
to achieve half-maximal reduction of plasma free fatty acids
through intraperitoneal administration. By way of illustration, if
said oral dose is twice said intraperitoneal dose, then the oral
bioavailability of the modulator is taken to be 50%. More
generally, if said oral dose is .theta. mg kg.sup.-1 and said
intraperitoneal dose is .rho. mg kg.sup.-1, then the oral
bioavailability of the modulator as a percentage is taken to be
[(.rho./.theta.).times.100].
[2591] It is readily envisioned that the reference route of
administration may be other than intraperitoneal. In some
embodiments, said reference route of administration is
intravenous.
[2592] It would be readily apparent to anyone of ordinary skill in
the art that the aforesaid determination could be modified to
utilize a different in vivo animal model other than normal Sprague
Dawley rats. It would also be readily apparent to anyone of
ordinary skill in the art that the bioactivity readout in the
aforesaid determination could be a parameter other than plasma free
fatty acids.
[2593] Alternative, physicochemico analytical approaches for
assessing oral bioavailability are well known to those of ordinary
skill in the art [see, e.g., without limitation: Wong P C et al.,
Cardiovasc Drug Rev (2002) 20:137-52; and Buchan P et al., Headache
(2002) Suppl 2:S54-62; the disclosure of each of which is hereby
incorporated by reference in its entirety]. By way of further
illustration and not limitation, said alternative analytical
approaches may comprise liquid chromatography-tandem mass
spectrometry [Chavez-Eng C M et al., J ChromatogrB Analyt Technol
Biomed Life Sci (2002) 767:117-29; Jetter A et al., Clin Pharmacol
Ther (2002) 71:21-9; Zimmerman J J et al., J Clin Pharmacol (1999)
39:1155-61; and Barrish A et al., Rapid Commun Mass Spectrom (1996)
10:1033-7; the disclosure of each of which is hereby incorporated
by reference in its entirety].
Sequence CWU 1
1
161 1 1155 DNA Homo sapiens 1 atggcagccc agaatggaaa caccagtttc
acacccaact ttaatccacc ccaagaccat 60 gcctcctccc tctcctttaa
cttcagttat ggtgattatg acctccctat ggatgaggat 120 gaggacatga
ccaagacccg gaccttcttc gcagccaaga tcgtcattgg cattgcactg 180
gcaggcatca tgctggtctg cggcatcggt aactttgtct ttatcgctgc cctcacccgc
240 tataagaagt tgcgcaacct caccaatctg ctcattgcca acctggccat
ctccgacttc 300 ctggtggcca tcatctgctg ccccttcgag atggactact
acgtggtacg gcagctctcc 360 tgggagcatg gccacgtgct ctgtgcctcc
gtcaactacc tgcgcaccgt ctccctctac 420 gtctccacca atgccttgct
ggccattgcc attgacagat atctcgccat cgttcacccc 480 ttgaaaccac
ggatgaatta tcaaacggcc tccttcctga tcgccttggt ctggatggtg 540
tccattctca ttgccatccc atcggcttac tttgcaacag aaacggtcct ctttattgtc
600 aagagccagg agaagatctt ctgtggccag atctggcctg tggatcagca
gctctactac 660 aagtcctact tcctcttcat ctttggtgtc gagttcgtgg
gccctgtggt caccatgacc 720 ctgtgctatg ccaggatctc ccgggagctc
tggttcaagg cagtccctgg gttccagacg 780 gagcagattc gcaagcggct
gcgctgccgc aggaagacgg tcctggtgct catgtgcatt 840 ctcacggcct
atgtgctgtg ctgggcaccc ttctacggtt tcaccatcgt tcgtgacttc 900
ttccccactg tgttcgtgaa ggaaaagcac tacctcactg ccttctacgt ggtcgagtgc
960 atcgccatga gcaacagcat gatcaacacc gtgtgcttcg tgacggtcaa
gaacaacacc 1020 atgaagtact tcaagaagat gatgctgctg cactggcgtc
cctcccagcg ggggagcaag 1080 tccagtgctg accttgacct cagaaccaac
ggggtgccca ccacagaaga ggtggactgt 1140 atcaggctga agtga 1155 2 384
PRT Homo sapiens 2 Met Ala Ala Gln Asn Gly Asn Thr Ser Phe Thr Pro
Asn Phe Asn Pro 1 5 10 15 Pro Gln Asp His Ala Ser Ser Leu Ser Phe
Asn Phe Ser Tyr Gly Asp 20 25 30 Tyr Asp Leu Pro Met Asp Glu Asp
Glu Asp Met Thr Lys Thr Arg Thr 35 40 45 Phe Phe Ala Ala Lys Ile
Val Ile Gly Ile Ala Leu Ala Gly Ile Met 50 55 60 Leu Val Cys Gly
Ile Gly Asn Phe Val Phe Ile Ala Ala Leu Thr Arg 65 70 75 80 Tyr Lys
Lys Leu Arg Asn Leu Thr Asn Leu Leu Ile Ala Asn Leu Ala 85 90 95
Ile Ser Asp Phe Leu Val Ala Ile Ile Cys Cys Pro Phe Glu Met Asp 100
105 110 Tyr Tyr Val Val Arg Gln Leu Ser Trp Glu His Gly His Val Leu
Cys 115 120 125 Ala Ser Val Asn Tyr Leu Arg Thr Val Ser Leu Tyr Val
Ser Thr Asn 130 135 140 Ala Leu Leu Ala Ile Ala Ile Asp Arg Tyr Leu
Ala Ile Val His Pro 145 150 155 160 Leu Lys Pro Arg Met Asn Tyr Gln
Thr Ala Ser Phe Leu Ile Ala Leu 165 170 175 Val Trp Met Val Ser Ile
Leu Ile Ala Ile Pro Ser Ala Tyr Phe Ala 180 185 190 Thr Glu Thr Val
Leu Phe Ile Val Lys Ser Gln Glu Lys Ile Phe Cys 195 200 205 Gly Gln
Ile Trp Pro Val Asp Gln Gln Leu Tyr Tyr Lys Ser Tyr Phe 210 215 220
Leu Phe Ile Phe Gly Val Glu Phe Val Gly Pro Val Val Thr Met Thr 225
230 235 240 Leu Cys Tyr Ala Arg Ile Ser Arg Glu Leu Trp Phe Lys Ala
Val Pro 245 250 255 Gly Phe Gln Thr Glu Gln Ile Arg Lys Arg Leu Arg
Cys Arg Arg Lys 260 265 270 Thr Val Leu Val Leu Met Cys Ile Leu Thr
Ala Tyr Val Leu Cys Trp 275 280 285 Ala Pro Phe Tyr Gly Phe Thr Ile
Val Arg Asp Phe Phe Pro Thr Val 290 295 300 Phe Val Lys Glu Lys His
Tyr Leu Thr Ala Phe Tyr Val Val Glu Cys 305 310 315 320 Ile Ala Met
Ser Asn Ser Met Ile Asn Thr Val Cys Phe Val Thr Val 325 330 335 Lys
Asn Asn Thr Met Lys Tyr Phe Lys Lys Met Met Leu Leu His Trp 340 345
350 Arg Pro Ser Gln Arg Gly Ser Lys Ser Ser Ala Asp Leu Asp Leu Arg
355 360 365 Thr Asn Gly Val Pro Thr Thr Glu Glu Val Asp Cys Ile Arg
Leu Lys 370 375 380 3 1260 DNA Homo sapiens 3 atgctggcag ctgcctttgc
agactctaac tccagcagca tgaatgtgtc ctttgctcac 60 ctccactttg
ccggagggta cctgccctct gattcccagg actggagaac catcatcccg 120
gctctcttgg tggctgtctg cctggtgggc ttcgtgggaa acctgtgtgt gattggcatc
180 ctccttcaca atgcttggaa aggaaagcca tccatgatcc actccctgat
tctgaatctc 240 agcctggctg atctctccct cctgctgttt tctgcaccta
tccgagctac ggcgtactcc 300 aaaagtgttt gggatctagg ctggtttgtc
tgcaagtcct ctgactggtt tatccacaca 360 tgcatggcag ccaagagcct
gacaatcgtt gtggtggcca aagtatgctt catgtatgca 420 agtgacccag
ccaagcaagt gagtatccac aactacacca tctggtcagt gctggtggcc 480
atctggactg tggctagcct gttacccctg ccggaatggt tctttagcac catcaggcat
540 catgaaggtg tggaaatgtg cctcgtggat gtaccagctg tggctgaaga
gtttatgtcg 600 atgtttggta agctctaccc actcctggca tttggccttc
cattattttt tgccagcttt 660 tatttctgga gagcttatga ccaatgtaaa
aaacgaggaa ctaagactca aaatcttaga 720 aaccagatac gctcaaagca
agtcacagtg atgctgctga gcattgccat catctctgct 780 ctcttgtggc
tccccgaatg ggtagcttgg ctgtgggtat ggcatctgaa ggctgcaggc 840
ccggccccac cacaaggttt catagccctg tctcaagtct tgatgttttc catctcttca
900 gcaaatcctc tcatttttct tgtgatgtcg gaagagttca gggaaggctt
gaaaggtgta 960 tggaaatgga tgataaccaa aaaacctcca actgtctcag
agtctcagga aacaccagct 1020 ggcaactcag agggtcttcc tgacaaggtt
ccatctccag aatccccagc atccatacca 1080 gaaaaagaga aacccagctc
tccctcctct ggcaaaggga aaactgagaa ggcagagatt 1140 cccatccttc
ctgacgtaga gcagttttgg catgagaggg acacagtccc ttctgtacag 1200
gacaatgacc ctatcccctg ggaacatgaa gatcaagaga caggggaagg tgttaaatag
1260 4 419 PRT Homo sapiens 4 Met Leu Ala Ala Ala Phe Ala Asp Ser
Asn Ser Ser Ser Met Asn Val 1 5 10 15 Ser Phe Ala His Leu His Phe
Ala Gly Gly Tyr Leu Pro Ser Asp Ser 20 25 30 Gln Asp Trp Arg Thr
Ile Ile Pro Ala Leu Leu Val Ala Val Cys Leu 35 40 45 Val Gly Phe
Val Gly Asn Leu Cys Val Ile Gly Ile Leu Leu His Asn 50 55 60 Ala
Trp Lys Gly Lys Pro Ser Met Ile His Ser Leu Ile Leu Asn Leu 65 70
75 80 Ser Leu Ala Asp Leu Ser Leu Leu Leu Phe Ser Ala Pro Ile Arg
Ala 85 90 95 Thr Ala Tyr Ser Lys Ser Val Trp Asp Leu Gly Trp Phe
Val Cys Lys 100 105 110 Ser Ser Asp Trp Phe Ile His Thr Cys Met Ala
Ala Lys Ser Leu Thr 115 120 125 Ile Val Val Val Ala Lys Val Cys Phe
Met Tyr Ala Ser Asp Pro Ala 130 135 140 Lys Gln Val Ser Ile His Asn
Tyr Thr Ile Trp Ser Val Leu Val Ala 145 150 155 160 Ile Trp Thr Val
Ala Ser Leu Leu Pro Leu Pro Glu Trp Phe Phe Ser 165 170 175 Thr Ile
Arg His His Glu Gly Val Glu Met Cys Leu Val Asp Val Pro 180 185 190
Ala Val Ala Glu Glu Phe Met Ser Met Phe Gly Lys Leu Tyr Pro Leu 195
200 205 Leu Ala Phe Gly Leu Pro Leu Phe Phe Ala Ser Phe Tyr Phe Trp
Arg 210 215 220 Ala Tyr Asp Gln Cys Lys Lys Arg Gly Thr Lys Thr Gln
Asn Leu Arg 225 230 235 240 Asn Gln Ile Arg Ser Lys Gln Val Thr Val
Met Leu Leu Ser Ile Ala 245 250 255 Ile Ile Ser Ala Leu Leu Trp Leu
Pro Glu Trp Val Ala Trp Leu Trp 260 265 270 Val Trp His Leu Lys Ala
Ala Gly Pro Ala Pro Pro Gln Gly Phe Ile 275 280 285 Ala Leu Ser Gln
Val Leu Met Phe Ser Ile Ser Ser Ala Asn Pro Leu 290 295 300 Ile Phe
Leu Val Met Ser Glu Glu Phe Arg Glu Gly Leu Lys Gly Val 305 310 315
320 Trp Lys Trp Met Ile Thr Lys Lys Pro Pro Thr Val Ser Glu Ser Gln
325 330 335 Glu Thr Pro Ala Gly Asn Ser Glu Gly Leu Pro Asp Lys Val
Pro Ser 340 345 350 Pro Glu Ser Pro Ala Ser Ile Pro Glu Lys Glu Lys
Pro Ser Ser Pro 355 360 365 Ser Ser Gly Lys Gly Lys Thr Glu Lys Ala
Glu Ile Pro Ile Leu Pro 370 375 380 Asp Val Glu Gln Phe Trp His Glu
Arg Asp Thr Val Pro Ser Val Gln 385 390 395 400 Asp Asn Asp Pro Ile
Pro Trp Glu His Glu Asp Gln Glu Thr Gly Glu 405 410 415 Gly Val Lys
5 1014 DNA Homo sapiens 5 atggggaacg attctgtcag ctacgagtat
ggggattaca gcgacctctc ggaccgccct 60 gtggactgcc tggatggcgc
ctgcctggcc atcgacccgc tgcgcgtggc cccgctccca 120 ctgtatgccg
ccatcttcct ggtgggggtg ccgggcaatg ccatggtggc ctgggtggct 180
gggaaggtgg cccgccggag ggtgggtgcc acctggttgc tccacctggc cgtggcggat
240 ttgctgtgct gtttgtctct gcccatcctg gcagtgccca ttgcccgtgg
aggccactgg 300 ccgtatggtg cagtgggctg tcgggcgctg ccctccatca
tcctgctgac catgtatgcc 360 agcgtcctgc tcctggcagc tctcagtgcc
gacctctgct tcctggctct cgggcctgcc 420 tggtggtcta cggttcagcg
ggcgtgcggg gtgcaggtgg cctgtggggc agcctggaca 480 ctggccttgc
tgctcaccgt gccctccgcc atctaccgcc ggctgcacca ggagcacttc 540
ccagcccggc tgcagtgtgt ggtggactac ggcggctcct ccagcaccga gaatgcggtg
600 actgccatcc ggtttctttt tggcttcctg gggcccctgg tggccgtggc
cagctgccac 660 agtgccctcc tgtgctgggc agcccgacgc tgccggccgc
tgggcacagc cattgtggtg 720 gggttttttg tctgctgggc accctaccac
ctgctggggc tggtgctcac tgtggcggcc 780 ccgaactccg cactcctggc
cagggccctg cgggctgaac ccctcatcgt gggccttgcc 840 ctcgctcaca
gctgcctcaa tcccatgctc ttcctgtatt ttgggagggc tcaactccgc 900
cggtcactgc cagctgcctg tcactgggcc ctgagggagt cccagggcca ggacgaaagt
960 gtggacagca agaaatccac cagccatgac ctggtctcgg agatggaggt gtag
1014 6 337 PRT Homo sapiens 6 Met Gly Asn Asp Ser Val Ser Tyr Glu
Tyr Gly Asp Tyr Ser Asp Leu 1 5 10 15 Ser Asp Arg Pro Val Asp Cys
Leu Asp Gly Ala Cys Leu Ala Ile Asp 20 25 30 Pro Leu Arg Val Ala
Pro Leu Pro Leu Tyr Ala Ala Ile Phe Leu Val 35 40 45 Gly Val Pro
Gly Asn Ala Met Val Ala Trp Val Ala Gly Lys Val Ala 50 55 60 Arg
Arg Arg Val Gly Ala Thr Trp Leu Leu His Leu Ala Val Ala Asp 65 70
75 80 Leu Leu Cys Cys Leu Ser Leu Pro Ile Leu Ala Val Pro Ile Ala
Arg 85 90 95 Gly Gly His Trp Pro Tyr Gly Ala Val Gly Cys Arg Ala
Leu Pro Ser 100 105 110 Ile Ile Leu Leu Thr Met Tyr Ala Ser Val Leu
Leu Leu Ala Ala Leu 115 120 125 Ser Ala Asp Leu Cys Phe Leu Ala Leu
Gly Pro Ala Trp Trp Ser Thr 130 135 140 Val Gln Arg Ala Cys Gly Val
Gln Val Ala Cys Gly Ala Ala Trp Thr 145 150 155 160 Leu Ala Leu Leu
Leu Thr Val Pro Ser Ala Ile Tyr Arg Arg Leu His 165 170 175 Gln Glu
His Phe Pro Ala Arg Leu Gln Cys Val Val Asp Tyr Gly Gly 180 185 190
Ser Ser Ser Thr Glu Asn Ala Val Thr Ala Ile Arg Phe Leu Phe Gly 195
200 205 Phe Leu Gly Pro Leu Val Ala Val Ala Ser Cys His Ser Ala Leu
Leu 210 215 220 Cys Trp Ala Ala Arg Arg Cys Arg Pro Leu Gly Thr Ala
Ile Val Val 225 230 235 240 Gly Phe Phe Val Cys Trp Ala Pro Tyr His
Leu Leu Gly Leu Val Leu 245 250 255 Thr Val Ala Ala Pro Asn Ser Ala
Leu Leu Ala Arg Ala Leu Arg Ala 260 265 270 Glu Pro Leu Ile Val Gly
Leu Ala Leu Ala His Ser Cys Leu Asn Pro 275 280 285 Met Leu Phe Leu
Tyr Phe Gly Arg Ala Gln Leu Arg Arg Ser Leu Pro 290 295 300 Ala Ala
Cys His Trp Ala Leu Arg Glu Ser Gln Gly Gln Asp Glu Ser 305 310 315
320 Val Asp Ser Lys Lys Ser Thr Ser His Asp Leu Val Ser Glu Met Glu
325 330 335 Val 7 1272 DNA Homo sapiens 7 atgttgtgtc accgtggtgg
ccagctgata gtgccaatca tcccactttg ccctgagcac 60 tcctgcaggg
gtagaagact ccagaacctt ctctcaggcc catggcccaa gcagcccatg 120
gaacttcata acctgagctc tccatctccc tctctctcct cctctgttct ccctccctcc
180 ttctctccct caccctcctc tgctccctct gcctttacca ctgtgggggg
gtcctctgga 240 gggccctgcc accccacctc ttcctcgctg gtgtctgcct
tcctggcacc aatcctggcc 300 ctggagtttg tcctgggcct ggtggggaac
agtttggccc tcttcatctt ctgcatccac 360 acgcggccct ggacctccaa
cacggtgttc ctggtcagcc tggtggccgc tgacttcctc 420 ctgatcagca
acctgcccct ccgcgtggac tactacctcc tccatgagac ctggcgcttt 480
ggggctgctg cctgcaaagt caacctcttc atgctgtcca ccaaccgcac ggccagcgtt
540 gtcttcctca cagccatcgc actcaaccgc tacctgaagg tggtgcagcc
ccaccacgtg 600 ctgagccgtg cttccgtggg ggcagctgcc cgggtggccg
ggggactctg ggtgggcatc 660 ctgctcctca acgggcacct gctcctgagc
accttctccg gcccctcctg cctcagctac 720 agggtgggca cgaagccctc
ggcctcgctc cgctggcacc aggcactgta cctgctggag 780 ttcttcctgc
cactggcgct catcctcttt gctattgtga gcattgggct caccatccgg 840
aaccgtggtc tgggcgggca ggcaggcccg cagagggcca tgcgtgtgct ggccatggtg
900 gtggccgtct acaccatctg cttcttgccc agcatcatct ttggcatggc
ttccatggtg 960 gctttctggc tgtccgcctg ccgatccctg gacctctgca
cacagctctt ccatggctcc 1020 ctggccttca cctacctcaa cagtgtcctg
gaccccgtgc tctactgctt ctctagcccc 1080 aacttcctcc accagagccg
ggccttgctg ggcctcacgc ggggccggca gggcccagtg 1140 agcgacgaga
gctcctacca accctccagg cagtggcgct accgggaggc ctctaggaag 1200
gcggaggcca tagggaagct gaaagtgcag ggcgaggtct ctctggaaaa ggaaggctcc
1260 tcccagggct ga 1272 8 423 PRT Homo sapiens 8 Met Leu Cys His
Arg Gly Gly Gln Leu Ile Val Pro Ile Ile Pro Leu 1 5 10 15 Cys Pro
Glu His Ser Cys Arg Gly Arg Arg Leu Gln Asn Leu Leu Ser 20 25 30
Gly Pro Trp Pro Lys Gln Pro Met Glu Leu His Asn Leu Ser Ser Pro 35
40 45 Ser Pro Ser Leu Ser Ser Ser Val Leu Pro Pro Ser Phe Ser Pro
Ser 50 55 60 Pro Ser Ser Ala Pro Ser Ala Phe Thr Thr Val Gly Gly
Ser Ser Gly 65 70 75 80 Gly Pro Cys His Pro Thr Ser Ser Ser Leu Val
Ser Ala Phe Leu Ala 85 90 95 Pro Ile Leu Ala Leu Glu Phe Val Leu
Gly Leu Val Gly Asn Ser Leu 100 105 110 Ala Leu Phe Ile Phe Cys Ile
His Thr Arg Pro Trp Thr Ser Asn Thr 115 120 125 Val Phe Leu Val Ser
Leu Val Ala Ala Asp Phe Leu Leu Ile Ser Asn 130 135 140 Leu Pro Leu
Arg Val Asp Tyr Tyr Leu Leu His Glu Thr Trp Arg Phe 145 150 155 160
Gly Ala Ala Ala Cys Lys Val Asn Leu Phe Met Leu Ser Thr Asn Arg 165
170 175 Thr Ala Ser Val Val Phe Leu Thr Ala Ile Ala Leu Asn Arg Tyr
Leu 180 185 190 Lys Val Val Gln Pro His His Val Leu Ser Arg Ala Ser
Val Gly Ala 195 200 205 Ala Ala Arg Val Ala Gly Gly Leu Trp Val Gly
Ile Leu Leu Leu Asn 210 215 220 Gly His Leu Leu Leu Ser Thr Phe Ser
Gly Pro Ser Cys Leu Ser Tyr 225 230 235 240 Arg Val Gly Thr Lys Pro
Ser Ala Ser Leu Arg Trp His Gln Ala Leu 245 250 255 Tyr Leu Leu Glu
Phe Phe Leu Pro Leu Ala Leu Ile Leu Phe Ala Ile 260 265 270 Val Ser
Ile Gly Leu Thr Ile Arg Asn Arg Gly Leu Gly Gly Gln Ala 275 280 285
Gly Pro Gln Arg Ala Met Arg Val Leu Ala Met Val Val Ala Val Tyr 290
295 300 Thr Ile Cys Phe Leu Pro Ser Ile Ile Phe Gly Met Ala Ser Met
Val 305 310 315 320 Ala Phe Trp Leu Ser Ala Cys Arg Ser Leu Asp Leu
Cys Thr Gln Leu 325 330 335 Phe His Gly Ser Leu Ala Phe Thr Tyr Leu
Asn Ser Val Leu Asp Pro 340 345 350 Val Leu Tyr Cys Phe Ser Ser Pro
Asn Phe Leu His Gln Ser Arg Ala 355 360 365 Leu Leu Gly Leu Thr Arg
Gly Arg Gln Gly Pro Val Ser Asp Glu Ser 370 375 380 Ser Tyr Gln Pro
Ser Arg Gln Trp Arg Tyr Arg Glu Ala Ser Arg Lys 385 390 395 400 Ala
Glu Ala Ile Gly Lys Leu Lys Val Gln Gly Glu Val Ser Leu Glu 405 410
415 Lys Glu Gly Ser Ser Gln Gly 420 9 966 DNA Homo sapiens 9
atgaaccaga ctttgaatag cagtgggacc gtggagtcag ccctaaacta ttccagaggg
60 agcacagtgc acacggccta cctggtgctg agctccctgg ccatgttcac
ctgcctgtgc 120 gggatggcag gcaacagcat ggtgatctgg ctgctgggct
ttcgaatgca caggaacccc 180 ttctgcatct atatcctcaa cctggcggca
gccgacctcc tcttcctctt cagcatggct 240 tccacgctca gcctggaaac
ccagcccctg gtcaatacca ctgacaaggt ccacgagctg 300 atgaagagac
tgatgtactt tgcctacaca gtgggcctga gcctgctgac ggccatcagc 360
acccagcgct gtctctctgt cctcttccct atctggttca agtgtcaccg gcccaggcac
420 ctgtcagcct gggtgtgtgg cctgctgtgg acactctgtc
tcctgatgaa cgggttgacc 480 tcttccttct gcagcaagtt cttgaaattc
aatgaagatc ggtgcttcag ggtggacatg 540 gtccaggccg ccctcatcat
gggggtctta accccagtga tgactctgtc cagcctgacc 600 ctctttgtct
gggtgcggag gagctcccag cagtggcggc ggcagcccac acggctgttc 660
gtggtggtcc tggcctctgt cctggtgttc ctcatctgtt ccctgcctct gagcatctac
720 tggtttgtgc tctactggtt gagcctgccg cccgagatgc aggtcctgtg
cttcagcttg 780 tcacgcctct cctcgtccgt aagcagcagc gccaaccccg
tcatctactt cctggtgggc 840 agccggagga gccacaggct gcccaccagg
tccctgggga ctgtgctcca acaggcgctt 900 cgcgaggagc ccgagctgga
aggtggggag acgcccaccg tgggcaccaa tgagatgggg 960 gcttga 966 10 321
PRT Homo sapiens 10 Met Asn Gln Thr Leu Asn Ser Ser Gly Thr Val Glu
Ser Ala Leu Asn 1 5 10 15 Tyr Ser Arg Gly Ser Thr Val His Thr Ala
Tyr Leu Val Leu Ser Ser 20 25 30 Leu Ala Met Phe Thr Cys Leu Cys
Gly Met Ala Gly Asn Ser Met Val 35 40 45 Ile Trp Leu Leu Gly Phe
Arg Met His Arg Asn Pro Phe Cys Ile Tyr 50 55 60 Ile Leu Asn Leu
Ala Ala Ala Asp Leu Leu Phe Leu Phe Ser Met Ala 65 70 75 80 Ser Thr
Leu Ser Leu Glu Thr Gln Pro Leu Val Asn Thr Thr Asp Lys 85 90 95
Val His Glu Leu Met Lys Arg Leu Met Tyr Phe Ala Tyr Thr Val Gly 100
105 110 Leu Ser Leu Leu Thr Ala Ile Ser Thr Gln Arg Cys Leu Ser Val
Leu 115 120 125 Phe Pro Ile Trp Phe Lys Cys His Arg Pro Arg His Leu
Ser Ala Trp 130 135 140 Val Cys Gly Leu Leu Trp Thr Leu Cys Leu Leu
Met Asn Gly Leu Thr 145 150 155 160 Ser Ser Phe Cys Ser Lys Phe Leu
Lys Phe Asn Glu Asp Arg Cys Phe 165 170 175 Arg Val Asp Met Val Gln
Ala Ala Leu Ile Met Gly Val Leu Thr Pro 180 185 190 Val Met Thr Leu
Ser Ser Leu Thr Leu Phe Val Trp Val Arg Arg Ser 195 200 205 Ser Gln
Gln Trp Arg Arg Gln Pro Thr Arg Leu Phe Val Val Val Leu 210 215 220
Ala Ser Val Leu Val Phe Leu Ile Cys Ser Leu Pro Leu Ser Ile Tyr 225
230 235 240 Trp Phe Val Leu Tyr Trp Leu Ser Leu Pro Pro Glu Met Gln
Val Leu 245 250 255 Cys Phe Ser Leu Ser Arg Leu Ser Ser Ser Val Ser
Ser Ser Ala Asn 260 265 270 Pro Val Ile Tyr Phe Leu Val Gly Ser Arg
Arg Ser His Arg Leu Pro 275 280 285 Thr Arg Ser Leu Gly Thr Val Leu
Gln Gln Ala Leu Arg Glu Glu Pro 290 295 300 Glu Leu Glu Gly Gly Glu
Thr Pro Thr Val Gly Thr Asn Glu Met Gly 305 310 315 320 Ala 11 1356
DNA Homo sapiens 11 atggagtcct cacccatccc ccagtcatca gggaactctt
ccactttggg gagggtccct 60 caaaccccag gtccctctac tgccagtggg
gtcccggagg tggggctacg ggatgttgct 120 tcggaatctg tggccctctt
cttcatgctc ctgctggact tgactgctgt ggctggcaat 180 gccgctgtga
tggccgtgat cgccaagacg cctgccctcc gaaaatttgt cttcgtcttc 240
cacctctgcc tggtggacct gctggctgcc ctgaccctca tgcccctggc catgctctcc
300 agctctgccc tctttgacca cgccctcttt ggggaggtgg cctgccgcct
ctacttgttt 360 ctgagcgtgt gctttgtcag cctggccatc ctctcggtgt
cagccatcaa tgtggagcgc 420 tactattacg tagtccaccc catgcgctac
gaggtgcgca tgacgctggg gctggtggcc 480 tctgtgctgg tgggtgtgtg
ggtgaaggcc ttggccatgg cttctgtgcc agtgttggga 540 agggtctcct
gggaggaagg agctcccagt gtccccccag gctgttcact ccagtggagc 600
cacagtgcct actgccagct ttttgtggtg gtctttgctg tcctttactt tctgttgccc
660 ctgctcctca tacttgtggt ctactgcagc atgttccgag tggcccgcgt
ggctgccatg 720 cagcacgggc cgctgcccac gtggatggag acaccccggc
aacgctccga atctctcagc 780 agccgctcca cgatggtcac cagctcgggg
gccccccaga ccaccccaca ccggacgttt 840 gggggaggga aagcagcagt
ggttctcctg gctgtggggg gacagttcct gctctgttgg 900 ttgccctact
tctctttcca cctctatgtt gccctgagtg ctcagcccat ttcaactggg 960
caggtggaga gtgtggtcac ctggattggc tacttttgct tcacttccaa ccctttcttc
1020 tatggatgtc tcaaccggca gatccggggg gagctcagca agcagtttgt
ctgcttcttc 1080 aagccagctc cagaggagga gctgaggctg cctagccggg
agggctccat tgaggagaac 1140 ttcctgcagt tccttcaggg gactggctgt
ccttctgagt cctgggtttc ccgaccccta 1200 cccagcccca agcaggagcc
acctgctgtt gactttcgaa tcccaggcca gatagctgag 1260 gagacctctg
agttcctgga gcagcaactc accagcgaca tcatcatgtc agacagctac 1320
ctccgtcctg ccgcctcacc ccggctggag tcatga 1356 12 451 PRT Homo
sapiens 12 Met Glu Ser Ser Pro Ile Pro Gln Ser Ser Gly Asn Ser Ser
Thr Leu 1 5 10 15 Gly Arg Val Pro Gln Thr Pro Gly Pro Ser Thr Ala
Ser Gly Val Pro 20 25 30 Glu Val Gly Leu Arg Asp Val Ala Ser Glu
Ser Val Ala Leu Phe Phe 35 40 45 Met Leu Leu Leu Asp Leu Thr Ala
Val Ala Gly Asn Ala Ala Val Met 50 55 60 Ala Val Ile Ala Lys Thr
Pro Ala Leu Arg Lys Phe Val Phe Val Phe 65 70 75 80 His Leu Cys Leu
Val Asp Leu Leu Ala Ala Leu Thr Leu Met Pro Leu 85 90 95 Ala Met
Leu Ser Ser Ser Ala Leu Phe Asp His Ala Leu Phe Gly Glu 100 105 110
Val Ala Cys Arg Leu Tyr Leu Phe Leu Ser Val Cys Phe Val Ser Leu 115
120 125 Ala Ile Leu Ser Val Ser Ala Ile Asn Val Glu Arg Tyr Tyr Tyr
Val 130 135 140 Val His Pro Met Arg Tyr Glu Val Arg Met Thr Leu Gly
Leu Val Ala 145 150 155 160 Ser Val Leu Val Gly Val Trp Val Lys Ala
Leu Ala Met Ala Ser Val 165 170 175 Pro Val Leu Gly Arg Val Ser Trp
Glu Glu Gly Ala Pro Ser Val Pro 180 185 190 Pro Gly Cys Ser Leu Gln
Trp Ser His Ser Ala Tyr Cys Gln Leu Phe 195 200 205 Val Val Val Phe
Ala Val Leu Tyr Phe Leu Leu Pro Leu Leu Leu Ile 210 215 220 Leu Val
Val Tyr Cys Ser Met Phe Arg Val Ala Arg Val Ala Ala Met 225 230 235
240 Gln His Gly Pro Leu Pro Thr Trp Met Glu Thr Pro Arg Gln Arg Ser
245 250 255 Glu Ser Leu Ser Ser Arg Ser Thr Met Val Thr Ser Ser Gly
Ala Pro 260 265 270 Gln Thr Thr Pro His Arg Thr Phe Gly Gly Gly Lys
Ala Ala Val Val 275 280 285 Leu Leu Ala Val Gly Gly Gln Phe Leu Leu
Cys Trp Leu Pro Tyr Phe 290 295 300 Ser Phe His Leu Tyr Val Ala Leu
Ser Ala Gln Pro Ile Ser Thr Gly 305 310 315 320 Gln Val Glu Ser Val
Val Thr Trp Ile Gly Tyr Phe Cys Phe Thr Ser 325 330 335 Asn Pro Phe
Phe Tyr Gly Cys Leu Asn Arg Gln Ile Arg Gly Glu Leu 340 345 350 Ser
Lys Gln Phe Val Cys Phe Phe Lys Pro Ala Pro Glu Glu Glu Leu 355 360
365 Arg Leu Pro Ser Arg Glu Gly Ser Ile Glu Glu Asn Phe Leu Gln Phe
370 375 380 Leu Gln Gly Thr Gly Cys Pro Ser Glu Ser Trp Val Ser Arg
Pro Leu 385 390 395 400 Pro Ser Pro Lys Gln Glu Pro Pro Ala Val Asp
Phe Arg Ile Pro Gly 405 410 415 Gln Ile Ala Glu Glu Thr Ser Glu Phe
Leu Glu Gln Gln Leu Thr Ser 420 425 430 Asp Ile Ile Met Ser Asp Ser
Tyr Leu Arg Pro Ala Ala Ser Pro Arg 435 440 445 Leu Glu Ser 450 13
1041 DNA Homo sapiens 13 atggagagaa aatttatgtc cttgcaacca
tccatctccg tatcagaaat ggaaccaaat 60 ggcaccttca gcaataacaa
cagcaggaac tgcacaattg aaaacttcaa gagagaattt 120 ttcccaattg
tatatctgat aatatttttc tggggagtct tgggaaatgg gttgtccata 180
tatgttttcc tgcagcctta taagaagtcc acatctgtga acgttttcat gctaaatctg
240 gccatttcag atctcctgtt cataagcacg cttcccttca gggctgacta
ttatcttaga 300 ggctccaatt ggatatttgg agacctggcc tgcaggatta
tgtcttattc cttgtatgtc 360 aacatgtaca gcagtattta tttcctgacc
gtgctgagtg ttgtgcgttt cctggcaatg 420 gttcacccct ttcggcttct
gcatgtcacc agcatcagga gtgcctggat cctctgtggg 480 atcatatgga
tccttatcat ggcttcctca ataatgctcc tggacagtgg ctctgagcag 540
aacggcagtg tcacatcatg cttagagctg aatctctata aaattgctaa gctgcagacc
600 atgaactata ttgccttggt ggtgggctgc ctgctgccat ttttcacact
cagcatctgt 660 tatctgctga tcattcgggt tctgttaaaa gtggaggtcc
cagaatcggg gctgcgggtt 720 tctcacagga aggcactgac caccatcatc
atcaccttga tcatcttctt cttgtgtttc 780 ctgccctatc acacactgag
gaccgtccac ttgacgacat ggaaagtggg tttatgcaaa 840 gacagactgc
ataaagcttt ggttatcaca ctggccttgg cagcagccaa tgcctgcttc 900
aatcctctgc tctattactt tgctggggag aattttaagg acagactaaa gtctgcactc
960 agaaaaggcc atccacagaa ggcaaagaca aagtgtgttt tccctgttag
tgtgtggttg 1020 agaaaggaaa caagagtata a 1041 14 346 PRT Homo
sapiens 14 Met Glu Arg Lys Phe Met Ser Leu Gln Pro Ser Ile Ser Val
Ser Glu 1 5 10 15 Met Glu Pro Asn Gly Thr Phe Ser Asn Asn Asn Ser
Arg Asn Cys Thr 20 25 30 Ile Glu Asn Phe Lys Arg Glu Phe Phe Pro
Ile Val Tyr Leu Ile Ile 35 40 45 Phe Phe Trp Gly Val Leu Gly Asn
Gly Leu Ser Ile Tyr Val Phe Leu 50 55 60 Gln Pro Tyr Lys Lys Ser
Thr Ser Val Asn Val Phe Met Leu Asn Leu 65 70 75 80 Ala Ile Ser Asp
Leu Leu Phe Ile Ser Thr Leu Pro Phe Arg Ala Asp 85 90 95 Tyr Tyr
Leu Arg Gly Ser Asn Trp Ile Phe Gly Asp Leu Ala Cys Arg 100 105 110
Ile Met Ser Tyr Ser Leu Tyr Val Asn Met Tyr Ser Ser Ile Tyr Phe 115
120 125 Leu Thr Val Leu Ser Val Val Arg Phe Leu Ala Met Val His Pro
Phe 130 135 140 Arg Leu Leu His Val Thr Ser Ile Arg Ser Ala Trp Ile
Leu Cys Gly 145 150 155 160 Ile Ile Trp Ile Leu Ile Met Ala Ser Ser
Ile Met Leu Leu Asp Ser 165 170 175 Gly Ser Glu Gln Asn Gly Ser Val
Thr Ser Cys Leu Glu Leu Asn Leu 180 185 190 Tyr Lys Ile Ala Lys Leu
Gln Thr Met Asn Tyr Ile Ala Leu Val Val 195 200 205 Gly Cys Leu Leu
Pro Phe Phe Thr Leu Ser Ile Cys Tyr Leu Leu Ile 210 215 220 Ile Arg
Val Leu Leu Lys Val Glu Val Pro Glu Ser Gly Leu Arg Val 225 230 235
240 Ser His Arg Lys Ala Leu Thr Thr Ile Ile Ile Thr Leu Ile Ile Phe
245 250 255 Phe Leu Cys Phe Leu Pro Tyr His Thr Leu Arg Thr Val His
Leu Thr 260 265 270 Thr Trp Lys Val Gly Leu Cys Lys Asp Arg Leu His
Lys Ala Leu Val 275 280 285 Ile Thr Leu Ala Leu Ala Ala Ala Asn Ala
Cys Phe Asn Pro Leu Leu 290 295 300 Tyr Tyr Phe Ala Gly Glu Asn Phe
Lys Asp Arg Leu Lys Ser Ala Leu 305 310 315 320 Arg Lys Gly His Pro
Gln Lys Ala Lys Thr Lys Cys Val Phe Pro Val 325 330 335 Ser Val Trp
Leu Arg Lys Glu Thr Arg Val 340 345 15 1527 DNA Homo sapiens 15
atgacgtcca cctgcaccaa cagcacgcgc gagagtaaca gcagccacac gtgcatgccc
60 ctctccaaaa tgcccatcag cctggcccac ggcatcatcc gctcaaccgt
gctggttatc 120 ttcctcgccg cctctttcgt cggcaacata gtgctggcgc
tagtgttgca gcgcaagccg 180 cagctgctgc aggtgaccaa ccgttttatc
tttaacctcc tcgtcaccga cctgctgcag 240 atttcgctcg tggccccctg
ggtggtggcc acctctgtgc ctctcttctg gcccctcaac 300 agccacttct
gcacggccct ggttagcctc acccacctgt tcgccttcgc cagcgtcaac 360
accattgtcg tggtgtcagt ggatcgctac ttgtccatca tccaccctct ctcctacccg
420 tccaagatga cccagcgccg cggttacctg ctcctctatg gcacctggat
tgtggccatc 480 ctgcagagca ctcctccact ctacggctgg ggccaggctg
cctttgatga gcgcaatgct 540 ctctgctcca tgatctgggg ggccagcccc
agctacacta ttctcagcgt ggtgtccttc 600 atcgtcattc cactgattgt
catgattgcc tgctactccg tggtgttctg tgcagcccgg 660 aggcagcatg
ctctgctgta caatgtcaag agacacagct tggaagtgcg agtcaaggac 720
tgtgtggaga atgaggatga agagggagca gagaagaagg aggagttcca ggatgagagt
780 gagtttcgcc gccagcatga aggtgaggtc aaggccaagg agggcagaat
ggaagccaag 840 gacggcagcc tgaaggccaa ggaaggaagc acggggacca
gtgagagtag tgtagaggcc 900 aggggcagcg aggaggtcag agagagcagc
acggtggcca gcgacggcag catggagggt 960 aaggaaggca gcaccaaagt
tgaggagaac agcatgaagg cagacaaggg tcgcacagag 1020 gtcaaccagt
gcagcattga cttgggtgaa gatgacatgg agtttggtga agacgacatc 1080
aatttcagtg aggatgacgt cgaggcagtg aacatcccgg agagcctccc acccagtcgt
1140 cgtaacagca acagcaaccc tcctctgccc aggtgctacc agtgcaaagc
tgctaaagtg 1200 atcttcatca tcattttctc ctatgtgcta tccctggggc
cctactgctt tttagcagtc 1260 ctggccgtgt gggtggatgt cgaaacccag
gtaccccagt gggtgatcac cataatcatc 1320 tggcttttct tcctgcagtg
ctgcatccac ccctatgtct atggctacat gcacaagacc 1380 attaagaagg
aaatccagga catgctgaag aagttcttct gcaaggaaaa gcccccgaaa 1440
gaagatagcc acccagacct gcccggaaca gagggtggga ctgaaggcaa gattgtccct
1500 tcctacgatt ctgctacttt tccttga 1527 16 508 PRT Homo sapiens 16
Met Thr Ser Thr Cys Thr Asn Ser Thr Arg Glu Ser Asn Ser Ser His 1 5
10 15 Thr Cys Met Pro Leu Ser Lys Met Pro Ile Ser Leu Ala His Gly
Ile 20 25 30 Ile Arg Ser Thr Val Leu Val Ile Phe Leu Ala Ala Ser
Phe Val Gly 35 40 45 Asn Ile Val Leu Ala Leu Val Leu Gln Arg Lys
Pro Gln Leu Leu Gln 50 55 60 Val Thr Asn Arg Phe Ile Phe Asn Leu
Leu Val Thr Asp Leu Leu Gln 65 70 75 80 Ile Ser Leu Val Ala Pro Trp
Val Val Ala Thr Ser Val Pro Leu Phe 85 90 95 Trp Pro Leu Asn Ser
His Phe Cys Thr Ala Leu Val Ser Leu Thr His 100 105 110 Leu Phe Ala
Phe Ala Ser Val Asn Thr Ile Val Val Val Ser Val Asp 115 120 125 Arg
Tyr Leu Ser Ile Ile His Pro Leu Ser Tyr Pro Ser Lys Met Thr 130 135
140 Gln Arg Arg Gly Tyr Leu Leu Leu Tyr Gly Thr Trp Ile Val Ala Ile
145 150 155 160 Leu Gln Ser Thr Pro Pro Leu Tyr Gly Trp Gly Gln Ala
Ala Phe Asp 165 170 175 Glu Arg Asn Ala Leu Cys Ser Met Ile Trp Gly
Ala Ser Pro Ser Tyr 180 185 190 Thr Ile Leu Ser Val Val Ser Phe Ile
Val Ile Pro Leu Ile Val Met 195 200 205 Ile Ala Cys Tyr Ser Val Val
Phe Cys Ala Ala Arg Arg Gln His Ala 210 215 220 Leu Leu Tyr Asn Val
Lys Arg His Ser Leu Glu Val Arg Val Lys Asp 225 230 235 240 Cys Val
Glu Asn Glu Asp Glu Glu Gly Ala Glu Lys Lys Glu Glu Phe 245 250 255
Gln Asp Glu Ser Glu Phe Arg Arg Gln His Glu Gly Glu Val Lys Ala 260
265 270 Lys Glu Gly Arg Met Glu Ala Lys Asp Gly Ser Leu Lys Ala Lys
Glu 275 280 285 Gly Ser Thr Gly Thr Ser Glu Ser Ser Val Glu Ala Arg
Gly Ser Glu 290 295 300 Glu Val Arg Glu Ser Ser Thr Val Ala Ser Asp
Gly Ser Met Glu Gly 305 310 315 320 Lys Glu Gly Ser Thr Lys Val Glu
Glu Asn Ser Met Lys Ala Asp Lys 325 330 335 Gly Arg Thr Glu Val Asn
Gln Cys Ser Ile Asp Leu Gly Glu Asp Asp 340 345 350 Met Glu Phe Gly
Glu Asp Asp Ile Asn Phe Ser Glu Asp Asp Val Glu 355 360 365 Ala Val
Asn Ile Pro Glu Ser Leu Pro Pro Ser Arg Arg Asn Ser Asn 370 375 380
Ser Asn Pro Pro Leu Pro Arg Cys Tyr Gln Cys Lys Ala Ala Lys Val 385
390 395 400 Ile Phe Ile Ile Ile Phe Ser Tyr Val Leu Ser Leu Gly Pro
Tyr Cys 405 410 415 Phe Leu Ala Val Leu Ala Val Trp Val Asp Val Glu
Thr Gln Val Pro 420 425 430 Gln Trp Val Ile Thr Ile Ile Ile Trp Leu
Phe Phe Leu Gln Cys Cys 435 440 445 Ile His Pro Tyr Val Tyr Gly Tyr
Met His Lys Thr Ile Lys Lys Glu 450 455 460 Ile Gln Asp Met Leu Lys
Lys Phe Phe Cys Lys Glu Lys Pro Pro Lys 465 470 475 480 Glu Asp Ser
His Pro Asp Leu Pro Gly Thr Glu Gly Gly Thr Glu Gly 485 490 495 Lys
Ile Val Pro Ser Tyr Asp Ser Ala Thr Phe Pro 500 505 17 1068 DNA
Homo sapiens 17 atgcccttga cggacggcat ttcttcattt gaggacctct
tggctaacaa tatcctcaga 60 atatttgtct gggttatagc tttcattacc
tgctttggaa atctttttgt cattggcatg 120 agatctttca ttaaagctga
aaatacaact cacgctatgt ccatcaaaat cctttgttgc 180 gctgattgcc
tgatgggtgt ttacttgttc tttgttggca ttttcgatat aaaataccga 240
gggcagtatc agaagtatgc cttgctgtgg atggagagcg tgcagtgccg
cctcatgggg 300 ttcctggcca tgctgtccac cgaagtctct gttctgctac
tgacctactt gactttggag 360 aagttcctgg tcattgtctt ccccttcagt
aacattcgac ctggaaaacg gcagacctca 420 gtcatcctca tttgcatctg
gatggcggga tttttaatag ctgtaattcc attttggaat 480 aaggattatt
ttggaaactt ttatgggaaa aatggagtat gtttcccact ttattatgac 540
caaacagaag atattggaag caaagggtat tctcttggaa ttttcctagg tgtgaacttg
600 ctggcttttc tcatcattgt gttttcctat attactatgt tctgttccat
tcaaaaaacc 660 gccttgcaga ccacagaagt aaggaattgt tttggaagag
aggtggctgt tgcaaatcgt 720 ttctttttta tagtgttctc tgatgccatc
tgctggattc ctgtatttgt agttaaaatc 780 ctttccctct tccgggtgga
aataccagac acaatgactt cctggatagt gatttttttc 840 cttccagtta
acagtgcttt gaatccaatc ctctatactc tcacaaccaa cttttttaag 900
gacaagttga aacagctgct gcacaaacat cagaggaaat caattttcaa aattaaaaaa
960 aaaagtttat ctacatccat tgtgtggata gaggactcct cttccctgaa
acttggggtt 1020 ttgaacaaaa taacacttgg agacagtata atgaaaccag
tttcctag 1068 18 355 PRT Homo sapiens 18 Met Pro Leu Thr Asp Gly
Ile Ser Ser Phe Glu Asp Leu Leu Ala Asn 1 5 10 15 Asn Ile Leu Arg
Ile Phe Val Trp Val Ile Ala Phe Ile Thr Cys Phe 20 25 30 Gly Asn
Leu Phe Val Ile Gly Met Arg Ser Phe Ile Lys Ala Glu Asn 35 40 45
Thr Thr His Ala Met Ser Ile Lys Ile Leu Cys Cys Ala Asp Cys Leu 50
55 60 Met Gly Val Tyr Leu Phe Phe Val Gly Ile Phe Asp Ile Lys Tyr
Arg 65 70 75 80 Gly Gln Tyr Gln Lys Tyr Ala Leu Leu Trp Met Glu Ser
Val Gln Cys 85 90 95 Arg Leu Met Gly Phe Leu Ala Met Leu Ser Thr
Glu Val Ser Val Leu 100 105 110 Leu Leu Thr Tyr Leu Thr Leu Glu Lys
Phe Leu Val Ile Val Phe Pro 115 120 125 Phe Ser Asn Ile Arg Pro Gly
Lys Arg Gln Thr Ser Val Ile Leu Ile 130 135 140 Cys Ile Trp Met Ala
Gly Phe Leu Ile Ala Val Ile Pro Phe Trp Asn 145 150 155 160 Lys Asp
Tyr Phe Gly Asn Phe Tyr Gly Lys Asn Gly Val Cys Phe Pro 165 170 175
Leu Tyr Tyr Asp Gln Thr Glu Asp Ile Gly Ser Lys Gly Tyr Ser Leu 180
185 190 Gly Ile Phe Leu Gly Val Asn Leu Leu Ala Phe Leu Ile Ile Val
Phe 195 200 205 Ser Tyr Ile Thr Met Phe Cys Ser Ile Gln Lys Thr Ala
Leu Gln Thr 210 215 220 Thr Glu Val Arg Asn Cys Phe Gly Arg Glu Val
Ala Val Ala Asn Arg 225 230 235 240 Phe Phe Phe Ile Val Phe Ser Asp
Ala Ile Cys Trp Ile Pro Val Phe 245 250 255 Val Val Lys Ile Leu Ser
Leu Phe Arg Val Glu Ile Pro Asp Thr Met 260 265 270 Thr Ser Trp Ile
Val Ile Phe Phe Leu Pro Val Asn Ser Ala Leu Asn 275 280 285 Pro Ile
Leu Tyr Thr Leu Thr Thr Asn Phe Phe Lys Asp Lys Leu Lys 290 295 300
Gln Leu Leu His Lys His Gln Arg Lys Ser Ile Phe Lys Ile Lys Lys 305
310 315 320 Lys Ser Leu Ser Thr Ser Ile Val Trp Ile Glu Asp Ser Ser
Ser Leu 325 330 335 Lys Leu Gly Val Leu Asn Lys Ile Thr Leu Gly Asp
Ser Ile Met Lys 340 345 350 Pro Val Ser 355 19 969 DNA Homo sapiens
19 atggatccaa ccatctcaac cttggacaca gaactgacac caatcaacgg
aactgaggag 60 actctttgct acaagcagac cttgagcctc acggtgctga
cgtgcatcgt ttcccttgtc 120 gggctgacag gaaacgcagt tgtgctctgg
ctcctgggct gccgcatgcg caggaacgcc 180 ttctccatct acatcctcaa
cttggccgca gcagacttcc tcttcctcag cggccgcctt 240 atatattccc
tgttaagctt catcagtatc ccccatacca tctctaaaat cctctatcct 300
gtgatgatgt tttcctactt tgcaggcctg agctttctga gtgccgtgag caccgagcgc
360 tgcctgtccg tcctgtggcc catctggtac cgctgccacc gccccacaca
cctgtcagcg 420 gtggtgtgtg tcctgctctg ggccctgtcc ctgctgcgga
gcatcctgga gtggatgtta 480 tgtggcttcc tgttcagtgg tgctgattct
gcttggtgtc aaacatcaga tttcatcaca 540 gtcgcgtggc tgattttttt
atgtgtggtt ctctgtgggt ccagcctggt cctgctgatc 600 aggattctct
gtggatcccg gaagataccg ctgaccaggc tgtacgtgac catcctgctc 660
acagtactgg tcttcctcct ctgtggcctg ccctttggca ttcagttttt cctattttta
720 tggatccacg tggacaggga agtcttattt tgtcatgttc atctagtttc
tattttcctg 780 tccgctctta acagcagtgc caaccccatc atttacttct
tcgtgggctc ctttaggcag 840 cgtcaaaata ggcagaacct gaagctggtt
ctccagaggg ctctgcagga cgcgtctgag 900 gtggatgaag gtggagggca
gcttcctgag gaaatcctgg agctgtcggg aagcagattg 960 gagcagtga 969 20
322 PRT Homo sapiens 20 Met Asp Pro Thr Ile Ser Thr Leu Asp Thr Glu
Leu Thr Pro Ile Asn 1 5 10 15 Gly Thr Glu Glu Thr Leu Cys Tyr Lys
Gln Thr Leu Ser Leu Thr Val 20 25 30 Leu Thr Cys Ile Val Ser Leu
Val Gly Leu Thr Gly Asn Ala Val Val 35 40 45 Leu Trp Leu Leu Gly
Cys Arg Met Arg Arg Asn Ala Phe Ser Ile Tyr 50 55 60 Ile Leu Asn
Leu Ala Ala Ala Asp Phe Leu Phe Leu Ser Gly Arg Leu 65 70 75 80 Ile
Tyr Ser Leu Leu Ser Phe Ile Ser Ile Pro His Thr Ile Ser Lys 85 90
95 Ile Leu Tyr Pro Val Met Met Phe Ser Tyr Phe Ala Gly Leu Ser Phe
100 105 110 Leu Ser Ala Val Ser Thr Glu Arg Cys Leu Ser Val Leu Trp
Pro Ile 115 120 125 Trp Tyr Arg Cys His Arg Pro Thr His Leu Ser Ala
Val Val Cys Val 130 135 140 Leu Leu Trp Ala Leu Ser Leu Leu Arg Ser
Ile Leu Glu Trp Met Leu 145 150 155 160 Cys Gly Phe Leu Phe Ser Gly
Ala Asp Ser Ala Trp Cys Gln Thr Ser 165 170 175 Asp Phe Ile Thr Val
Ala Trp Leu Ile Phe Leu Cys Val Val Leu Cys 180 185 190 Gly Ser Ser
Leu Val Leu Leu Ile Arg Ile Leu Cys Gly Ser Arg Lys 195 200 205 Ile
Pro Leu Thr Arg Leu Tyr Val Thr Ile Leu Leu Thr Val Leu Val 210 215
220 Phe Leu Leu Cys Gly Leu Pro Phe Gly Ile Gln Phe Phe Leu Phe Leu
225 230 235 240 Trp Ile His Val Asp Arg Glu Val Leu Phe Cys His Val
His Leu Val 245 250 255 Ser Ile Phe Leu Ser Ala Leu Asn Ser Ser Ala
Asn Pro Ile Ile Tyr 260 265 270 Phe Phe Val Gly Ser Phe Arg Gln Arg
Gln Asn Arg Gln Asn Leu Lys 275 280 285 Leu Val Leu Gln Arg Ala Leu
Gln Asp Ala Ser Glu Val Asp Glu Gly 290 295 300 Gly Gly Gln Leu Pro
Glu Glu Ile Leu Glu Leu Ser Gly Ser Arg Leu 305 310 315 320 Glu Gln
21 1305 DNA Homo sapiens 21 atggaggatc tctttagccc ctcaattctg
ccgccggcgc ccaacatttc cgtgcccatc 60 ttgctgggct ggggtctcaa
cctgaccttg gggcaaggag cccctgcctc tgggccgccc 120 agccgccgcg
tccgcctggt gttcctgggg gtcatcctgg tggtggcggt ggcaggcaac 180
accacagtgc tgtgccgcct gtgcggcggc ggcgggccct gggcgggccc caagcgtcgc
240 aagatggact tcctgctggt gcagctggcc ctggcggacc tgtacgcgtg
cgggggcacg 300 gcgctgtcac agctggcctg ggaactgctg ggcgagcccc
gcgcggccac gggggacctg 360 gcgtgccgct tcctgcagct gctgcaggca
tccgggcggg gcgcctcggc ccacctcgtg 420 gtgctcatcg ccctcgagcg
ccggcgcgcg gtgcgtcttc cgcacggccg gccgctgccc 480 gcgcgtgccc
tcgccgccct gggctggctg ctggcactgc tgctggcgct gcccccggcc 540
ttcgtggtgc gcggggactc cccctcgccg ctgccgccgc cgccgccgcc aacgtccctg
600 cagccaggcg cgcccccggc cgcccgcgcc tggccggggg agcgtcgctg
ccacgggatc 660 ttcgcgcccc tgccgcgctg gcacctgcag gtctacgcgt
tctacgaggc cgtcgcgggc 720 ttcgtcgcgc ctgttacggt cctgggcgtc
gcttgcggcc acctactctc cgtctggtgg 780 cggcaccggc cgcaggcccc
cgcggctgca gcgccctggt cggcgagccc aggtcgagcc 840 cctgcgccca
gcgcgctgcc ccgcgccaag gtgcagagcc tgaagatgag cctgctgctg 900
gcgctgctgt tcgtgggctg cgagctgccc tactttgccg cccggctggc ggccgcgtgg
960 tcgtccgggc ccgcgggaga ctgggaggga gagggcctgt cggcggcgct
gcgcgtggtg 1020 gcgatggcca acagcgctct caatcccttc gtctacctct
tcttccaggc gggcgactgc 1080 cggctccggc gacagctgcg gaagcggctg
ggctctctgt gctgcgcgcc gcagggaggc 1140 gcggaggacg aggaggggcc
ccggggccac caggcgctct accgccaacg ctggccccac 1200 cctcattatc
accatgctcg gcgggaaccg ctggacgagg gcggcttgcg cccaccccct 1260
ccgcgcccca gacccctgcc ttgctcctgc gaaagtgcct tctag 1305 22 434 PRT
Homo sapiens 22 Met Glu Asp Leu Phe Ser Pro Ser Ile Leu Pro Pro Ala
Pro Asn Ile 1 5 10 15 Ser Val Pro Ile Leu Leu Gly Trp Gly Leu Asn
Leu Thr Leu Gly Gln 20 25 30 Gly Ala Pro Ala Ser Gly Pro Pro Ser
Arg Arg Val Arg Leu Val Phe 35 40 45 Leu Gly Val Ile Leu Val Val
Ala Val Ala Gly Asn Thr Thr Val Leu 50 55 60 Cys Arg Leu Cys Gly
Gly Gly Gly Pro Trp Ala Gly Pro Lys Arg Arg 65 70 75 80 Lys Met Asp
Phe Leu Leu Val Gln Leu Ala Leu Ala Asp Leu Tyr Ala 85 90 95 Cys
Gly Gly Thr Ala Leu Ser Gln Leu Ala Trp Glu Leu Leu Gly Glu 100 105
110 Pro Arg Ala Ala Thr Gly Asp Leu Ala Cys Arg Phe Leu Gln Leu Leu
115 120 125 Gln Ala Ser Gly Arg Gly Ala Ser Ala His Leu Val Val Leu
Ile Ala 130 135 140 Leu Glu Arg Arg Arg Ala Val Arg Leu Pro His Gly
Arg Pro Leu Pro 145 150 155 160 Ala Arg Ala Leu Ala Ala Leu Gly Trp
Leu Leu Ala Leu Leu Leu Ala 165 170 175 Leu Pro Pro Ala Phe Val Val
Arg Gly Asp Ser Pro Ser Pro Leu Pro 180 185 190 Pro Pro Pro Pro Pro
Thr Ser Leu Gln Pro Gly Ala Pro Pro Ala Ala 195 200 205 Arg Ala Trp
Pro Gly Glu Arg Arg Cys His Gly Ile Phe Ala Pro Leu 210 215 220 Pro
Arg Trp His Leu Gln Val Tyr Ala Phe Tyr Glu Ala Val Ala Gly 225 230
235 240 Phe Val Ala Pro Val Thr Val Leu Gly Val Ala Cys Gly His Leu
Leu 245 250 255 Ser Val Trp Trp Arg His Arg Pro Gln Ala Pro Ala Ala
Ala Ala Pro 260 265 270 Trp Ser Ala Ser Pro Gly Arg Ala Pro Ala Pro
Ser Ala Leu Pro Arg 275 280 285 Ala Lys Val Gln Ser Leu Lys Met Ser
Leu Leu Leu Ala Leu Leu Phe 290 295 300 Val Gly Cys Glu Leu Pro Tyr
Phe Ala Ala Arg Leu Ala Ala Ala Trp 305 310 315 320 Ser Ser Gly Pro
Ala Gly Asp Trp Glu Gly Glu Gly Leu Ser Ala Ala 325 330 335 Leu Arg
Val Val Ala Met Ala Asn Ser Ala Leu Asn Pro Phe Val Tyr 340 345 350
Leu Phe Phe Gln Ala Gly Asp Cys Arg Leu Arg Arg Gln Leu Arg Lys 355
360 365 Arg Leu Gly Ser Leu Cys Cys Ala Pro Gln Gly Gly Ala Glu Asp
Glu 370 375 380 Glu Gly Pro Arg Gly His Gln Ala Leu Tyr Arg Gln Arg
Trp Pro His 385 390 395 400 Pro His Tyr His His Ala Arg Arg Glu Pro
Leu Asp Glu Gly Gly Leu 405 410 415 Arg Pro Pro Pro Pro Arg Pro Arg
Pro Leu Pro Cys Ser Cys Glu Ser 420 425 430 Ala Phe 23 1041 DNA
Homo sapiens 23 atgtacaacg ggtcgtgctg ccgcatcgag ggggacacca
tctcccaggt gatgccgccg 60 ctgctcattg tggcctttgt gctgggcgca
ctaggcaatg gggtcgccct gtgtggtttc 120 tgcttccaca tgaagacctg
gaagcccagc actgtttacc ttttcaattt ggccgtggct 180 gatttcctcc
ttatgatctg cctgcctttt cggacagact attacctcag acgtagacac 240
tgggcttttg gggacattcc ctgccgagtg gggctcttca cgttggccat gaacagggcc
300 gggagcatcg tgttccttac ggtggtggct gcggacaggt atttcaaagt
ggtccacccc 360 caccacgcgg tgaacactat ctccacccgg gtggcggctg
gcatcgtctg caccctgtgg 420 gccctggtca tcctgggaac agtgtatctt
ttgctggaga accatctctg cgtgcaagag 480 acggccgtct cctgtgagag
cttcatcatg gagtcggcca atggctggca tgacatcatg 540 ttccagctgg
agttctttat gcccctcggc atcatcttat tttgctcctt caagattgtt 600
tggagcctga ggcggaggca gcagctggcc agacaggctc ggatgaagaa ggcgacccgg
660 ttcatcatgg tggtggcaat tgtgttcatc acatgctacc tgcccagcgt
gtctgctaga 720 ctctatttcc tctggacggt gccctcgagt gcctgcgatc
cctctgtcca tggggccctg 780 cacataaccc tcagcttcac ctacatgaac
agcatgctgg atcccctggt gtattatttt 840 tcaagcccct cctttcccaa
attctacaac aagctcaaaa tctgcagtct gaaacccaag 900 cagccaggac
actcaaaaac acaaaggccg gaagagatgc caatttcgaa cctcggtcgc 960
aggagttgca tcagtgtggc aaatagtttc caaagccagt ctgatgggca atgggatccc
1020 cacattgttg agtggcactg a 1041 24 346 PRT Homo sapiens 24 Met
Tyr Asn Gly Ser Cys Cys Arg Ile Glu Gly Asp Thr Ile Ser Gln 1 5 10
15 Val Met Pro Pro Leu Leu Ile Val Ala Phe Val Leu Gly Ala Leu Gly
20 25 30 Asn Gly Val Ala Leu Cys Gly Phe Cys Phe His Met Lys Thr
Trp Lys 35 40 45 Pro Ser Thr Val Tyr Leu Phe Asn Leu Ala Val Ala
Asp Phe Leu Leu 50 55 60 Met Ile Cys Leu Pro Phe Arg Thr Asp Tyr
Tyr Leu Arg Arg Arg His 65 70 75 80 Trp Ala Phe Gly Asp Ile Pro Cys
Arg Val Gly Leu Phe Thr Leu Ala 85 90 95 Met Asn Arg Ala Gly Ser
Ile Val Phe Leu Thr Val Val Ala Ala Asp 100 105 110 Arg Tyr Phe Lys
Val Val His Pro His His Ala Val Asn Thr Ile Ser 115 120 125 Thr Arg
Val Ala Ala Gly Ile Val Cys Thr Leu Trp Ala Leu Val Ile 130 135 140
Leu Gly Thr Val Tyr Leu Leu Leu Glu Asn His Leu Cys Val Gln Glu 145
150 155 160 Thr Ala Val Ser Cys Glu Ser Phe Ile Met Glu Ser Ala Asn
Gly Trp 165 170 175 His Asp Ile Met Phe Gln Leu Glu Phe Phe Met Pro
Leu Gly Ile Ile 180 185 190 Leu Phe Cys Ser Phe Lys Ile Val Trp Ser
Leu Arg Arg Arg Gln Gln 195 200 205 Leu Ala Arg Gln Ala Arg Met Lys
Lys Ala Thr Arg Phe Ile Met Val 210 215 220 Val Ala Ile Val Phe Ile
Thr Cys Tyr Leu Pro Ser Val Ser Ala Arg 225 230 235 240 Leu Tyr Phe
Leu Trp Thr Val Pro Ser Ser Ala Cys Asp Pro Ser Val 245 250 255 His
Gly Ala Leu His Ile Thr Leu Ser Phe Thr Tyr Met Asn Ser Met 260 265
270 Leu Asp Pro Leu Val Tyr Tyr Phe Ser Ser Pro Ser Phe Pro Lys Phe
275 280 285 Tyr Asn Lys Leu Lys Ile Cys Ser Leu Lys Pro Lys Gln Pro
Gly His 290 295 300 Ser Lys Thr Gln Arg Pro Glu Glu Met Pro Ile Ser
Asn Leu Gly Arg 305 310 315 320 Arg Ser Cys Ile Ser Val Ala Asn Ser
Phe Gln Ser Gln Ser Asp Gly 325 330 335 Gln Trp Asp Pro His Ile Val
Glu Trp His 340 345 25 1011 DNA Homo sapiens 25 atgaacaaca
atacaacatg tattcaacca tctatgatct cttccatggc tttaccaatc 60
atttacatcc tcctttgtat tgttggtgtt tttggaaaca ctctctctca atggatattt
120 ttaacaaaaa taggtaaaaa aacatcaacg cacatctacc tgtcacacct
tgtgactgca 180 aacttacttg tgtgcagtgc catgcctttc atgagtatct
atttcctgaa aggtttccaa 240 tgggaatatc aatctgctca atgcagagtg
gtcaattttc tgggaactct atccatgcat 300 gcaagtatgt ttgtcagtct
cttaatttta agttggattg ccataagccg ctatgctacc 360 ttaatgcaaa
aggattcctc gcaagagact acttcatgct atgagaaaat attttatggc 420
catttactga aaaaatttcg ccagcccaac tttgctagaa aactatgcat ttacatatgg
480 ggagttgtac tgggcataat cattccagtt accgtatact actcagtcat
agaggctaca 540 gaaggagaag agagcctatg ctacaatcgg cagatggaac
taggagccat gatctctcag 600 attgcaggtc tcattggaac cacatttatt
ggattttcct ttttagtagt actaacatca 660 tactactctt ttgtaagcca
tctgagaaaa ataagaacct gtacgtccat tatggagaaa 720 gatttgactt
acagttctgt gaaaagacat cttttggtca tccagattct actaatagtt 780
tgcttccttc cttatagtat ttttaaaccc attttttatg ttctacacca aagagataac
840 tgtcagcaat tgaattattt aatagaaaca aaaaacattc tcacctgtct
tgcttcggcc 900 agaagtagca cagaccccat tatatttctt ttattagata
aaacattcaa gaagacacta 960 tataatctct ttacaaagtc taattcagca
catatgcaat catatggttg a 1011 26 336 PRT Homo sapiens 26 Met Asn Asn
Asn Thr Thr Cys Ile Gln Pro Ser Met Ile Ser Ser Met 1 5 10 15 Ala
Leu Pro Ile Ile Tyr Ile Leu Leu Cys Ile Val Gly Val Phe Gly 20 25
30 Asn Thr Leu Ser Gln Trp Ile Phe Leu Thr Lys Ile Gly Lys Lys Thr
35 40 45 Ser Thr His Ile Tyr Leu Ser His Leu Val Thr Ala Asn Leu
Leu Val 50 55 60 Cys Ser Ala Met Pro Phe Met Ser Ile Tyr Phe Leu
Lys Gly Phe Gln 65 70 75 80 Trp Glu Tyr Gln Ser Ala Gln Cys Arg Val
Val Asn Phe Leu Gly Thr 85 90 95 Leu Ser Met His Ala Ser Met Phe
Val Ser Leu Leu Ile Leu Ser Trp
100 105 110 Ile Ala Ile Ser Arg Tyr Ala Thr Leu Met Gln Lys Asp Ser
Ser Gln 115 120 125 Glu Thr Thr Ser Cys Tyr Glu Lys Ile Phe Tyr Gly
His Leu Leu Lys 130 135 140 Lys Phe Arg Gln Pro Asn Phe Ala Arg Lys
Leu Cys Ile Tyr Ile Trp 145 150 155 160 Gly Val Val Leu Gly Ile Ile
Ile Pro Val Thr Val Tyr Tyr Ser Val 165 170 175 Ile Glu Ala Thr Glu
Gly Glu Glu Ser Leu Cys Tyr Asn Arg Gln Met 180 185 190 Glu Leu Gly
Ala Met Ile Ser Gln Ile Ala Gly Leu Ile Gly Thr Thr 195 200 205 Phe
Ile Gly Phe Ser Phe Leu Val Val Leu Thr Ser Tyr Tyr Ser Phe 210 215
220 Val Ser His Leu Arg Lys Ile Arg Thr Cys Thr Ser Ile Met Glu Lys
225 230 235 240 Asp Leu Thr Tyr Ser Ser Val Lys Arg His Leu Leu Val
Ile Gln Ile 245 250 255 Leu Leu Ile Val Cys Phe Leu Pro Tyr Ser Ile
Phe Lys Pro Ile Phe 260 265 270 Tyr Val Leu His Gln Arg Asp Asn Cys
Gln Gln Leu Asn Tyr Leu Ile 275 280 285 Glu Thr Lys Asn Ile Leu Thr
Cys Leu Ala Ser Ala Arg Ser Ser Thr 290 295 300 Asp Pro Ile Ile Phe
Leu Leu Leu Asp Lys Thr Phe Lys Lys Thr Leu 305 310 315 320 Tyr Asn
Leu Phe Thr Lys Ser Asn Ser Ala His Met Gln Ser Tyr Gly 325 330 335
27 1014 DNA Homo sapiens 27 atgaatgagc cactagacta tttagcaaat
gcttctgatt tccccgatta tgcagctgct 60 tttggaaatt gcactgatga
aaacatccca ctcaagatgc actacctccc tgttatttat 120 ggcattatct
tcctcgtggg atttccaggc aatgcagtag tgatatccac ttacattttc 180
aaaatgagac cttggaagag cagcaccatc attatgctga acctggcctg cacagatctg
240 ctgtatctga ccagcctccc cttcctgatt cactactatg ccagtggcga
aaactggatc 300 tttggagatt tcatgtgtaa gtttatccgc ttcagcttcc
atttcaacct gtatagcagc 360 atcctcttcc tcacctgttt cagcatcttc
cgctactgtg tgatcattca cccaatgagc 420 tgcttttcca ttcacaaaac
tcgatgtgca gttgtagcct gtgctgtggt gtggatcatt 480 tcactggtag
ctgtcattcc gatgaccttc ttgatcacat caaccaacag gaccaacaga 540
tcagcctgtc tcgacctcac cagttcggat gaactcaata ctattaagtg gtacaacctg
600 attttgactg caactacttt ctgcctcccc ttggtgatag tgacactttg
ctataccacg 660 attatccaca ctctgaccca tggactgcaa actgacagct
gccttaagca gaaagcacga 720 aggctaacca ttctgctact ccttgcattt
tacgtatgtt ttttaccctt ccatatcttg 780 agggtcattc ggatcgaatc
tcgcctgctt tcaatcagtt gttccattga gaatcagatc 840 catgaagctt
acatcgtttc tagaccatta gctgctctga acacctttgg taacctgtta 900
ctatatgtgg tggtcagcga caactttcag caggctgtct gctcaacagt gagatgcaaa
960 gtaagcggga accttgagca agcaaagaaa attagttact caaacaaccc ttga
1014 28 337 PRT Homo sapiens 28 Met Asn Glu Pro Leu Asp Tyr Leu Ala
Asn Ala Ser Asp Phe Pro Asp 1 5 10 15 Tyr Ala Ala Ala Phe Gly Asn
Cys Thr Asp Glu Asn Ile Pro Leu Lys 20 25 30 Met His Tyr Leu Pro
Val Ile Tyr Gly Ile Ile Phe Leu Val Gly Phe 35 40 45 Pro Gly Asn
Ala Val Val Ile Ser Thr Tyr Ile Phe Lys Met Arg Pro 50 55 60 Trp
Lys Ser Ser Thr Ile Ile Met Leu Asn Leu Ala Cys Thr Asp Leu 65 70
75 80 Leu Tyr Leu Thr Ser Leu Pro Phe Leu Ile His Tyr Tyr Ala Ser
Gly 85 90 95 Glu Asn Trp Ile Phe Gly Asp Phe Met Cys Lys Phe Ile
Arg Phe Ser 100 105 110 Phe His Phe Asn Leu Tyr Ser Ser Ile Leu Phe
Leu Thr Cys Phe Ser 115 120 125 Ile Phe Arg Tyr Cys Val Ile Ile His
Pro Met Ser Cys Phe Ser Ile 130 135 140 His Lys Thr Arg Cys Ala Val
Val Ala Cys Ala Val Val Trp Ile Ile 145 150 155 160 Ser Leu Val Ala
Val Ile Pro Met Thr Phe Leu Ile Thr Ser Thr Asn 165 170 175 Arg Thr
Asn Arg Ser Ala Cys Leu Asp Leu Thr Ser Ser Asp Glu Leu 180 185 190
Asn Thr Ile Lys Trp Tyr Asn Leu Ile Leu Thr Ala Thr Thr Phe Cys 195
200 205 Leu Pro Leu Val Ile Val Thr Leu Cys Tyr Thr Thr Ile Ile His
Thr 210 215 220 Leu Thr His Gly Leu Gln Thr Asp Ser Cys Leu Lys Gln
Lys Ala Arg 225 230 235 240 Arg Leu Thr Ile Leu Leu Leu Leu Ala Phe
Tyr Val Cys Phe Leu Pro 245 250 255 Phe His Ile Leu Arg Val Ile Arg
Ile Glu Ser Arg Leu Leu Ser Ile 260 265 270 Ser Cys Ser Ile Glu Asn
Gln Ile His Glu Ala Tyr Ile Val Ser Arg 275 280 285 Pro Leu Ala Ala
Leu Asn Thr Phe Gly Asn Leu Leu Leu Tyr Val Val 290 295 300 Val Ser
Asp Asn Phe Gln Gln Ala Val Cys Ser Thr Val Arg Cys Lys 305 310 315
320 Val Ser Gly Asn Leu Glu Gln Ala Lys Lys Ile Ser Tyr Ser Asn Asn
325 330 335 Pro 29 993 DNA Homo sapiens 29 atggatccaa ccaccccggc
ctggggaaca gaaagtacaa cagtgaatgg aaatgaccaa 60 gcccttcttc
tgctttgtgg caaggagacc ctgatcccgg tcttcctgat ccttttcatt 120
gccctggtcg ggctggtagg aaacgggttt gtgctctggc tcctgggctt ccgcatgcgc
180 aggaacgcct tctctgtcta cgtcctcagc ctggccgggg ccgacttcct
cttcctctgc 240 ttccagatta taaattgcct ggtgtacctc agtaacttct
tctgttccat ctccatcaat 300 ttccctagct tcttcaccac tgtgatgacc
tgtgcctacc ttgcaggcct gagcatgctg 360 agcaccgtca gcaccgagcg
ctgcctgtcc gtcctgtggc ccatctggta tcgctgccgc 420 cgccccagac
acctgtcagc ggtcgtgtgt gtcctgctct gggccctgtc cctactgctg 480
agcatcttgg aagggaagtt ctgtggcttc ttatttagtg atggtgactc tggttggtgt
540 cagacatttg atttcatcac tgcagcgtgg ctgatttttt tattcatggt
tctctgtggg 600 tccagtctgg ccctgctggt caggatcctc tgtggctcca
ggggtctgcc actgaccagg 660 ctgtacctga ccatcctgct cacagtgctg
gtgttcctcc tctgcggcct gccctttggc 720 attcagtggt tcctaatatt
atggatctgg aaggattctg atgtcttatt ttgtcatatt 780 catccagttt
cagttgtcct gtcatctctt aacagcagtg ccaaccccat catttacttc 840
ttcgtgggct cttttaggaa gcagtggcgg ctgcagcagc cgatcctcaa gctggctctc
900 cagagggctc tgcaggacat tgctgaggtg gatcacagtg aaggatgctt
ccgtcagggc 960 accccggaga tgtcgagaag cagtctggtg tag 993 30 330 PRT
Homo sapiens 30 Met Asp Pro Thr Thr Pro Ala Trp Gly Thr Glu Ser Thr
Thr Val Asn 1 5 10 15 Gly Asn Asp Gln Ala Leu Leu Leu Leu Cys Gly
Lys Glu Thr Leu Ile 20 25 30 Pro Val Phe Leu Ile Leu Phe Ile Ala
Leu Val Gly Leu Val Gly Asn 35 40 45 Gly Phe Val Leu Trp Leu Leu
Gly Phe Arg Met Arg Arg Asn Ala Phe 50 55 60 Ser Val Tyr Val Leu
Ser Leu Ala Gly Ala Asp Phe Leu Phe Leu Cys 65 70 75 80 Phe Gln Ile
Ile Asn Cys Leu Val Tyr Leu Ser Asn Phe Phe Cys Ser 85 90 95 Ile
Ser Ile Asn Phe Pro Ser Phe Phe Thr Thr Val Met Thr Cys Ala 100 105
110 Tyr Leu Ala Gly Leu Ser Met Leu Ser Thr Val Ser Thr Glu Arg Cys
115 120 125 Leu Ser Val Leu Trp Pro Ile Trp Tyr Arg Cys Arg Arg Pro
Arg His 130 135 140 Leu Ser Ala Val Val Cys Val Leu Leu Trp Ala Leu
Ser Leu Leu Leu 145 150 155 160 Ser Ile Leu Glu Gly Lys Phe Cys Gly
Phe Leu Phe Ser Asp Gly Asp 165 170 175 Ser Gly Trp Cys Gln Thr Phe
Asp Phe Ile Thr Ala Ala Trp Leu Ile 180 185 190 Phe Leu Phe Met Val
Leu Cys Gly Ser Ser Leu Ala Leu Leu Val Arg 195 200 205 Ile Leu Cys
Gly Ser Arg Gly Leu Pro Leu Thr Arg Leu Tyr Leu Thr 210 215 220 Ile
Leu Leu Thr Val Leu Val Phe Leu Leu Cys Gly Leu Pro Phe Gly 225 230
235 240 Ile Gln Trp Phe Leu Ile Leu Trp Ile Trp Lys Asp Ser Asp Val
Leu 245 250 255 Phe Cys His Ile His Pro Val Ser Val Val Leu Ser Ser
Leu Asn Ser 260 265 270 Ser Ala Asn Pro Ile Ile Tyr Phe Phe Val Gly
Ser Phe Arg Lys Gln 275 280 285 Trp Arg Leu Gln Gln Pro Ile Leu Lys
Leu Ala Leu Gln Arg Ala Leu 290 295 300 Gln Asp Ile Ala Glu Val Asp
His Ser Glu Gly Cys Phe Arg Gln Gly 305 310 315 320 Thr Pro Glu Met
Ser Arg Ser Ser Leu Val 325 330 31 1092 DNA Homo sapiens 31
atgggccccg gcgaggcgct gctggcgggt ctcctggtga tggtactggc cgtggcgctg
60 ctatccaacg cactggtgct gctttgttgc gcctacagcg ctgagctccg
cactcgagcc 120 tcaggcgtcc tcctggtgaa tctgtcgctg ggccacctgc
tgctggcggc gctggacatg 180 cccttcacgc tgctcggtgt gatgcgcggg
cggacaccgt cggcgcccgg cgcatgccaa 240 gtcattggct tcctggacac
cttcctggcg tccaacgcgg cgctgagcgt ggcggcgctg 300 agcgcagacc
agtggctggc agtgggcttc ccactgcgct acgccggacg cctgcgaccg 360
cgctatgccg gcctgctgct gggctgtgcc tggggacagt cgctggcctt ctcaggcgct
420 gcacttggct gctcgtggct tggctacagc agcgccttcg cgtcctgttc
gctgcgcctg 480 ccgcccgagc ctgagcgtcc gcgcttcgca gccttcaccg
ccacgctcca tgccgtgggc 540 ttcgtgctgc cgctggcggt gctctgcctc
acctcgctcc aggtgcaccg ggtggcacgc 600 agccactgcc agcgcatgga
caccgtcacc atgaaggcgc tcgcgctgct cgccgacctg 660 caccccagtg
tgcggcagcg ctgcctcatc cagcagaagc ggcgccgcca ccgcgccacc 720
aggaagattg gcattgctat tgcgaccttc ctcatctgct ttgccccgta tgtcatgacc
780 aggctggcgg agctcgtgcc cttcgtcacc gtgaacgccc agtggggcat
cctcagcaag 840 tgcctgacct acagcaaggc ggtggccgac ccgttcacgt
actctctgct ccgccggccg 900 ttccgccaag tcctggccgg catggtgcac
cggctgctga agagaacccc gcgcccagca 960 tccacccatg acagctctct
ggatgtggcc ggcatggtgc accagctgct gaagagaacc 1020 ccgcgcccag
cgtccaccca caacggctct gtggacacag agaatgattc ctgcctgcag 1080
cagacacact ga 1092 32 363 PRT Homo sapiens 32 Met Gly Pro Gly Glu
Ala Leu Leu Ala Gly Leu Leu Val Met Val Leu 1 5 10 15 Ala Val Ala
Leu Leu Ser Asn Ala Leu Val Leu Leu Cys Cys Ala Tyr 20 25 30 Ser
Ala Glu Leu Arg Thr Arg Ala Ser Gly Val Leu Leu Val Asn Leu 35 40
45 Ser Leu Gly His Leu Leu Leu Ala Ala Leu Asp Met Pro Phe Thr Leu
50 55 60 Leu Gly Val Met Arg Gly Arg Thr Pro Ser Ala Pro Gly Ala
Cys Gln 65 70 75 80 Val Ile Gly Phe Leu Asp Thr Phe Leu Ala Ser Asn
Ala Ala Leu Ser 85 90 95 Val Ala Ala Leu Ser Ala Asp Gln Trp Leu
Ala Val Gly Phe Pro Leu 100 105 110 Arg Tyr Ala Gly Arg Leu Arg Pro
Arg Tyr Ala Gly Leu Leu Leu Gly 115 120 125 Cys Ala Trp Gly Gln Ser
Leu Ala Phe Ser Gly Ala Ala Leu Gly Cys 130 135 140 Ser Trp Leu Gly
Tyr Ser Ser Ala Phe Ala Ser Cys Ser Leu Arg Leu 145 150 155 160 Pro
Pro Glu Pro Glu Arg Pro Arg Phe Ala Ala Phe Thr Ala Thr Leu 165 170
175 His Ala Val Gly Phe Val Leu Pro Leu Ala Val Leu Cys Leu Thr Ser
180 185 190 Leu Gln Val His Arg Val Ala Arg Ser His Cys Gln Arg Met
Asp Thr 195 200 205 Val Thr Met Lys Ala Leu Ala Leu Leu Ala Asp Leu
His Pro Ser Val 210 215 220 Arg Gln Arg Cys Leu Ile Gln Gln Lys Arg
Arg Arg His Arg Ala Thr 225 230 235 240 Arg Lys Ile Gly Ile Ala Ile
Ala Thr Phe Leu Ile Cys Phe Ala Pro 245 250 255 Tyr Val Met Thr Arg
Leu Ala Glu Leu Val Pro Phe Val Thr Val Asn 260 265 270 Ala Gln Trp
Gly Ile Leu Ser Lys Cys Leu Thr Tyr Ser Lys Ala Val 275 280 285 Ala
Asp Pro Phe Thr Tyr Ser Leu Leu Arg Arg Pro Phe Arg Gln Val 290 295
300 Leu Ala Gly Met Val His Arg Leu Leu Lys Arg Thr Pro Arg Pro Ala
305 310 315 320 Ser Thr His Asp Ser Ser Leu Asp Val Ala Gly Met Val
His Gln Leu 325 330 335 Leu Lys Arg Thr Pro Arg Pro Ala Ser Thr His
Asn Gly Ser Val Asp 340 345 350 Thr Glu Asn Asp Ser Cys Leu Gln Gln
Thr His 355 360 33 1125 DNA Homo sapiens 33 atgcccacac tcaatacttc
tgcctctcca cccacattct tctgggccaa tgcctccgga 60 ggcagtgtgc
tgagtgctga tgatgctccg atgcctgtca aattcctagc cctgaggctc 120
atggttgccc tggcctatgg gcttgtgggg gccattggct tgctgggaaa tttggcggtg
180 ctgtgggtac tgagtaactg tgcccggaga gcccctggcc caccttcaga
caccttcgtc 240 ttcaacctgg ctctggcgga cctgggactg gcactcactc
tccccttttg ggcagccgag 300 tcggcactgg actttcactg gcccttcgga
ggtgccctct gcaagatggt tctgacggcc 360 actgtcctca acgtctatgc
cagcatcttc ctcatcacag cgctgagcgt tgctcgctac 420 tgggtggtgg
ccatggctgc ggggccaggc acccacctct cactcttctg ggcccgaata 480
gccaccctgg cagtgtgggc ggcggctgcc ctggtgacgg tgcccacagc tgtcttcggg
540 gtggagggtg aggtgtgtgg tgtgcgcctt tgcctgctgc gtttccccag
caggtactgg 600 ctgggggcct accagctgca gagggtggtg ctggctttca
tggtgccctt gggcgtcatc 660 accaccagct acctgctgct gctggccttc
ctgcagcggc ggcaacggcg gcggcaggac 720 agcagggtcg tggcccgctc
tgtccgcatc ctggtggctt ccttcttcct ctgctggttt 780 cccaaccatg
tggtcactct ctggggtgtc ctggtgaagt ttgacctggt gccctggaac 840
agtactttct atactatcca gacgtatgtc ttccctgtca ctacttgctt ggcacacagc
900 aatagctgcc tcaaccctgt gctgtactgt ctcctgaggc gggagccccg
gcaggctctg 960 gcaggcacct tcagggatct gcggtcgagg ctgtggcccc
agggcggagg ctgggtgcaa 1020 caggtggccc taaagcaggt aggcaggcgg
tgggtcgcaa gcaacccccg ggagagccgc 1080 ccttctaccc tgctcaccaa
cctggacaga gggacacccg ggtga 1125 34 374 PRT Homo sapiens 34 Met Pro
Thr Leu Asn Thr Ser Ala Ser Pro Pro Thr Phe Phe Trp Ala 1 5 10 15
Asn Ala Ser Gly Gly Ser Val Leu Ser Ala Asp Asp Ala Pro Met Pro 20
25 30 Val Lys Phe Leu Ala Leu Arg Leu Met Val Ala Leu Ala Tyr Gly
Leu 35 40 45 Val Gly Ala Ile Gly Leu Leu Gly Asn Leu Ala Val Leu
Trp Val Leu 50 55 60 Ser Asn Cys Ala Arg Arg Ala Pro Gly Pro Pro
Ser Asp Thr Phe Val 65 70 75 80 Phe Asn Leu Ala Leu Ala Asp Leu Gly
Leu Ala Leu Thr Leu Pro Phe 85 90 95 Trp Ala Ala Glu Ser Ala Leu
Asp Phe His Trp Pro Phe Gly Gly Ala 100 105 110 Leu Cys Lys Met Val
Leu Thr Ala Thr Val Leu Asn Val Tyr Ala Ser 115 120 125 Ile Phe Leu
Ile Thr Ala Leu Ser Val Ala Arg Tyr Trp Val Val Ala 130 135 140 Met
Ala Ala Gly Pro Gly Thr His Leu Ser Leu Phe Trp Ala Arg Ile 145 150
155 160 Ala Thr Leu Ala Val Trp Ala Ala Ala Ala Leu Val Thr Val Pro
Thr 165 170 175 Ala Val Phe Gly Val Glu Gly Glu Val Cys Gly Val Arg
Leu Cys Leu 180 185 190 Leu Arg Phe Pro Ser Arg Tyr Trp Leu Gly Ala
Tyr Gln Leu Gln Arg 195 200 205 Val Val Leu Ala Phe Met Val Pro Leu
Gly Val Ile Thr Thr Ser Tyr 210 215 220 Leu Leu Leu Leu Ala Phe Leu
Gln Arg Arg Gln Arg Arg Arg Gln Asp 225 230 235 240 Ser Arg Val Val
Ala Arg Ser Val Arg Ile Leu Val Ala Ser Phe Phe 245 250 255 Leu Cys
Trp Phe Pro Asn His Val Val Thr Leu Trp Gly Val Leu Val 260 265 270
Lys Phe Asp Leu Val Pro Trp Asn Ser Thr Phe Tyr Thr Ile Gln Thr 275
280 285 Tyr Val Phe Pro Val Thr Thr Cys Leu Ala His Ser Asn Ser Cys
Leu 290 295 300 Asn Pro Val Leu Tyr Cys Leu Leu Arg Arg Glu Pro Arg
Gln Ala Leu 305 310 315 320 Ala Gly Thr Phe Arg Asp Leu Arg Ser Arg
Leu Trp Pro Gln Gly Gly 325 330 335 Gly Trp Val Gln Gln Val Ala Leu
Lys Gln Val Gly Arg Arg Trp Val 340 345 350 Ala Ser Asn Pro Arg Glu
Ser Arg Pro Ser Thr Leu Leu Thr Asn Leu 355 360 365 Asp Arg Gly Thr
Pro Gly 370 35 1092 DNA Homo sapiens 35 atgaatcggc accatctgca
ggatcacttt ctggaaatag acaagaagaa ctgctgtgtg 60 ttccgagatg
acttcattgt caaggtgttg ccgccggtgt tggggctgga gtttatcttc 120
gggcttctgg gcaatggcct tgccctgtgg attttctgtt tccacctcaa gtcctggaaa
180 tccagccgga ttttcctgtt caacctggca gtggctgact ttctactgat
catctgcctg 240 cccttcctga tggacaacta tgtgaggcgt tgggactgga
agtttgggga catcccttgc 300 cggctgatgc tcttcatgtt ggctatgaac
cgccagggca gcatcatctt cctcacggtg 360 gtggcggtag acaggtattt
ccgggtggtc catccccacc acgccctgaa caagatctcc 420 aatcggacag
cagccatcat ctcttgcctt ctgtggggca tcactattgg cctgacagtc 480
cacctcctga
agaagaagat gccgatccag aatggcggtg caaatttgtg cagcagcttc 540
agcatctgcc ataccttcca gtggcacgaa gccatgttcc tcctggagtt cttcctgccc
600 ctgggcatca tcctgttctg ctcagccaga attatctgga gcctgcggca
gagacaaatg 660 gaccggcatg ccaagatcaa gagagccatc accttcatca
tggtggtggc catcgtcttt 720 gtcatctgct tccttcccag cgtggttgtg
cggatccgca tcttctggct cctgcacact 780 tcgggcacgc agaattgtga
agtgtaccgc tcggtggacc tggcgttctt tatcactctc 840 agcttcacct
acatgaacag catgctggac cccgtggtgt actacttctc cagcccatcc 900
tttcccaact tcttctccac tttgatcaac cgctgcctcc agaggaagat gacaggtgag
960 ccagataata accgcagcac gagcgtcgag ctcacagggg accccaacaa
aaccagaggc 1020 gctccagagg cgttaatggc caactccggt gagccatgga
gcccctctta tctgggccca 1080 acctctcctt aa 1092 36 363 PRT Homo
sapiens 36 Met Asn Arg His His Leu Gln Asp His Phe Leu Glu Ile Asp
Lys Lys 1 5 10 15 Asn Cys Cys Val Phe Arg Asp Asp Phe Ile Val Lys
Val Leu Pro Pro 20 25 30 Val Leu Gly Leu Glu Phe Ile Phe Gly Leu
Leu Gly Asn Gly Leu Ala 35 40 45 Leu Trp Ile Phe Cys Phe His Leu
Lys Ser Trp Lys Ser Ser Arg Ile 50 55 60 Phe Leu Phe Asn Leu Ala
Val Ala Asp Phe Leu Leu Ile Ile Cys Leu 65 70 75 80 Pro Phe Leu Met
Asp Asn Tyr Val Arg Arg Trp Asp Trp Lys Phe Gly 85 90 95 Asp Ile
Pro Cys Arg Leu Met Leu Phe Met Leu Ala Met Asn Arg Gln 100 105 110
Gly Ser Ile Ile Phe Leu Thr Val Val Ala Val Asp Arg Tyr Phe Arg 115
120 125 Val Val His Pro His His Ala Leu Asn Lys Ile Ser Asn Arg Thr
Ala 130 135 140 Ala Ile Ile Ser Cys Leu Leu Trp Gly Ile Thr Ile Gly
Leu Thr Val 145 150 155 160 His Leu Leu Lys Lys Lys Met Pro Ile Gln
Asn Gly Gly Ala Asn Leu 165 170 175 Cys Ser Ser Phe Ser Ile Cys His
Thr Phe Gln Trp His Glu Ala Met 180 185 190 Phe Leu Leu Glu Phe Phe
Leu Pro Leu Gly Ile Ile Leu Phe Cys Ser 195 200 205 Ala Arg Ile Ile
Trp Ser Leu Arg Gln Arg Gln Met Asp Arg His Ala 210 215 220 Lys Ile
Lys Arg Ala Ile Thr Phe Ile Met Val Val Ala Ile Val Phe 225 230 235
240 Val Ile Cys Phe Leu Pro Ser Val Val Val Arg Ile Arg Ile Phe Trp
245 250 255 Leu Leu His Thr Ser Gly Thr Gln Asn Cys Glu Val Tyr Arg
Ser Val 260 265 270 Asp Leu Ala Phe Phe Ile Thr Leu Ser Phe Thr Tyr
Met Asn Ser Met 275 280 285 Leu Asp Pro Val Val Tyr Tyr Phe Ser Ser
Pro Ser Phe Pro Asn Phe 290 295 300 Phe Ser Thr Leu Ile Asn Arg Cys
Leu Gln Arg Lys Met Thr Gly Glu 305 310 315 320 Pro Asp Asn Asn Arg
Ser Thr Ser Val Glu Leu Thr Gly Asp Pro Asn 325 330 335 Lys Thr Arg
Gly Ala Pro Glu Ala Leu Met Ala Asn Ser Gly Glu Pro 340 345 350 Trp
Ser Pro Ser Tyr Leu Gly Pro Thr Ser Pro 355 360 37 1044 DNA Homo
sapiens 37 atgggggatg agctggcacc ttgccctgtg ggcactacag cttggccggc
cctgatccag 60 ctcatcagca agacaccctg catgccccaa gcagccagca
acacttcctt gggcctgggg 120 gacctcaggg tgcccagctc catgctgtac
tggcttttcc ttccctcaag cctgctggct 180 gcagccacac tggctgtcag
ccccctgctg ctggtgacca tcctgcggaa ccaacggctg 240 cgacaggagc
cccactacct gctcccggct aacatcctgc tctcagacct ggcctacatt 300
ctcctccaca tgctcatctc ctccagcagc ctgggtggct gggagctggg ccgcatggcc
360 tgtggcattc tcactgatgc tgtcttcgcc gcctgcacca gcaccatcct
gtccttcacc 420 gccattgtgc tgcacaccta cctggcagtc atccatccac
tgcgctacct ctccttcatg 480 tcccatgggg ctgcctggaa ggcagtggcc
ctcatctggc tggtggcctg ctgcttcccc 540 acattcctta tttggctcag
caagtggcag gatgcccagc tggaggagca aggagcttca 600 tacatcctac
caccaagcat gggcacccag ccgggatgtg gcctcctggt cattgttacc 660
tacacctcca ttctgtgcgt tctgttcctc tgcacagctc tcattgccaa ctgtttctgg
720 aggatctatg cagaggccaa gacttcaggc atctgggggc agggctattc
ccgggccagg 780 ggcaccctgc tgatccactc agtgctgatc acattgtacg
tgagcacagg ggtggtgttc 840 tccctggaca tggtgctgac caggtaccac
cacattgact ctgggactca cacatggctc 900 ctggcagcta acagtgaggt
actcatgatg cttccccgtg ccatgctccc atacctgtac 960 ctgctccgct
accggcagct gttgggcatg gtccggggcc acctcccatc caggaggcac 1020
caggccatct ttaccatttc ctag 1044 38 347 PRT Homo sapiens 38 Met Gly
Asp Glu Leu Ala Pro Cys Pro Val Gly Thr Thr Ala Trp Pro 1 5 10 15
Ala Leu Ile Gln Leu Ile Ser Lys Thr Pro Cys Met Pro Gln Ala Ala 20
25 30 Ser Asn Thr Ser Leu Gly Leu Gly Asp Leu Arg Val Pro Ser Ser
Met 35 40 45 Leu Tyr Trp Leu Phe Leu Pro Ser Ser Leu Leu Ala Ala
Ala Thr Leu 50 55 60 Ala Val Ser Pro Leu Leu Leu Val Thr Ile Leu
Arg Asn Gln Arg Leu 65 70 75 80 Arg Gln Glu Pro His Tyr Leu Leu Pro
Ala Asn Ile Leu Leu Ser Asp 85 90 95 Leu Ala Tyr Ile Leu Leu His
Met Leu Ile Ser Ser Ser Ser Leu Gly 100 105 110 Gly Trp Glu Leu Gly
Arg Met Ala Cys Gly Ile Leu Thr Asp Ala Val 115 120 125 Phe Ala Ala
Cys Thr Ser Thr Ile Leu Ser Phe Thr Ala Ile Val Leu 130 135 140 His
Thr Tyr Leu Ala Val Ile His Pro Leu Arg Tyr Leu Ser Phe Met 145 150
155 160 Ser His Gly Ala Ala Trp Lys Ala Val Ala Leu Ile Trp Leu Val
Ala 165 170 175 Cys Cys Phe Pro Thr Phe Leu Ile Trp Leu Ser Lys Trp
Gln Asp Ala 180 185 190 Gln Leu Glu Glu Gln Gly Ala Ser Tyr Ile Leu
Pro Pro Ser Met Gly 195 200 205 Thr Gln Pro Gly Cys Gly Leu Leu Val
Ile Val Thr Tyr Thr Ser Ile 210 215 220 Leu Cys Val Leu Phe Leu Cys
Thr Ala Leu Ile Ala Asn Cys Phe Trp 225 230 235 240 Arg Ile Tyr Ala
Glu Ala Lys Thr Ser Gly Ile Trp Gly Gln Gly Tyr 245 250 255 Ser Arg
Ala Arg Gly Thr Leu Leu Ile His Ser Val Leu Ile Thr Leu 260 265 270
Tyr Val Ser Thr Gly Val Val Phe Ser Leu Asp Met Val Leu Thr Arg 275
280 285 Tyr His His Ile Asp Ser Gly Thr His Thr Trp Leu Leu Ala Ala
Asn 290 295 300 Ser Glu Val Leu Met Met Leu Pro Arg Ala Met Leu Pro
Tyr Leu Tyr 305 310 315 320 Leu Leu Arg Tyr Arg Gln Leu Leu Gly Met
Val Arg Gly His Leu Pro 325 330 335 Ser Arg Arg His Gln Ala Ile Phe
Thr Ile Ser 340 345 39 1023 DNA Homo sapiens 39 atgaatccat
ttcatgcatc ttgttggaac acctctgccg aacttttaaa caaatcctgg 60
aataaagagt ttgcttatca aactgccagt gtggtagata cagtcatcct cccttccatg
120 attgggatta tctgttcaac agggctggtt ggcaacatcc tcattgtatt
cactataata 180 agatccagga aaaaaacagt ccctgacatc tatatctgca
acctggctgt ggctgatttg 240 gtccacatag ttggaatgcc ttttcttatt
caccaatggg cccgaggggg agagtgggtg 300 tttggggggc ctctctgcac
catcatcaca tccctggata cttgtaacca atttgcctgt 360 agtgccatca
tgactgtaat gagtgtggac aggtactttg ccctcgtcca accatttcga 420
ctgacacgtt ggagaacaag gtacaagacc atccggatca atttgggcct ttgggcagct
480 tcctttatcc tggcattgcc tgtctgggtc tactcgaagg tcatcaaatt
taaagacggt 540 gttgagagtt gtgcttttga tttgacatcc cctgacgatg
tactctggta tacactttat 600 ttgacgataa caactttttt tttccctcta
cccttgattt tggtgtgcta tattttaatt 660 ttatgctata cttgggagat
gtatcaacag aataaggatg ccagatgctg caatcccagt 720 gtaccaaaac
agagagtgat gaagttgaca aagatggtgc tggtgctggt ggtagtcttt 780
atcctgagtg ctgcccctta tcatgtgata caactggtga acttacagat ggaacagccc
840 acactggcct tctatgtggg ttattacctc tccatctgtc tcagctatgc
cagcagcagc 900 attaaccctt ttctctacat cctgctgagt ggaaatttcc
agaaacgtct gcctcaaatc 960 caaagaagag cgactgagaa ggaaatcaac
aatatgggaa acactctgaa atcacacttt 1020 tag 1023 40 340 PRT Homo
sapiens 40 Met Asn Pro Phe His Ala Ser Cys Trp Asn Thr Ser Ala Glu
Leu Leu 1 5 10 15 Asn Lys Ser Trp Asn Lys Glu Phe Ala Tyr Gln Thr
Ala Ser Val Val 20 25 30 Asp Thr Val Ile Leu Pro Ser Met Ile Gly
Ile Ile Cys Ser Thr Gly 35 40 45 Leu Val Gly Asn Ile Leu Ile Val
Phe Thr Ile Ile Arg Ser Arg Lys 50 55 60 Lys Thr Val Pro Asp Ile
Tyr Ile Cys Asn Leu Ala Val Ala Asp Leu 65 70 75 80 Val His Ile Val
Gly Met Pro Phe Leu Ile His Gln Trp Ala Arg Gly 85 90 95 Gly Glu
Trp Val Phe Gly Gly Pro Leu Cys Thr Ile Ile Thr Ser Leu 100 105 110
Asp Thr Cys Asn Gln Phe Ala Cys Ser Ala Ile Met Thr Val Met Ser 115
120 125 Val Asp Arg Tyr Phe Ala Leu Val Gln Pro Phe Arg Leu Thr Arg
Trp 130 135 140 Arg Thr Arg Tyr Lys Thr Ile Arg Ile Asn Leu Gly Leu
Trp Ala Ala 145 150 155 160 Ser Phe Ile Leu Ala Leu Pro Val Trp Val
Tyr Ser Lys Val Ile Lys 165 170 175 Phe Lys Asp Gly Val Glu Ser Cys
Ala Phe Asp Leu Thr Ser Pro Asp 180 185 190 Asp Val Leu Trp Tyr Thr
Leu Tyr Leu Thr Ile Thr Thr Phe Phe Phe 195 200 205 Pro Leu Pro Leu
Ile Leu Val Cys Tyr Ile Leu Ile Leu Cys Tyr Thr 210 215 220 Trp Glu
Met Tyr Gln Gln Asn Lys Asp Ala Arg Cys Cys Asn Pro Ser 225 230 235
240 Val Pro Lys Gln Arg Val Met Lys Leu Thr Lys Met Val Leu Val Leu
245 250 255 Val Val Val Phe Ile Leu Ser Ala Ala Pro Tyr His Val Ile
Gln Leu 260 265 270 Val Asn Leu Gln Met Glu Gln Pro Thr Leu Ala Phe
Tyr Val Gly Tyr 275 280 285 Tyr Leu Ser Ile Cys Leu Ser Tyr Ala Ser
Ser Ser Ile Asn Pro Phe 290 295 300 Leu Tyr Ile Leu Leu Ser Gly Asn
Phe Gln Lys Arg Leu Pro Gln Ile 305 310 315 320 Gln Arg Arg Ala Thr
Glu Lys Glu Ile Asn Asn Met Gly Asn Thr Leu 325 330 335 Lys Ser His
Phe 340 41 24 DNA Artificial Sequence Novel Sequence misc_feature
Novel Sequence 41 cttgcagaca tcaccatggc agcc 24 42 24 DNA
Artificial Sequence Novel Sequence 42 gtgatgctct gagtactgga ctgg 24
43 20 DNA Artificial Sequence Novel Sequence 43 gaagctgtga
agagtgatgc 20 44 24 DNA Artificial Sequence Novel Sequence 44
gtcagcaata ttgataagca gcag 24 45 27 DNA Artificial Sequence Novel
Sequence 45 ccatggggaa cgattctgtc agctacg 27 46 24 DNA Artificial
Sequence Novel Sequence 46 gctatgcctg aagccagtct tgtg 24 47 26 DNA
Artificial Sequence Novel Sequence 47 ccaggatgtt gtgtcaccgt ggtggc
26 48 26 DNA Artificial Sequence Novel Sequence 48 cacagcgctg
cagccctgca gctggc 26 49 26 DNA Artificial Sequence Novel Sequence
49 cttcctctcg tagggatgaa ccagac 26 50 26 DNA Artificial Sequence
Novel Sequence 50 ctcgcacagg tgggaagcac ctgtgg 26 51 23 DNA
Artificial Sequence Novel Sequence 51 gcctgtgaca ggaggtaccc tgg 23
52 25 DNA Artificial Sequence Novel Sequence 52 catatccctc
cgagtgtcca gcggc 25 53 31 DNA Artificial Sequence Novel Sequence 53
gcatggagag aaaatttatg tccttgcaac c 31 54 27 DNA Artificial Sequence
Novel Sequence misc_feature Novel Sequence 54 caagaacagg tctcatctaa
gagctcc 27 55 26 DNA Artificial Sequence Novel Sequence 55
gctgttgcca tgacgtccac ctgcac 26 56 26 DNA Artificial Sequence Novel
Sequence 56 ggacagttca aggtttgcct tagaac 26 57 23 DNA Artificial
Sequence Novel Sequence 57 ctttcgatac tgctcctatg ctc 23 58 26 DNA
Artificial Sequence Novel Sequence 58 gtagtccact gaaagtccag tgatcc
26 59 26 DNA Artificial Sequence Novel Sequence 59 tttctgagca
tggatccaac catctc 26 60 25 DNA Artificial Sequence Novel Sequence
60 ctgtctgaca gggcagaggc tcttc 25 61 28 DNA Artificial Sequence
Novel Sequence 61 ggaactcgta tagacccagc gtcgctcc 28 62 28 DNA
Artificial Sequence Novel Sequence 62 ggaggttgcg ccttagcgac
agatgacc 28 63 22 DNA Artificial Sequence Novel Sequence 63
ctgcacccgg acacttgctc tg 22 64 25 DNA Artificial Sequence Novel
Sequence 64 gtctgcttgt tcagtgccac tcaac 25 65 26 DNA Artificial
Sequence Novel Sequence 65 tatctgcaat tctattctag ctcctg 26 66 26
DNA Artificial Sequence Novel Sequence 66 tgtccctaat aaagtcacat
gaatgc 26 67 23 DNA Artificial Sequence Novel Sequence 67
ggagacaacc atgaatgagc cac 23 68 24 DNA Artificial Sequence Novel
Sequence 68 tatttcaagg gttgtttgag taac 24 69 27 DNA Artificial
Sequence Novel Sequence 69 ggcaccagtg gaggttttct gagcatg 27 70 27
DNA Artificial Sequence Novel Sequence 70 ctgatggaag tagaggctgt
ccatctc 27 71 23 DNA Artificial Sequence Novel Sequence 71
cctggcgagc cgctagcgcc atg 23 72 23 DNA Artificial Sequence Novel
Sequence 72 atgagccctg ccaggccctc agt 23 73 27 DNA Artificial
Sequence Novel Sequence 73 ctgcgatgcc cacactcaat acttctg 27 74 27
DNA Artificial Sequence Novel Sequence 74 aaggatccta cacttggtgg
atctcag 27 75 22 DNA Artificial Sequence Novel Sequence 75
gctggagcat tcactaggcg ag 22 76 24 DNA Artificial Sequence Novel
Sequence 76 agatcctggt tcttggtgac aatg 24 77 24 DNA Artificial
Sequence Novel Sequence 77 agccatccct gccaggaagc atgg 24 78 27 DNA
Artificial Sequence Novel Sequence 78 ccagactgtg gactcaagaa ctctagg
27 79 28 DNA Artificial Sequence Novel Sequence 79 agtccacgaa
caatgaatcc atttcatg 28 80 25 DNA Artificial Sequence Novel Sequence
80 atcatgtcta gactcatggt gatcc 25 81 30 DNA Artificial Sequence
Novel Sequence 81 ggggagggaa agcaaaggtg gtcctcctgg 30 82 30 DNA
Artificial Sequence Novel Sequence 82 ccaggagaac cacctttgct
ttccctcccc 30 83 1356 DNA Homo sapiens 83 atggagtcct cacccatccc
ccagtcatca gggaactctt ccactttggg gagggtccct 60 caaaccccag
gtccctctac tgccagtggg gtcccggagg tggggctacg ggatgttgct 120
tcggaatctg tggccctctt cttcatgctc ctgctggact tgactgctgt ggctggcaat
180 gccgctgtga tggccgtgat cgccaagacg cctgccctcc gaaaatttgt
cttcgtcttc 240 cacctctgcc tggtggacct gctggctgcc ctgaccctca
tgcccctggc catgctctcc 300 agctctgccc tctttgacca cgccctcttt
ggggaggtgg cctgccgcct ctacttgttt 360 ctgagcgtgt gctttgtcag
cctggccatc ctctcggtgt cagccatcaa tgtggagcgc 420 tactattacg
tagtccaccc catgcgctac gaggtgcgca tgacgctggg gctggtggcc 480
tctgtgctgg tgggtgtgtg ggtgaaggcc ttggccatgg cttctgtgcc agtgttggga
540 agggtctcct gggaggaagg agctcccagt gtccccccag gctgttcact
ccagtggagc 600 cacagtgcct actgccagct ttttgtggtg gtctttgctg
tcctttactt tctgttgccc 660 ctgctcctca tacttgtggt ctactgcagc
atgttccgag tggcccgcgt ggctgccatg 720 cagcacgggc cgctgcccac
gtggatggag acaccccggc aacgctccga atctctcagc 780 agccgctcca
cgatggtcac cagctcgggg gccccccaga ccaccccaca ccggacgttt 840
gggggaggga aagcaaaggt ggttctcctg gctgtggggg gacagttcct gctctgttgg
900 ttgccctact tctctttcca cctctatgtt gccctgagtg ctcagcccat
ttcaactggg 960 caggtggaga gtgtggtcac ctggattggc tacttttgct
tcacttccaa ccctttcttc 1020 tatggatgtc tcaaccggca gatccggggg
gagctcagca agcagtttgt ctgcttcttc 1080 aagccagctc cagaggagga
gctgaggctg cctagccggg agggctccat tgaggagaac 1140 ttcctgcagt
tccttcaggg gactggctgt ccttctgagt cctgggtttc ccgaccccta 1200
cccagcccca agcaggagcc acctgctgtt gactttcgaa tcccaggcca gatagctgag
1260 gagacctctg agttcctgga gcagcaactc accagcgaca tcatcatgtc
agacagctac 1320 ctccgtcctg ccgcctcacc ccggctggag tcatga 1356
84 451 PRT Homo sapiens 84 Met Glu Ser Ser Pro Ile Pro Gln Ser Ser
Gly Asn Ser Ser Thr Leu 1 5 10 15 Gly Arg Val Pro Gln Thr Pro Gly
Pro Ser Thr Ala Ser Gly Val Pro 20 25 30 Glu Val Gly Leu Arg Asp
Val Ala Ser Glu Ser Val Ala Leu Phe Phe 35 40 45 Met Leu Leu Leu
Asp Leu Thr Ala Val Ala Gly Asn Ala Ala Val Met 50 55 60 Ala Val
Ile Ala Lys Thr Pro Ala Leu Arg Lys Phe Val Phe Val Phe 65 70 75 80
His Leu Cys Leu Val Asp Leu Leu Ala Ala Leu Thr Leu Met Pro Leu 85
90 95 Ala Met Leu Ser Ser Ser Ala Leu Phe Asp His Ala Leu Phe Gly
Glu 100 105 110 Val Ala Cys Arg Leu Tyr Leu Phe Leu Ser Val Cys Phe
Val Ser Leu 115 120 125 Ala Ile Leu Ser Val Ser Ala Ile Asn Val Glu
Arg Tyr Tyr Tyr Val 130 135 140 Val His Pro Met Arg Tyr Glu Val Arg
Met Thr Leu Gly Leu Val Ala 145 150 155 160 Ser Val Leu Val Gly Val
Trp Val Lys Ala Leu Ala Met Ala Ser Val 165 170 175 Pro Val Leu Gly
Arg Val Ser Trp Glu Glu Gly Ala Pro Ser Val Pro 180 185 190 Pro Gly
Cys Ser Leu Gln Trp Ser His Ser Ala Tyr Cys Gln Leu Phe 195 200 205
Val Val Val Phe Ala Val Leu Tyr Phe Leu Leu Pro Leu Leu Leu Ile 210
215 220 Leu Val Val Tyr Cys Ser Met Phe Arg Val Ala Arg Val Ala Ala
Met 225 230 235 240 Gln His Gly Pro Leu Pro Thr Trp Met Glu Thr Pro
Arg Gln Arg Ser 245 250 255 Glu Ser Leu Ser Ser Arg Ser Thr Met Val
Thr Ser Ser Gly Ala Pro 260 265 270 Gln Thr Thr Pro His Arg Thr Phe
Gly Gly Gly Lys Ala Lys Val Val 275 280 285 Leu Leu Ala Val Gly Gly
Gln Phe Leu Leu Cys Trp Leu Pro Tyr Phe 290 295 300 Ser Phe His Leu
Tyr Val Ala Leu Ser Ala Gln Pro Ile Ser Thr Gly 305 310 315 320 Gln
Val Glu Ser Val Val Thr Trp Ile Gly Tyr Phe Cys Phe Thr Ser 325 330
335 Asn Pro Phe Phe Tyr Gly Cys Leu Asn Arg Gln Ile Arg Gly Glu Leu
340 345 350 Ser Lys Gln Phe Val Cys Phe Phe Lys Pro Ala Pro Glu Glu
Glu Leu 355 360 365 Arg Leu Pro Ser Arg Glu Gly Ser Ile Glu Glu Asn
Phe Leu Gln Phe 370 375 380 Leu Gln Gly Thr Gly Cys Pro Ser Glu Ser
Trp Val Ser Arg Pro Leu 385 390 395 400 Pro Ser Pro Lys Gln Glu Pro
Pro Ala Val Asp Phe Arg Ile Pro Gly 405 410 415 Gln Ile Ala Glu Glu
Thr Ser Glu Phe Leu Glu Gln Gln Leu Thr Ser 420 425 430 Asp Ile Ile
Met Ser Asp Ser Tyr Leu Arg Pro Ala Ala Ser Pro Arg 435 440 445 Leu
Glu Ser 450 85 28 DNA Homo sapiens 85 caggaaggca aagaccacca
tcatcatc 28 86 28 DNA Homo sapiens 86 gatgatgatg gtggtctttg
ccttcctg 28 87 1041 DNA Homo sapiens 87 atggagagaa aatttatgtc
cttgcaacca tccatctccg tatcagaaat ggaaccaaat 60 ggcaccttca
gcaataacaa cagcaggaac tgcacaattg aaaacttcaa gagagaattt 120
ttcccaattg tatatctgat aatatttttc tggggagtct tgggaaatgg gttgtccata
180 tatgttttcc tgcagcctta taagaagtcc acatctgtga acgttttcat
gctaaatctg 240 gccatttcag atctcctgtt cataagcacg cttcccttca
gggctgacta ttatcttaga 300 ggctccaatt ggatatttgg agacctggcc
tgcaggatta tgtcttattc cttgtatgtc 360 aacatgtaca gcagtattta
tttcctgacc gtgctgagtg ttgtgcgttt cctggcaatg 420 gttcacccct
ttcggcttct gcatgtcacc agcatcagga gtgcctggat cctctgtggg 480
atcatatgga tccttatcat ggcttcctca ataatgctcc tggacagtgg ctctgagcag
540 aacggcagtg tcacatcatg cttagagctg aatctctata aaattgctaa
gctgcagacc 600 atgaactata ttgccttggt ggtgggctgc ctgctgccat
ttttcacact cagcatctgt 660 tatctgctga tcattcgggt tctgttaaaa
gtggaggtcc cagaatcggg gctgcgggtt 720 tctcacagga aggcaaagac
caccatcatc atcaccttga tcatcttctt cttgtgtttc 780 ctgccctatc
acacactgag gaccgtccac ttgacgacat ggaaagtggg tttatgcaaa 840
gacagactgc ataaagcttt ggttatcaca ctggccttgg cagcagccaa tgcctgcttc
900 aatcctctgc tctattactt tgctggggag aattttaagg acagactaaa
gtctgcactc 960 agaaaaggcc atccacagaa ggcaaagaca aagtgtgttt
tccctgttag tgtgtggttg 1020 agaaaggaaa caagagtata a 1041 88 346 PRT
Homo sapiens 88 Met Glu Arg Lys Phe Met Ser Leu Gln Pro Ser Ile Ser
Val Ser Glu 1 5 10 15 Met Glu Pro Asn Gly Thr Phe Ser Asn Asn Asn
Ser Arg Asn Cys Thr 20 25 30 Ile Glu Asn Phe Lys Arg Glu Phe Phe
Pro Ile Val Tyr Leu Ile Ile 35 40 45 Phe Phe Trp Gly Val Leu Gly
Asn Gly Leu Ser Ile Tyr Val Phe Leu 50 55 60 Gln Pro Tyr Lys Lys
Ser Thr Ser Val Asn Val Phe Met Leu Asn Leu 65 70 75 80 Ala Ile Ser
Asp Leu Leu Phe Ile Ser Thr Leu Pro Phe Arg Ala Asp 85 90 95 Tyr
Tyr Leu Arg Gly Ser Asn Trp Ile Phe Gly Asp Leu Ala Cys Arg 100 105
110 Ile Met Ser Tyr Ser Leu Tyr Val Asn Met Tyr Ser Ser Ile Tyr Phe
115 120 125 Leu Thr Val Leu Ser Val Val Arg Phe Leu Ala Met Val His
Pro Phe 130 135 140 Arg Leu Leu His Val Thr Ser Ile Arg Ser Ala Trp
Ile Leu Cys Gly 145 150 155 160 Ile Ile Trp Ile Leu Ile Met Ala Ser
Ser Ile Met Leu Leu Asp Ser 165 170 175 Gly Ser Glu Gln Asn Gly Ser
Val Thr Ser Cys Leu Glu Leu Asn Leu 180 185 190 Tyr Lys Ile Ala Lys
Leu Gln Thr Met Asn Tyr Ile Ala Leu Val Val 195 200 205 Gly Cys Leu
Leu Pro Phe Phe Thr Leu Ser Ile Cys Tyr Leu Leu Ile 210 215 220 Ile
Arg Val Leu Leu Lys Val Glu Val Pro Glu Ser Gly Leu Arg Val 225 230
235 240 Ser His Arg Lys Ala Lys Thr Thr Ile Ile Ile Thr Leu Ile Ile
Phe 245 250 255 Phe Leu Cys Phe Leu Pro Tyr His Thr Leu Arg Thr Val
His Leu Thr 260 265 270 Thr Trp Lys Val Gly Leu Cys Lys Asp Arg Leu
His Lys Ala Leu Val 275 280 285 Ile Thr Leu Ala Leu Ala Ala Ala Asn
Ala Cys Phe Asn Pro Leu Leu 290 295 300 Tyr Tyr Phe Ala Gly Glu Asn
Phe Lys Asp Arg Leu Lys Ser Ala Leu 305 310 315 320 Arg Lys Gly His
Pro Gln Lys Ala Lys Thr Lys Cys Val Phe Pro Val 325 330 335 Ser Val
Trp Leu Arg Lys Glu Thr Arg Val 340 345 89 28 DNA Artificial
Sequence Novel Sequence 89 ccagtgcaaa gctaagaaag tgatcttc 28 90 28
DNA Artificial Sequence Novel Sequence misc_feature Novel Sequence
90 gaagatcact ttcttagctt tgcactgg 28 91 1527 DNA Homo sapiens 91
atgacgtcca cctgcaccaa cagcacgcgc gagagtaaca gcagccacac gtgcatgccc
60 ctctccaaaa tgcccatcag cctggcccac ggcatcatcc gctcaaccgt
gctggttatc 120 ttcctcgccg cctctttcgt cggcaacata gtgctggcgc
tagtgttgca gcgcaagccg 180 cagctgctgc aggtgaccaa ccgttttatc
tttaacctcc tcgtcaccga cctgctgcag 240 atttcgctcg tggccccctg
ggtggtggcc acctctgtgc ctctcttctg gcccctcaac 300 agccacttct
gcacggccct ggttagcctc acccacctgt tcgccttcgc cagcgtcaac 360
accattgtcg tggtgtcagt ggatcgctac ttgtccatca tccaccctct ctcctacccg
420 tccaagatga cccagcgccg cggttacctg ctcctctatg gcacctggat
tgtggccatc 480 ctgcagagca ctcctccact ctacggctgg ggccaggctg
cctttgatga gcgcaatgct 540 ctctgctcca tgatctgggg ggccagcccc
agctacacta ttctcagcgt ggtgtccttc 600 atcgtcattc cactgattgt
catgattgcc tgctactccg tggtgttctg tgcagcccgg 660 aggcagcatg
ctctgctgta caatgtcaag agacacagct tggaagtgcg agtcaaggac 720
tgtgtggaga atgaggatga agagggagca gagaagaagg aggagttcca ggatgagagt
780 gagtttcgcc gccagcatga aggtgaggtc aaggccaagg agggcagaat
ggaagccaag 840 gacggcagcc tgaaggccaa ggaaggaagc acggggacca
gtgagagtag tgtagaggcc 900 aggggcagcg aggaggtcag agagagcagc
acggtggcca gcgacggcag catggagggt 960 aaggaaggca gcaccaaagt
tgaggagaac agcatgaagg cagacaaggg tcgcacagag 1020 gtcaaccagt
gcagcattga cttgggtgaa gatgacatgg agtttggtga agacgacatc 1080
aatttcagtg aggatgacgt cgaggcagtg aacatcccgg agagcctccc acccagtcgt
1140 cgtaacagca acagcaaccc tcctctgccc aggtgctacc agtgcaaagc
taagaaagtg 1200 atcttcatca tcattttctc ctatgtgcta tccctggggc
cctactgctt tttagcagtc 1260 ctggccgtgt gggtggatgt cgaaacccag
gtaccccagt gggtgatcac cataatcatc 1320 tggcttttct tcctgcagtg
ctgcatccac ccctatgtct atggctacat gcacaagacc 1380 attaagaagg
aaatccagga catgctgaag aagttcttct gcaaggaaaa gcccccgaaa 1440
gaagatagcc acccagacct gcccggaaca gagggtggga ctgaaggcaa gattgtccct
1500 tcctacgatt ctgctacttt tccttga 1527 92 508 PRT Homo sapiens 92
Met Thr Ser Thr Cys Thr Asn Ser Thr Arg Glu Ser Asn Ser Ser His 1 5
10 15 Thr Cys Met Pro Leu Ser Lys Met Pro Ile Ser Leu Ala His Gly
Ile 20 25 30 Ile Arg Ser Thr Val Leu Val Ile Phe Leu Ala Ala Ser
Phe Val Gly 35 40 45 Asn Ile Val Leu Ala Leu Val Leu Gln Arg Lys
Pro Gln Leu Leu Gln 50 55 60 Val Thr Asn Arg Phe Ile Phe Asn Leu
Leu Val Thr Asp Leu Leu Gln 65 70 75 80 Ile Ser Leu Val Ala Pro Trp
Val Val Ala Thr Ser Val Pro Leu Phe 85 90 95 Trp Pro Leu Asn Ser
His Phe Cys Thr Ala Leu Val Ser Leu Thr His 100 105 110 Leu Phe Ala
Phe Ala Ser Val Asn Thr Ile Val Val Val Ser Val Asp 115 120 125 Arg
Tyr Leu Ser Ile Ile His Pro Leu Ser Tyr Pro Ser Lys Met Thr 130 135
140 Gln Arg Arg Gly Tyr Leu Leu Leu Tyr Gly Thr Trp Ile Val Ala Ile
145 150 155 160 Leu Gln Ser Thr Pro Pro Leu Tyr Gly Trp Gly Gln Ala
Ala Phe Asp 165 170 175 Glu Arg Asn Ala Leu Cys Ser Met Ile Trp Gly
Ala Ser Pro Ser Tyr 180 185 190 Thr Ile Leu Ser Val Val Ser Phe Ile
Val Ile Pro Leu Ile Val Met 195 200 205 Ile Ala Cys Tyr Ser Val Val
Phe Cys Ala Ala Arg Arg Gln His Ala 210 215 220 Leu Leu Tyr Asn Val
Lys Arg His Ser Leu Glu Val Arg Val Lys Asp 225 230 235 240 Cys Val
Glu Asn Glu Asp Glu Glu Gly Ala Glu Lys Lys Glu Glu Phe 245 250 255
Gln Asp Glu Ser Glu Phe Arg Arg Gln His Glu Gly Glu Val Lys Ala 260
265 270 Lys Glu Gly Arg Met Glu Ala Lys Asp Gly Ser Leu Lys Ala Lys
Glu 275 280 285 Gly Ser Thr Gly Thr Ser Glu Ser Ser Val Glu Ala Arg
Gly Ser Glu 290 295 300 Glu Val Arg Glu Ser Ser Thr Val Ala Ser Asp
Gly Ser Met Glu Gly 305 310 315 320 Lys Glu Gly Ser Thr Lys Val Glu
Glu Asn Ser Met Lys Ala Asp Lys 325 330 335 Gly Arg Thr Glu Val Asn
Gln Cys Ser Ile Asp Leu Gly Glu Asp Asp 340 345 350 Met Glu Phe Gly
Glu Asp Asp Ile Asn Phe Ser Glu Asp Asp Val Glu 355 360 365 Ala Val
Asn Ile Pro Glu Ser Leu Pro Pro Ser Arg Arg Asn Ser Asn 370 375 380
Ser Asn Pro Pro Leu Pro Arg Cys Tyr Gln Cys Lys Ala Lys Lys Val 385
390 395 400 Ile Phe Ile Ile Ile Phe Ser Tyr Val Leu Ser Leu Gly Pro
Tyr Cys 405 410 415 Phe Leu Ala Val Leu Ala Val Trp Val Asp Val Glu
Thr Gln Val Pro 420 425 430 Gln Trp Val Ile Thr Ile Ile Ile Trp Leu
Phe Phe Leu Gln Cys Cys 435 440 445 Ile His Pro Tyr Val Tyr Gly Tyr
Met His Lys Thr Ile Lys Lys Glu 450 455 460 Ile Gln Asp Met Leu Lys
Lys Phe Phe Cys Lys Glu Lys Pro Pro Lys 465 470 475 480 Glu Asp Ser
His Pro Asp Leu Pro Gly Thr Glu Gly Gly Thr Glu Gly 485 490 495 Lys
Ile Val Pro Ser Tyr Asp Ser Ala Thr Phe Pro 500 505 93 29 DNA
Artificial Sequence Novel Sequence 93 gccgccaccg cgccaagagg
aagattggc 29 94 29 DNA Artificial Sequence Novel Sequence 94
gccaatcttc ctcttggcgc ggtggcggc 29 95 1092 DNA Homo sapiens 95
atgggccccg gcgaggcgct gctggcgggt ctcctggtga tggtactggc cgtggcgctg
60 ctatccaacg cactggtgct gctttgttgc gcctacagcg ctgagctccg
cactcgagcc 120 tcaggcgtcc tcctggtgaa tctgtcgctg ggccacctgc
tgctggcggc gctggacatg 180 cccttcacgc tgctcggtgt gatgcgcggg
cggacaccgt cggcgcccgg cgcatgccaa 240 gtcattggct tcctggacac
cttcctggcg tccaacgcgg cgctgagcgt ggcggcgctg 300 agcgcagacc
agtggctggc agtgggcttc ccactgcgct acgccggacg cctgcgaccg 360
cgctatgccg gcctgctgct gggctgtgcc tggggacagt cgctggcctt ctcaggcgct
420 gcacttggct gctcgtggct tggctacagc agcgccttcg cgtcctgttc
gctgcgcctg 480 ccgcccgagc ctgagcgtcc gcgcttcgca gccttcaccg
ccacgctcca tgccgtgggc 540 ttcgtgctgc cgctggcggt gctctgcctc
acctcgctcc aggtgcaccg ggtggcacgc 600 agccactgcc agcgcatgga
caccgtcacc atgaaggcgc tcgcgctgct cgccgacctg 660 caccccagtg
tgcggcagcg ctgcctcatc cagcagaagc ggcgccgcca ccgcgccacc 720
aggaagattg gcattgctat tgcgaccttc ctcatctgct ttgccccgta tgtcatgacc
780 aggctggcgg agctcgtgcc cttcgtcacc gtgaacgccc agaagggcat
cctcagcaag 840 tgcctgacct acagcaaggc ggtggccgac ccgttcacgt
actctctgct ccgccggccg 900 ttccgccaag tcctggccgg catggtgcac
cggctgctga agagaacccc gcgcccagca 960 tccacccatg acagctctct
ggatgtggcc ggcatggtgc accagctgct gaagagaacc 1020 ccgcgcccag
cgtccaccca caacggctct gtggacacag agaatgattc ctgcctgcag 1080
cagacacact ga 1092 96 363 PRT Homo sapiens 96 Met Gly Pro Gly Glu
Ala Leu Leu Ala Gly Leu Leu Val Met Val Leu 1 5 10 15 Ala Val Ala
Leu Leu Ser Asn Ala Leu Val Leu Leu Cys Cys Ala Tyr 20 25 30 Ser
Ala Glu Leu Arg Thr Arg Ala Ser Gly Val Leu Leu Val Asn Leu 35 40
45 Ser Leu Gly His Leu Leu Leu Ala Ala Leu Asp Met Pro Phe Thr Leu
50 55 60 Leu Gly Val Met Arg Gly Arg Thr Pro Ser Ala Pro Gly Ala
Cys Gln 65 70 75 80 Val Ile Gly Phe Leu Asp Thr Phe Leu Ala Ser Asn
Ala Ala Leu Ser 85 90 95 Val Ala Ala Leu Ser Ala Asp Gln Trp Leu
Ala Val Gly Phe Pro Leu 100 105 110 Arg Tyr Ala Gly Arg Leu Arg Pro
Arg Tyr Ala Gly Leu Leu Leu Gly 115 120 125 Cys Ala Trp Gly Gln Ser
Leu Ala Phe Ser Gly Ala Ala Leu Gly Cys 130 135 140 Ser Trp Leu Gly
Tyr Ser Ser Ala Phe Ala Ser Cys Ser Leu Arg Leu 145 150 155 160 Pro
Pro Glu Pro Glu Arg Pro Arg Phe Ala Ala Phe Thr Ala Thr Leu 165 170
175 His Ala Val Gly Phe Val Leu Pro Leu Ala Val Leu Cys Leu Thr Ser
180 185 190 Leu Gln Val His Arg Val Ala Arg Ser His Cys Gln Arg Met
Asp Thr 195 200 205 Val Thr Met Lys Ala Leu Ala Leu Leu Ala Asp Leu
His Pro Ser Val 210 215 220 Arg Gln Arg Cys Leu Ile Gln Gln Lys Arg
Arg Arg His Arg Ala Thr 225 230 235 240 Arg Lys Ile Gly Ile Ala Ile
Ala Thr Phe Leu Ile Cys Phe Ala Pro 245 250 255 Tyr Val Met Thr Arg
Leu Ala Glu Leu Val Pro Phe Val Thr Val Asn 260 265 270 Ala Gln Lys
Gly Ile Leu Ser Lys Cys Leu Thr Tyr Ser Lys Ala Val 275 280 285 Ala
Asp Pro Phe Thr Tyr Ser Leu Leu Arg Arg Pro Phe Arg Gln Val 290 295
300 Leu Ala Gly Met Val His Arg Leu Leu Lys Arg Thr Pro Arg Pro Ala
305 310 315 320 Ser Thr His Asp Ser Ser Leu Asp Val Ala Gly Met Val
His Gln Leu 325 330 335 Leu Lys Arg Thr Pro Arg Pro Ala Ser Thr His
Asn Gly Ser Val Asp 340 345 350 Thr Glu Asn Asp Ser Cys Leu Gln Gln
Thr His 355 360 97 34 DNA Artificial Sequence Novel Sequence 97
gatctctaga atggagtcct cacccatccc ccag 34 98 36 DNA Artificial
Sequence Novel Sequence 98 gatcgatatc cgtgactcca gccggggtga ggcggc
36 99 2610 DNA Homo sapiens and Rat 99 atggagtcct
cacccatccc ccagtcatca gggaactctt ccactttggg gagggtccct 60
caaaccccag gtccctctac tgccagtggg gtcccggagg tggggctacg ggatgttgct
120 tcggaatctg tggccctctt cttcatgctc ctgctggact tgactgctgt
ggctggcaat 180 gccgctgtga tggccgtgat cgccaagacg cctgccctcc
gaaaatttgt cttcgtcttc 240 cacctctgcc tggtggacct gctggctgcc
ctgaccctca tgcccctggc catgctctcc 300 agctctgccc tctttgacca
cgccctcttt ggggaggtgg cctgccgcct ctacttgttt 360 ctgagcgtgt
gctttgtcag cctggccatc ctctcggtgt cagccatcaa tgtggagcgc 420
tactattacg tagtccaccc catgcgctac gaggtgcgca tgacgctggg gctggtggcc
480 tctgtgctgg tgggtgtgtg ggtgaaggcc ttggccatgg cttctgtgcc
agtgttggga 540 agggtctcct gggaggaagg agctcccagt gtccccccag
gctgttcact ccagtggagc 600 cacagtgcct actgccagct ttttgtggtg
gtctttgctg tcctttactt tctgttgccc 660 ctgctcctca tacttgtggt
ctactgcagc atgttccgag tggcccgcgt ggctgccatg 720 cagcacgggc
cgctgcccac gtggatggag acaccccggc aacgctccga atctctcagc 780
agccgctcca cgatggtcac cagctcgggg gccccccaga ccaccccaca ccggacgttt
840 gggggaggga aagcagcagt ggttctcctg gctgtggggg gacagttcct
gctctgttgg 900 ttgccctact tctctttcca cctctatgtt gccctgagtg
ctcagcccat ttcaactggg 960 caggtggaga gtgtggtcac ctggattggc
tacttttgct tcacttccaa ccctttcttc 1020 tatggatgtc tcaaccggca
gatccggggg gagctcagca agcagtttgt ctgcttcttc 1080 aagccagctc
cagaggagga gctgaggctg cctagccggg agggctccat tgaggagaac 1140
ttcctgcagt tccttcaggg gactggctgt ccttctgagt cctgggtttc ccgaccccta
1200 cccagcccca agcaggagcc acctgctgtt gactttcgaa tcccaggcca
gatagctgag 1260 gagacctctg agttcctgga gcagcaactc accagcgaca
tcatcatgtc agacagctac 1320 ctccgtcctg ccgcctcacc ccggctggag
tcagcgatat ctgcagaatt ccaccacact 1380 ggactagtgg atccgagctc
ggtaccaagc ttgggctgca ggtcgatggg ctgcctcggc 1440 aacagtaaga
ccgaggacca gcgcaacgag gagaaggcgc agcgcgaggc caacaaaaag 1500
atcgagaagc agctgcagaa ggacaagcag gtctaccggg ccacgcaccg cctgctgctg
1560 ctgggtgctg gagagtctgg caaaagcacc attgtgaagc agatgaggat
cctacatgtt 1620 aatgggttta acggagaggg cggcgaagag gacccgcagg
ctgcaaggag caacagcgat 1680 ggtgagaagg ccaccaaagt gcaggacatc
aaaaacaacc tgaaggaggc cattgaaacc 1740 attgtggccg ccatgagcaa
cctggtgccc cccgtggagc tggccaaccc tgagaaccag 1800 ttcagagtgg
actacattct gagcgtgatg aacgtgccaa actttgactt cccacctgaa 1860
ttctatgagc atgccaaggc tctgtgggag gatgagggag ttcgtgcctg ctacgagcgc
1920 tccaacgagt accagctgat cgactgtgcc cagtacttcc tggacaagat
tgatgtgatc 1980 aagcaggccg actacgtgcc aagtgaccag gacctgcttc
gctgccgcgt cctgacctct 2040 ggaatctttg agaccaagtt ccaggtggac
aaagtcaact tccacatgtt cgatgtgggc 2100 ggccagcgcg atgaacgccg
caagtggatc cagtgcttca atgatgtgac tgccatcatc 2160 ttcgtggtgg
ccagcagcag ctacaacatg gtcatccggg aggacaacca gaccaaccgt 2220
ctgcaggagg ctctgaacct cttcaagagc atctggaaca acagatggct gcgtaccatc
2280 tctgtgatcc tcttcctcaa caagcaagat ctgcttgctg agaaggtcct
cgctgggaaa 2340 tcgaagattg aggactactt tccagagttc gctcgctaca
ccactcctga ggatgcgact 2400 cccgagcccg gagaggaccc acgcgtgacc
cgggccaagt acttcatccg ggatgagttt 2460 ctgagaatca gcactgctag
tggagatgga cgtcactact gctaccctca ctttacctgc 2520 gccgtggaca
ctgagaacat ccgccgtgtc ttcaacgact gccgtgacat catccagcgc 2580
atgcatcttc gccaatacga gctgctctaa 2610 100 869 PRT Homo sapiens and
Rat 100 Met Glu Ser Ser Pro Ile Pro Gln Ser Ser Gly Asn Ser Ser Thr
Leu 1 5 10 15 Gly Arg Val Pro Gln Thr Pro Gly Pro Ser Thr Ala Ser
Gly Val Pro 20 25 30 Glu Val Gly Leu Arg Asp Val Ala Ser Glu Ser
Val Ala Leu Phe Phe 35 40 45 Met Leu Leu Leu Asp Leu Thr Ala Val
Ala Gly Asn Ala Ala Val Met 50 55 60 Ala Val Ile Ala Lys Thr Pro
Ala Leu Arg Lys Phe Val Phe Val Phe 65 70 75 80 His Leu Cys Leu Val
Asp Leu Leu Ala Ala Leu Thr Leu Met Pro Leu 85 90 95 Ala Met Leu
Ser Ser Ser Ala Leu Phe Asp His Ala Leu Phe Gly Glu 100 105 110 Val
Ala Cys Arg Leu Tyr Leu Phe Leu Ser Val Cys Phe Val Ser Leu 115 120
125 Ala Ile Leu Ser Val Ser Ala Ile Asn Val Glu Arg Tyr Tyr Tyr Val
130 135 140 Val His Pro Met Arg Tyr Glu Val Arg Met Thr Leu Gly Leu
Val Ala 145 150 155 160 Ser Val Leu Val Gly Val Trp Val Lys Ala Leu
Ala Met Ala Ser Val 165 170 175 Pro Val Leu Gly Arg Val Ser Trp Glu
Glu Gly Ala Pro Ser Val Pro 180 185 190 Pro Gly Cys Ser Leu Gln Trp
Ser His Ser Ala Tyr Cys Gln Leu Phe 195 200 205 Val Val Val Phe Ala
Val Leu Tyr Phe Leu Leu Pro Leu Leu Leu Ile 210 215 220 Leu Val Val
Tyr Cys Ser Met Phe Arg Val Ala Arg Val Ala Ala Met 225 230 235 240
Gln His Gly Pro Leu Pro Thr Trp Met Glu Thr Pro Arg Gln Arg Ser 245
250 255 Glu Ser Leu Ser Ser Arg Ser Thr Met Val Thr Ser Ser Gly Ala
Pro 260 265 270 Gln Thr Thr Pro His Arg Thr Phe Gly Gly Gly Lys Ala
Ala Val Val 275 280 285 Leu Leu Ala Val Gly Gly Gln Phe Leu Leu Cys
Trp Leu Pro Tyr Phe 290 295 300 Ser Phe His Leu Tyr Val Ala Leu Ser
Ala Gln Pro Ile Ser Thr Gly 305 310 315 320 Gln Val Glu Ser Val Val
Thr Trp Ile Gly Tyr Phe Cys Phe Thr Ser 325 330 335 Asn Pro Phe Phe
Tyr Gly Cys Leu Asn Arg Gln Ile Arg Gly Glu Leu 340 345 350 Ser Lys
Gln Phe Val Cys Phe Phe Lys Pro Ala Pro Glu Glu Glu Leu 355 360 365
Arg Leu Pro Ser Arg Glu Gly Ser Ile Glu Glu Asn Phe Leu Gln Phe 370
375 380 Leu Gln Gly Thr Gly Cys Pro Ser Glu Ser Trp Val Ser Arg Pro
Leu 385 390 395 400 Pro Ser Pro Lys Gln Glu Pro Pro Ala Val Asp Phe
Arg Ile Pro Gly 405 410 415 Gln Ile Ala Glu Glu Thr Ser Glu Phe Leu
Glu Gln Gln Leu Thr Ser 420 425 430 Asp Ile Ile Met Ser Asp Ser Tyr
Leu Arg Pro Ala Ala Ser Pro Arg 435 440 445 Leu Glu Ser Ala Ile Ser
Ala Glu Phe His His Thr Gly Leu Val Asp 450 455 460 Pro Ser Ser Val
Pro Ser Leu Gly Cys Arg Ser Met Gly Cys Leu Gly 465 470 475 480 Asn
Ser Lys Thr Glu Asp Gln Arg Asn Glu Glu Lys Ala Gln Arg Glu 485 490
495 Ala Asn Lys Lys Ile Glu Lys Gln Leu Gln Lys Asp Lys Gln Val Tyr
500 505 510 Arg Ala Thr His Arg Leu Leu Leu Leu Gly Ala Gly Glu Ser
Gly Lys 515 520 525 Ser Thr Ile Val Lys Gln Met Arg Ile Leu His Val
Asn Gly Phe Asn 530 535 540 Gly Glu Gly Gly Glu Glu Asp Pro Gln Ala
Ala Arg Ser Asn Ser Asp 545 550 555 560 Gly Glu Lys Ala Thr Lys Val
Gln Asp Ile Lys Asn Asn Leu Lys Glu 565 570 575 Ala Ile Glu Thr Ile
Val Ala Ala Met Ser Asn Leu Val Pro Pro Val 580 585 590 Glu Leu Ala
Asn Pro Glu Asn Gln Phe Arg Val Asp Tyr Ile Leu Ser 595 600 605 Val
Met Asn Val Pro Asn Phe Asp Phe Pro Pro Glu Phe Tyr Glu His 610 615
620 Ala Lys Ala Leu Trp Glu Asp Glu Gly Val Arg Ala Cys Tyr Glu Arg
625 630 635 640 Ser Asn Glu Tyr Gln Leu Ile Asp Cys Ala Gln Tyr Phe
Leu Asp Lys 645 650 655 Ile Asp Val Ile Lys Gln Ala Asp Tyr Val Pro
Ser Asp Gln Asp Leu 660 665 670 Leu Arg Cys Arg Val Leu Thr Ser Gly
Ile Phe Glu Thr Lys Phe Gln 675 680 685 Val Asp Lys Val Asn Phe His
Met Phe Asp Val Gly Gly Gln Arg Asp 690 695 700 Glu Arg Arg Lys Trp
Ile Gln Cys Phe Asn Asp Val Thr Ala Ile Ile 705 710 715 720 Phe Val
Val Ala Ser Ser Ser Tyr Asn Met Val Ile Arg Glu Asp Asn 725 730 735
Gln Thr Asn Arg Leu Gln Glu Ala Leu Asn Leu Phe Lys Ser Ile Trp 740
745 750 Asn Asn Arg Trp Leu Arg Thr Ile Ser Val Ile Leu Phe Leu Asn
Lys 755 760 765 Gln Asp Leu Leu Ala Glu Lys Val Leu Ala Gly Lys Ser
Lys Ile Glu 770 775 780 Asp Tyr Phe Pro Glu Phe Ala Arg Tyr Thr Thr
Pro Glu Asp Ala Thr 785 790 795 800 Pro Glu Pro Gly Glu Asp Pro Arg
Val Thr Arg Ala Lys Tyr Phe Ile 805 810 815 Arg Asp Glu Phe Leu Arg
Ile Ser Thr Ala Ser Gly Asp Gly Arg His 820 825 830 Tyr Cys Tyr Pro
His Phe Thr Cys Ala Val Asp Thr Glu Asn Ile Arg 835 840 845 Arg Val
Phe Asn Asp Cys Arg Asp Ile Ile Gln Arg Met His Leu Arg 850 855 860
Gln Tyr Glu Leu Leu 865 101 30 DNA Artificial Sequence Novel
Sequence 101 tctagaatga cgtccacctg caccaacagc 30 102 34 DNA
Artificial Sequence Novel Sequence 102 gatatcgcag gaaaagtagc
agaatcgtag gaag 34 103 2781 DNA Homo Sapiens and Rat 103 atgacgtcca
cctgcaccaa cagcacgcgc gagagtaaca gcagccacac gtgcatgccc 60
ctctccaaaa tgcccatcag cctggcccac ggcatcatcc gctcaaccgt gctggttatc
120 ttcctcgccg cctctttcgt cggcaacata gtgctggcgc tagtgttgca
gcgcaagccg 180 cagctgctgc aggtgaccaa ccgttttatc tttaacctcc
tcgtcaccga cctgctgcag 240 atttcgctcg tggccccctg ggtggtggcc
acctctgtgc ctctcttctg gcccctcaac 300 agccacttct gcacggccct
ggttagcctc acccacctgt tcgccttcgc cagcgtcaac 360 accattgtcg
tggtgtcagt ggatcgctac ttgtccatca tccaccctct ctcctacccg 420
tccaagatga cccagcgccg cggttacctg ctcctctatg gcacctggat tgtggccatc
480 ctgcagagca ctcctccact ctacggctgg ggccaggctg cctttgatga
gcgcaatgct 540 ctctgctcca tgatctgggg ggccagcccc agctacacta
ttctcagcgt ggtgtccttc 600 atcgtcattc cactgattgt catgattgcc
tgctactccg tggtgttctg tgcagcccgg 660 aggcagcatg ctctgctgta
caatgtcaag agacacagct tggaagtgcg agtcaaggac 720 tgtgtggaga
atgaggatga agagggagca gagaagaagg aggagttcca ggatgagagt 780
gagtttcgcc gccagcatga aggtgaggtc aaggccaagg agggcagaat ggaagccaag
840 gacggcagcc tgaaggccaa ggaaggaagc acggggacca gtgagagtag
tgtagaggcc 900 aggggcagcg aggaggtcag agagagcagc acggtggcca
gcgacggcag catggagggt 960 aaggaaggca gcaccaaagt tgaggagaac
agcatgaagg cagacaaggg tcgcacagag 1020 gtcaaccagt gcagcattga
cttgggtgaa gatgacatgg agtttggtga agacgacatc 1080 aatttcagtg
aggatgacgt cgaggcagtg aacatcccgg agagcctccc acccagtcgt 1140
cgtaacagca acagcaaccc tcctctgccc aggtgctacc agtgcaaagc tgctaaagtg
1200 atcttcatca tcattttctc ctatgtgcta tccctggggc cctactgctt
tttagcagtc 1260 ctggccgtgt gggtggatgt cgaaacccag gtaccccagt
gggtgatcac cataatcatc 1320 tggcttttct tcctgcagtg ctgcatccac
ccctatgtct atggctacat gcacaagacc 1380 attaagaagg aaatccagga
catgctgaag aagttcttct gcaaggaaaa gcccccgaaa 1440 gaagatagcc
acccagacct gcccggaaca gagggtggga ctgaaggcaa gattgtccct 1500
tcctacgatt ctgctacttt tcctgcgata tctgcagaat tccaccacac tggactagtg
1560 gatccgagct cggtaccaag cttgggctgc aggtcgatgg gctgcctcgg
caacagtaag 1620 accgaggacc agcgcaacga ggagaaggcg cagcgcgagg
ccaacaaaaa gatcgagaag 1680 cagctgcaga aggacaagca ggtctaccgg
gccacgcacc gcctgctgct gctgggtgct 1740 ggagagtctg gcaaaagcac
cattgtgaag cagatgagga tcctacatgt taatgggttt 1800 aacggagagg
gcggcgaaga ggacccgcag gctgcaagga gcaacagcga tggtgagaag 1860
gccaccaaag tgcaggacat caaaaacaac ctgaaggagg ccattgaaac cattgtggcc
1920 gccatgagca acctggtgcc ccccgtggag ctggccaacc ctgagaacca
gttcagagtg 1980 gactacattc tgagcgtgat gaacgtgcca aactttgact
tcccacctga attctatgag 2040 catgccaagg ctctgtggga ggatgaggga
gttcgtgcct gctacgagcg ctccaacgag 2100 taccagctga tcgactgtgc
ccagtacttc ctggacaaga ttgatgtgat caagcaggcc 2160 gactacgtgc
caagtgacca ggacctgctt cgctgccgcg tcctgacctc tggaatcttt 2220
gagaccaagt tccaggtgga caaagtcaac ttccacatgt tcgatgtggg cggccagcgc
2280 gatgaacgcc gcaagtggat ccagtgcttc aatgatgtga ctgccatcat
cttcgtggtg 2340 gccagcagca gctacaacat ggtcatccgg gaggacaacc
agaccaaccg tctgcaggag 2400 gctctgaacc tcttcaagag catctggaac
aacagatggc tgcgtaccat ctctgtgatc 2460 ctcttcctca acaagcaaga
tctgcttgct gagaaggtcc tcgctgggaa atcgaagatt 2520 gaggactact
ttccagagtt cgctcgctac accactcctg aggatgcgac tcccgagccc 2580
ggagaggacc cacgcgtgac ccgggccaag tacttcatcc gggatgagtt tctgagaatc
2640 agcactgcta gtggagatgg acgtcactac tgctaccctc actttacctg
cgccgtggac 2700 actgagaaca tccgccgtgt cttcaacgac tgccgtgaca
tcatccagcg catgcatctt 2760 cgccaatacg agctgctcta a 2781 104 926 PRT
Homo sapiens and Rat 104 Met Thr Ser Thr Cys Thr Asn Ser Thr Arg
Glu Ser Asn Ser Ser His 1 5 10 15 Thr Cys Met Pro Leu Ser Lys Met
Pro Ile Ser Leu Ala His Gly Ile 20 25 30 Ile Arg Ser Thr Val Leu
Val Ile Phe Leu Ala Ala Ser Phe Val Gly 35 40 45 Asn Ile Val Leu
Ala Leu Val Leu Gln Arg Lys Pro Gln Leu Leu Gln 50 55 60 Val Thr
Asn Arg Phe Ile Phe Asn Leu Leu Val Thr Asp Leu Leu Gln 65 70 75 80
Ile Ser Leu Val Ala Pro Trp Val Val Ala Thr Ser Val Pro Leu Phe 85
90 95 Trp Pro Leu Asn Ser His Phe Cys Thr Ala Leu Val Ser Leu Thr
His 100 105 110 Leu Phe Ala Phe Ala Ser Val Asn Thr Ile Val Val Val
Ser Val Asp 115 120 125 Arg Tyr Leu Ser Ile Ile His Pro Leu Ser Tyr
Pro Ser Lys Met Thr 130 135 140 Gln Arg Arg Gly Tyr Leu Leu Leu Tyr
Gly Thr Trp Ile Val Ala Ile 145 150 155 160 Leu Gln Ser Thr Pro Pro
Leu Tyr Gly Trp Gly Gln Ala Ala Phe Asp 165 170 175 Glu Arg Asn Ala
Leu Cys Ser Met Ile Trp Gly Ala Ser Pro Ser Tyr 180 185 190 Thr Ile
Leu Ser Val Val Ser Phe Ile Val Ile Pro Leu Ile Val Met 195 200 205
Ile Ala Cys Tyr Ser Val Val Phe Cys Ala Ala Arg Arg Gln His Ala 210
215 220 Leu Leu Tyr Asn Val Lys Arg His Ser Leu Glu Val Arg Val Lys
Asp 225 230 235 240 Cys Val Glu Asn Glu Asp Glu Glu Gly Ala Glu Lys
Lys Glu Glu Phe 245 250 255 Gln Asp Glu Ser Glu Phe Arg Arg Gln His
Glu Gly Glu Val Lys Ala 260 265 270 Lys Glu Gly Arg Met Glu Ala Lys
Asp Gly Ser Leu Lys Ala Lys Glu 275 280 285 Gly Ser Thr Gly Thr Ser
Glu Ser Ser Val Glu Ala Arg Gly Ser Glu 290 295 300 Glu Val Arg Glu
Ser Ser Thr Val Ala Ser Asp Gly Ser Met Glu Gly 305 310 315 320 Lys
Glu Gly Ser Thr Lys Val Glu Glu Asn Ser Met Lys Ala Asp Lys 325 330
335 Gly Arg Thr Glu Val Asn Gln Cys Ser Ile Asp Leu Gly Glu Asp Asp
340 345 350 Met Glu Phe Gly Glu Asp Asp Ile Asn Phe Ser Glu Asp Asp
Val Glu 355 360 365 Ala Val Asn Ile Pro Glu Ser Leu Pro Pro Ser Arg
Arg Asn Ser Asn 370 375 380 Ser Asn Pro Pro Leu Pro Arg Cys Tyr Gln
Cys Lys Ala Ala Lys Val 385 390 395 400 Ile Phe Ile Ile Ile Phe Ser
Tyr Val Leu Ser Leu Gly Pro Tyr Cys 405 410 415 Phe Leu Ala Val Leu
Ala Val Trp Val Asp Val Glu Thr Gln Val Pro 420 425 430 Gln Trp Val
Ile Thr Ile Ile Ile Trp Leu Phe Phe Leu Gln Cys Cys 435 440 445 Ile
His Pro Tyr Val Tyr Gly Tyr Met His Lys Thr Ile Lys Lys Glu 450 455
460 Ile Gln Asp Met Leu Lys Lys Phe Phe Cys Lys Glu Lys Pro Pro Lys
465 470 475 480 Glu Asp Ser His Pro Asp Leu Pro Gly Thr Glu Gly Gly
Thr Glu Gly 485 490 495 Lys Ile Val Pro Ser Tyr Asp Ser Ala Thr Phe
Pro Ala Ile Ser Ala 500 505 510 Glu Phe His His Thr Gly Leu Val Asp
Pro Ser Ser Val Pro Ser Leu 515 520 525 Gly Cys Arg Ser Met Gly Cys
Leu Gly Asn Ser Lys Thr Glu Asp Gln 530 535 540 Arg Asn Glu Glu Lys
Ala Gln Arg Glu Ala Asn Lys Lys Ile Glu Lys 545 550 555 560 Gln Leu
Gln Lys Asp Lys Gln Val Tyr Arg Ala Thr His Arg Leu Leu 565 570 575
Leu Leu Gly Ala Gly Glu Ser Gly Lys Ser Thr Ile Val Lys Gln Met 580
585 590 Arg Ile Leu His Val Asn Gly Phe Asn Gly Glu Gly Gly Glu Glu
Asp 595 600 605 Pro Gln Ala Ala Arg Ser Asn Ser Asp Gly Glu Lys Ala
Thr Lys Val 610 615 620 Gln Asp Ile Lys Asn Asn Leu Lys Glu Ala Ile
Glu Thr Ile Val Ala 625 630 635 640 Ala Met Ser Asn Leu Val Pro Pro
Val Glu Leu Ala Asn Pro
Glu Asn 645 650 655 Gln Phe Arg Val Asp Tyr Ile Leu Ser Val Met Asn
Val Pro Asn Phe 660 665 670 Asp Phe Pro Pro Glu Phe Tyr Glu His Ala
Lys Ala Leu Trp Glu Asp 675 680 685 Glu Gly Val Arg Ala Cys Tyr Glu
Arg Ser Asn Glu Tyr Gln Leu Ile 690 695 700 Asp Cys Ala Gln Tyr Phe
Leu Asp Lys Ile Asp Val Ile Lys Gln Ala 705 710 715 720 Asp Tyr Val
Pro Ser Asp Gln Asp Leu Leu Arg Cys Arg Val Leu Thr 725 730 735 Ser
Gly Ile Phe Glu Thr Lys Phe Gln Val Asp Lys Val Asn Phe His 740 745
750 Met Phe Asp Val Gly Gly Gln Arg Asp Glu Arg Arg Lys Trp Ile Gln
755 760 765 Cys Phe Asn Asp Val Thr Ala Ile Ile Phe Val Val Ala Ser
Ser Ser 770 775 780 Tyr Asn Met Val Ile Arg Glu Asp Asn Gln Thr Asn
Arg Leu Gln Glu 785 790 795 800 Ala Leu Asn Leu Phe Lys Ser Ile Trp
Asn Asn Arg Trp Leu Arg Thr 805 810 815 Ile Ser Val Ile Leu Phe Leu
Asn Lys Gln Asp Leu Leu Ala Glu Lys 820 825 830 Val Leu Ala Gly Lys
Ser Lys Ile Glu Asp Tyr Phe Pro Glu Phe Ala 835 840 845 Arg Tyr Thr
Thr Pro Glu Asp Ala Thr Pro Glu Pro Gly Glu Asp Pro 850 855 860 Arg
Val Thr Arg Ala Lys Tyr Phe Ile Arg Asp Glu Phe Leu Arg Ile 865 870
875 880 Ser Thr Ala Ser Gly Asp Gly Arg His Tyr Cys Tyr Pro His Phe
Thr 885 890 895 Cys Ala Val Asp Thr Glu Asn Ile Arg Arg Val Phe Asn
Asp Cys Arg 900 905 910 Asp Ile Ile Gln Arg Met His Leu Arg Gln Tyr
Glu Leu Leu 915 920 925 105 23 DNA Artificial Sequence Novel
Sequence 105 catgtatgcc agcgtcctgc tcc 23 106 24 DNA Artificial
Sequence Novel Sequence 106 gctatgcctg aagccagtct tgtg 24 107 25
DNA Artificial Sequence Novel Sequence 107 gcacctgctc ctgagcacct
tctcc 25 108 26 DNA Artificial Sequence misc_feature Novel Sequence
108 cacagcgctg cagccctgca gctggc 26 109 24 DNA Artificial Sequence
Novel Sequence 109 ccagtgatga ctctgtccag cctg 24 110 24 DNA
Artificial Sequence 4926> 110 cagacacttg gcagggacga ggtg 24 111
26 DNA Artificial Sequence Novel Sequence 111 cttgtggtct actgcagcat
gttccg 26 112 25 DNA Artificial Sequence Novel Sequence 112
catatccctc cgagtgtcca gcggc 25 113 24 DNA Artificial Sequence Novel
Sequence 113 atggatcctt atcatggctt cctc 24 114 27 DNA Artificial
Sequence Novel Sequence 114 caagaacagg tctcatctaa gagctcc 27 115 26
DNA Artificial Sequence Novel Sequence 115 ctctgatgcc atctgctgga
ttcctg 26 116 26 DNA Artificial Sequence Novel Sequence 116
gtagtccact gaaagtccag tgatcc 26 117 24 DNA Artificial Sequence
Novel Sequence 117 tggtggcgat ggccaacagc gctc 24 118 24 DNA
Artificial Sequence Novel Sequence 118 gttgcgcctt agcgacagat gacc
24 119 23 DNA Artificial Sequence Novel Sequence 119 tcaacctgta
tagcagcatc ctc 23 120 23 DNA Artificial Sequence Novel Sequence 120
aaggagtagc agaatggtta gcc 23 121 24 DNA Artificial Sequence Novel
Sequence 121 gacacctgtc agcggtcgtg tgtg 24 122 27 DNA Artificial
Sequence Novel Sequence 122 ctgatggaag tagaggctgt ccatctc 27 123 24
DNA Artificial Sequence Novel Sequence 123 gcgctgagcg cagaccagtg
gctg 24 124 24 DNA Artificial Sequence Novel Sequence 124
cacggtgacg aagggcacga gctc 24 125 24 DNA Artificial Sequence Novel
Sequence 125 agccatccct gccaggaagc atgg 24 126 25 DNA Artificial
Sequence Novel Sequence 126 ccaggtaggt gtgcagcaca atggc 25 127 25
DNA Artificial Sequence Novel Sequence 127 ctgttcaaca gggctggttg
gcaac 25 128 25 DNA Artificial Sequence Novel Sequence 128
atcatgtcta gactcatggt gatcc 25 129 6 PRT Artificial Sequence Novel
Sequence 129 Thr Leu Glu Ser Ile Met 1 5 130 5 PRT Artificial
Sequence Novel Sequence 130 Glu Tyr Asn Leu Val 1 5 131 5 PRT
Artificial Sequence Novel Sequence 131 Asp Cys Gly Leu Phe 1 5 132
36 PRT Artificial Sequence Novel Sequence 132 Gly Ala Thr Cys Ala
Ala Gly Cys Thr Thr Cys Cys Ala Thr Gly Gly 1 5 10 15 Cys Gly Thr
Gly Cys Thr Gly Cys Cys Thr Gly Ala Gly Cys Gly Ala 20 25 30 Gly
Gly Ala Gly 35 133 53 PRT Artificial Sequence Novel Sequence 133
Gly Ala Thr Cys Gly Gly Ala Thr Cys Cys Thr Thr Ala Gly Ala Ala 1 5
10 15 Cys Ala Gly Gly Cys Cys Gly Cys Ala Gly Thr Cys Cys Thr Thr
Cys 20 25 30 Ala Gly Gly Thr Thr Cys Ala Gly Cys Thr Gly Cys Ala
Gly Gly Ala 35 40 45 Thr Gly Gly Thr Gly 50 134 1164 DNA Homo
Sapien 134 atgaatcggc accatctgca ggatcacttt ctggaaatag acaagaagaa
ctgctgtgtg 60 ttccgagatg acttcattgc caaggtgttg ccgccggtgt
tggggctgga gtttatcttt 120 gggcttctgg gcaatggcct tgccctgtgg
attttctgtt tccacctcaa gtcctggaaa 180 tccagccgga ttttcctgtt
caacctggca gtagctgact ttctactgat catctgcctg 240 ccgttcgtga
tggactacta tgtgcggcgt tcagactgga actttgggga catcccttgc 300
cggctggtgc tcttcatgtt tgccatgaac cgccagggca gcatcatctt cctcacggtg
360 gtggcggtag acaggtattt ccgggtggtc catccccacc acgccctgaa
caagatctcc 420 aattggacag cagccatcat ctcttgcctt ctgtggggca
tcactgttgg cctaacagtc 480 cacctcctga agaagaagtt gctgatccag
aatggccctg caaatgtgtg catcagcttc 540 agcatctgcc ataccttccg
gtggcacgaa gctatgttcc tcctggagtt cctcctgccc 600 ctgggcatca
tcctgttctg ctcagccaga attatctgga gcctgcggca gagacaaatg 660
gaccggcatg ccaagatcaa gagagccatc accttcatca tggtggtggc catcgtcttt
720 gtcatctgct tccttcccag cgtggttgtg cggatccgca tcttctggct
cctgcacact 780 tcgggcacgc agaattgtga agtgtaccgc tcggtggacc
tggcgttctt tatcactctc 840 agcttcacct acatgaacag catgctggac
cccgtggtgt actacttctc cagcccatcc 900 tttcccaact tcttctccac
tttgatcaac cgctgcctcc agaggaagat gacaggtgag 960 ccagataata
accgcagcac gagcgtcgag ctcacagggg accccaacaa aaccagaggc 1020
gctccagagg cgttaatggc caactccggt gagccatgga gcccctctta tctgggccca
1080 acctcaaata accattccaa gaagggacat tgtcaccaag aaccagcatc
tctggagaaa 1140 cagttgggct gttgcatcga gtaa 1164 135 387 PRT Homo
sapiens 135 Met Asn Arg His His Leu Gln Asp His Phe Leu Glu Ile Asp
Lys Lys 1 5 10 15 Asn Cys Cys Val Phe Arg Asp Asp Phe Ile Ala Lys
Val Leu Pro Pro 20 25 30 Val Leu Gly Leu Glu Phe Ile Phe Gly Leu
Leu Gly Asn Gly Leu Ala 35 40 45 Leu Trp Ile Phe Cys Phe His Leu
Lys Ser Trp Lys Ser Ser Arg Ile 50 55 60 Phe Leu Phe Asn Leu Ala
Val Ala Asp Phe Leu Leu Ile Ile Cys Leu 65 70 75 80 Pro Phe Val Met
Asp Tyr Tyr Val Arg Arg Ser Asp Trp Asn Phe Gly 85 90 95 Asp Ile
Pro Cys Arg Leu Val Leu Phe Met Phe Ala Met Asn Arg Gln 100 105 110
Gly Ser Ile Ile Phe Leu Thr Val Val Ala Val Asp Arg Tyr Phe Arg 115
120 125 Val Val His Pro His His Ala Leu Asn Lys Ile Ser Asn Trp Thr
Ala 130 135 140 Ala Ile Ile Ser Cys Leu Leu Trp Gly Ile Thr Val Gly
Leu Thr Val 145 150 155 160 His Leu Leu Lys Lys Lys Leu Leu Ile Gln
Asn Gly Pro Ala Asn Val 165 170 175 Cys Ile Ser Phe Ser Ile Cys His
Thr Phe Arg Trp His Glu Ala Met 180 185 190 Phe Leu Leu Glu Phe Leu
Leu Pro Leu Gly Ile Ile Leu Phe Cys Ser 195 200 205 Ala Arg Ile Ile
Trp Ser Leu Arg Gln Arg Gln Met Asp Arg His Ala 210 215 220 Lys Ile
Lys Arg Ala Ile Thr Phe Ile Met Val Val Ala Ile Val Phe 225 230 235
240 Val Ile Cys Phe Leu Pro Ser Val Val Val Arg Ile Arg Ile Phe Trp
245 250 255 Leu Leu His Thr Ser Gly Thr Gln Asn Cys Glu Val Tyr Arg
Ser Val 260 265 270 Asp Leu Ala Phe Phe Ile Thr Leu Ser Phe Thr Tyr
Met Asn Ser Met 275 280 285 Leu Asp Pro Val Val Tyr Tyr Phe Ser Ser
Pro Ser Phe Pro Asn Phe 290 295 300 Phe Ser Thr Leu Ile Asn Arg Cys
Leu Gln Arg Lys Met Thr Gly Glu 305 310 315 320 Pro Asp Asn Asn Arg
Ser Thr Ser Val Glu Leu Thr Gly Asp Pro Asn 325 330 335 Lys Thr Arg
Gly Ala Pro Glu Ala Leu Met Ala Asn Ser Gly Glu Pro 340 345 350 Trp
Ser Pro Ser Tyr Leu Gly Pro Thr Ser Asn Asn His Ser Lys Lys 355 360
365 Gly His Cys His Gln Glu Pro Ala Ser Leu Glu Lys Gln Leu Gly Cys
370 375 380 Cys Ile Glu 385 136 1083 DNA Mouse 136 atgagcaagt
cagaccattt tctagtgata aacggcaaga actgctgtgt gttccgagat 60
gaaaacatcg ccaaggtctt gccaccggtg ttggggctgg aatttgtgtt cggactcctg
120 ggcaatggcc ttgccttgtg gattttctgt ttccacctca agtcctggaa
atccagccgg 180 attttcttgt tcaacttggc cgtggctgac tttctcctga
tcatctgcct gccgttcctg 240 acggacaact atgtccataa ctgggactgg
aggttcggag gcatcccttg ccgtgtgatg 300 ctcttcatgt tggctatgaa
ccgacagggc agcatcatct tcctcaccgt ggtggctgtg 360 gaccgctact
tccgggtggt ccatccacac cacttcttga acaagatctc caaccggacg 420
gcggccatca tttcttgctt cttgtggggt ctcaccatcg gcctgactgt ccacctcctc
480 tatacaaaca tgatgaccaa aaatggcgag gcatatctgt gtagcagctt
cagcatctgt 540 tacaacttca ggtggcacga tgctatgttc ctcttggaat
tcttcttgcc cctggccatc 600 atcttgttct gctcaggcag gatcatctgg
agcctgaggc agagacagat ggacagacat 660 gccaagatca agagggccat
caacttcatc atggtggtgg ctattgtatt catcatttgc 720 ttcctaccca
gtgtggctgt gcgcatccgc atcttctggc ttctctacaa atataacgta 780
cgcaactgtg acatctactc ctcggtggac ctggctttct ttaccaccct tagctttacc
840 tacatgaaca gcatgctgga ccctgtggtc tactatttct ccagcccatc
tttccccaac 900 ttcttctcca cgtgtatcaa ccgctgcctt cgaaagaaaa
cattgggtga acccgataat 960 aaccgaagca ctagtgtgga gctcacgggg
gaccccagca caaccagaag tattccaggg 1020 gcgctaatgg ctgaccccag
tgagccaggc agcccccctt atctggcttc cacatctcgt 1080 taa 1083 137 360
PRT Mouse 137 Met Ser Lys Ser Asp His Phe Leu Val Ile Asn Gly Lys
Asn Cys Cys 1 5 10 15 Val Phe Arg Asp Glu Asn Ile Ala Lys Val Leu
Pro Pro Val Leu Gly 20 25 30 Leu Glu Phe Val Phe Gly Leu Leu Gly
Asn Gly Leu Ala Leu Trp Ile 35 40 45 Phe Cys Phe His Leu Lys Ser
Trp Lys Ser Ser Arg Ile Phe Leu Phe 50 55 60 Asn Leu Ala Val Ala
Asp Phe Leu Leu Ile Ile Cys Leu Pro Phe Leu 65 70 75 80 Thr Asp Asn
Tyr Val His Asn Trp Asp Trp Arg Phe Gly Gly Ile Pro 85 90 95 Cys
Arg Val Met Leu Phe Met Leu Ala Met Asn Arg Gln Gly Ser Ile 100 105
110 Ile Phe Leu Thr Val Val Ala Val Asp Arg Tyr Phe Arg Val Val His
115 120 125 Pro His His Phe Leu Asn Lys Ile Ser Asn Arg Thr Ala Ala
Ile Ile 130 135 140 Ser Cys Phe Leu Trp Gly Leu Thr Ile Gly Leu Thr
Val His Leu Leu 145 150 155 160 Tyr Thr Asn Met Met Thr Lys Asn Gly
Glu Ala Tyr Leu Cys Ser Ser 165 170 175 Phe Ser Ile Cys Tyr Asn Phe
Arg Trp His Asp Ala Met Phe Leu Leu 180 185 190 Glu Phe Phe Leu Pro
Leu Ala Ile Ile Leu Phe Cys Ser Gly Arg Ile 195 200 205 Ile Trp Ser
Leu Arg Gln Arg Gln Met Asp Arg His Ala Lys Ile Lys 210 215 220 Arg
Ala Ile Asn Phe Ile Met Val Val Ala Ile Val Phe Ile Ile Cys 225 230
235 240 Phe Leu Pro Ser Val Ala Val Arg Ile Arg Ile Phe Trp Leu Leu
Tyr 245 250 255 Lys Tyr Asn Val Arg Asn Cys Asp Ile Tyr Ser Ser Val
Asp Leu Ala 260 265 270 Phe Phe Thr Thr Leu Ser Phe Thr Tyr Met Asn
Ser Met Leu Asp Pro 275 280 285 Val Val Tyr Tyr Phe Ser Ser Pro Ser
Phe Pro Asn Phe Phe Ser Thr 290 295 300 Cys Ile Asn Arg Cys Leu Arg
Lys Lys Thr Leu Gly Glu Pro Asp Asn 305 310 315 320 Asn Arg Ser Thr
Ser Val Glu Leu Thr Gly Asp Pro Ser Thr Thr Arg 325 330 335 Ser Ile
Pro Gly Ala Leu Met Ala Asp Pro Ser Glu Pro Gly Ser Pro 340 345 350
Pro Tyr Leu Ala Ser Thr Ser Arg 355 360 138 1086 DNA Rat 138
atgagcaagt cagaccattt tctagtgata aacggcaaga actgctgtgt gttccgagat
60 gaaaacatcg ccaaggtcct gccgccggtg ttggggctgg agtttgtgtt
tggactcctg 120 ggtaatggcc ttgccttgtg gatcttctgt ttccatctca
aatcctggaa atccagccgg 180 attttcttgt tcaacctggc cgtggctgac
tttctcctga tcatttgctt gccgttcttg 240 acggacaact atgtccagaa
ctgggactgg aggttcggga gcatcccctg ccgcgtgatg 300 ctcttcatgt
tggccatgaa ccgacagggc agcatcatct tcctcacggt ggtggctgtg 360
gacaggtact tcagggtggt ccacccgcac cacttcctga acaagatctc caaccggacg
420 gcggccatca tctcttgctt cctgtggggc atcaccatcg ggcctggaca
gtccaccttc 480 ctctacacgg acatgatgac ccgaaacggc gatgcaaacc
tgtgcagcag ttttagcatc 540 tgctacactt tcaggtggca cgatgcaatg
ttcctcttgg aattcttcct gcccctgggc 600 atcatcctgt tctgctctgg
caggatcatt tggagcctaa ggcagagaca gatggacagg 660 cacgtcaaga
tcaagagggc catcaacttc atcatggtgg ttgccattgt gtttgtcatc 720
tgcttcctgc ccagtgtggc cgtgaggatc cgcatcttct ggctcctcta caaacacaac
780 gtgaggaact gtgacatcta ctcctctgtg gacttggcct tcttcaccac
ccttagcttt 840 acctacatga acagcatgct cgacccggtg gtctactatt
tctccagccc atctttcccc 900 aacttcttct ccacgtgcat caaccgttgc
cttcgaagga aaaccttggg cgaaccagat 960 aataaccgga gcacgagtgt
ggagctcacg ggggacccca gcacaatcag aagtattcca 1020 ggggcattaa
tgactgaccc cagtgagcca ggcagccccc cttatctggc ttccacatct 1080 cgttaa
1086 139 361 PRT Rat 139 Met Ser Lys Ser Asp His Phe Leu Val Ile
Asn Gly Lys Asn Cys Cys 1 5 10 15 Val Phe Arg Asp Glu Asn Ile Ala
Lys Val Leu Pro Pro Val Leu Gly 20 25 30 Leu Glu Phe Val Phe Gly
Leu Leu Gly Asn Gly Leu Ala Leu Trp Ile 35 40 45 Phe Cys Phe His
Leu Lys Ser Trp Lys Ser Ser Arg Ile Phe Leu Phe 50 55 60 Asn Leu
Ala Val Ala Asp Phe Leu Leu Ile Ile Cys Leu Pro Phe Leu 65 70 75 80
Thr Asp Asn Tyr Val Gln Asn Trp Asp Trp Arg Phe Gly Ser Ile Pro 85
90 95 Cys Arg Val Met Leu Phe Met Leu Ala Met Asn Arg Gln Gly Ser
Ile 100 105 110 Ile Phe Leu Thr Val Val Ala Val Asp Arg Tyr Phe Arg
Val Val His 115 120 125 Pro His His Phe Leu Asn Lys Ile Ser Asn Arg
Thr Ala Ala Ile Ile 130 135 140 Ser Cys Phe Leu Trp Gly Ile Thr Ile
Gly Pro Gly Gln Ser Thr Phe 145 150 155 160 Leu Tyr Thr Asp Met Met
Thr Arg Asn Gly Asp Ala Asn Leu Cys Ser 165 170 175 Ser Phe Ser Ile
Cys Tyr Thr Phe Arg Trp His Asp Ala Met Phe Leu 180 185 190 Leu Glu
Phe Phe Leu Pro Leu Gly Ile Ile Leu Phe Cys Ser Gly Arg 195 200 205
Ile Ile Trp Ser Leu Arg Gln Arg Gln Met Asp Arg His Val Lys Ile 210
215 220 Lys Arg Ala Ile Asn Phe Ile Met Val Val Ala Ile Val Phe Val
Ile 225 230 235 240 Cys Phe Leu Pro Ser Val Ala Val Arg Ile Arg Ile
Phe Trp Leu Leu 245 250 255 Tyr Lys His Asn Val Arg Asn Cys Asp Ile
Tyr Ser Ser Val Asp Leu 260 265 270 Ala Phe Phe Thr Thr Leu Ser Phe
Thr Tyr Met Asn Ser Met Leu Asp 275 280 285 Pro Val Val Tyr Tyr Phe
Ser Ser Pro Ser Phe Pro Asn Phe Phe Ser 290
295 300 Thr Cys Ile Asn Arg Cys Leu Arg Arg Lys Thr Leu Gly Glu Pro
Asp 305 310 315 320 Asn Asn Arg Ser Thr Ser Val Glu Leu Thr Gly Asp
Pro Ser Thr Ile 325 330 335 Arg Ser Ile Pro Gly Ala Leu Met Thr Asp
Pro Ser Glu Pro Gly Ser 340 345 350 Pro Pro Tyr Leu Ala Ser Thr Ser
Arg 355 360 140 30 DNA Artificial Novel Sequence 140 atgagcaagt
cagaccattt tctagtgata 30 141 25 DNA Artificial Novel Sequence 141
ttatctggct tccacatctc gttaa 25 142 30 DNA Artificial Novel Sequence
142 atgagcaagt cagaccattt tctagtgata 30 143 25 DNA Artificial Novel
Sequence 143 ttatctggct tccacatctc gttaa 25 144 27 DNA Artificial
Novel Sequence 144 ctgatggaca actatgtgag gcgttgg 27 145 27 DNA
Artificial Novel Sequence 145 gctgaagctg ctgcacaaat ttgcacc 27 146
19 DNA Artificial Novel Sequence 146 ctactatgtg cggcgttca 19 147 21
DNA Artificial Novel Sequence 147 cccttcttgg aatggttatt t 21 148 20
DNA Artificial Novel Sequence 148 gcactcatga atcggcacca 20 149 20
DNA Artificial Novel Sequence 149 cagtgacatt actcgatgca 20 150 1032
DNA Mouse 150 atggacaacg ggtcgtgctg tctcatcgag ggggagccca
tctcccaagt gatgcctcct 60 ctactcatcc tggtcttcgt gcttggcgcc
ctgggcaacg gcatagccct gtgcggcttc 120 tgctttcaca tgaagacctg
gaagtcaagc actatttacc ttttcaactt ggctgtggcc 180 gattttctcc
tcatgatctg cttacccctt cggacagact actacctcag acgcagacac 240
tggatttttg gagatatcgc ctgtcgcctg gtcctcttca agctggccat gaatagggcc
300 gggagcattg tcttcctcac tgtggtggct gtggataggt atttcaaagt
ggtccacccc 360 caccatatgg tgaatgccat ctccaaccgg actgccgccg
ccaccgcctg tgtcctctgg 420 actttggtca tcttggggac tgtgtatctt
ctgatggaga gtcacctgtg tgtgcagggg 480 acactgtcgt cctgtgagag
cttcatcatg gagtcagcca acgggtggca cgatgtcatg 540 ttccagctgg
agttcttcct gcccctgaca atcatcttgt tctgctcggt caacgttgtt 600
tggagcctga gacggaggca gcagctgacc agacaggctc ggatgaggag ggccacccgg
660 ttcatcatgg tggtggcttc tgtgttcatc acgtgttacc tgcccagcgt
gctggctagg 720 ctctacttcc tctggacggt gcccactagt gcctgtgacc
cctctgtcca cacagccctc 780 cacgtcaccc tgagcttcac ctacctgaac
agtatgctgg atccccttgt atattacttc 840 tcaagcccct cgctccccaa
attctacacc aagctcacaa tctgcagcct gaagcccaaa 900 cgcccaggac
gcacgaagac gcggaggtca gaagagatgc caatttcgaa cctctgcagt 960
aagagctcca tcgatggggc aaatcgttcc cagaggccat ctgacgggca gtgggatctc
1020 caagtgtgtt ga 1032 151 343 PRT Mouse 151 Met Asp Asn Gly Ser
Cys Cys Leu Ile Glu Gly Glu Pro Ile Ser Gln 1 5 10 15 Val Met Pro
Pro Leu Leu Ile Leu Val Phe Val Leu Gly Ala Leu Gly 20 25 30 Asn
Gly Ile Ala Leu Cys Gly Phe Cys Phe His Met Lys Thr Trp Lys 35 40
45 Ser Ser Thr Ile Tyr Leu Phe Asn Leu Ala Val Ala Asp Phe Leu Leu
50 55 60 Met Ile Cys Leu Pro Leu Arg Thr Asp Tyr Tyr Leu Arg Arg
Arg His 65 70 75 80 Trp Ile Phe Gly Asp Ile Ala Cys Arg Leu Val Leu
Phe Lys Leu Ala 85 90 95 Met Asn Arg Ala Gly Ser Ile Val Phe Leu
Thr Val Val Ala Val Asp 100 105 110 Arg Tyr Phe Lys Val Val His Pro
His His Met Val Asn Ala Ile Ser 115 120 125 Asn Arg Thr Ala Ala Ala
Thr Ala Cys Val Leu Trp Thr Leu Val Ile 130 135 140 Leu Gly Thr Val
Tyr Leu Leu Met Glu Ser His Leu Cys Val Gln Gly 145 150 155 160 Thr
Leu Ser Ser Cys Glu Ser Phe Ile Met Glu Ser Ala Asn Gly Trp 165 170
175 His Asp Val Met Phe Gln Leu Glu Phe Phe Leu Pro Leu Thr Ile Ile
180 185 190 Leu Phe Cys Ser Val Asn Val Val Trp Ser Leu Arg Arg Arg
Gln Gln 195 200 205 Leu Thr Arg Gln Ala Arg Met Arg Arg Ala Thr Arg
Phe Ile Met Val 210 215 220 Val Ala Ser Val Phe Ile Thr Cys Tyr Leu
Pro Ser Val Leu Ala Arg 225 230 235 240 Leu Tyr Phe Leu Trp Thr Val
Pro Thr Ser Ala Cys Asp Pro Ser Val 245 250 255 His Thr Ala Leu His
Val Thr Leu Ser Phe Thr Tyr Leu Asn Ser Met 260 265 270 Leu Asp Pro
Leu Val Tyr Tyr Phe Ser Ser Pro Ser Leu Pro Lys Phe 275 280 285 Tyr
Thr Lys Leu Thr Ile Cys Ser Leu Lys Pro Lys Arg Pro Gly Arg 290 295
300 Thr Lys Thr Arg Arg Ser Glu Glu Met Pro Ile Ser Asn Leu Cys Ser
305 310 315 320 Lys Ser Ser Ile Asp Gly Ala Asn Arg Ser Gln Arg Pro
Ser Asp Gly 325 330 335 Gln Trp Asp Leu Gln Val Cys 340 152 24 DNA
Artificial Novel Sequence 152 ggccgtggct gatttcctcc ttat 24 153 22
DNA Artificial Novel Sequence 153 aaccgggtcg ccttcttcat cc 22 154
23 DNA Artificial Novel Sequence 154 actgtggtgg ctgtggatag gta 23
155 24 DNA Artificial Novel Sequence 155 gcagattgtg agcttggcgt agaa
24 156 1056 DNA Rat 156 atgctcttcc tctctccgag tgctatggac aacgggtcgt
gctgtctcat cgagggggaa 60 cccatcaccc aggtaatgcc acctttactc
atcctggcct tcctgcttgg agccctgggc 120 aacggcctag ccctgtgtgg
tttctgcttt cacatgaaga cctggaagtc gagcactatt 180 taccttttca
acttggctgt agccgatttt ctcctcatga tctgcctacc ccttcggaca 240
gactactacc tcagacgtag gcattggatt ttgggggata ttccctgccg cctggtcctc
300 ttcatgctgg ccatgaatag ggccggaagc attgtcttcc tcactgtggt
ggccgtggac 360 aggtatttca aagtggtcca cccccaccat atggtgaacg
ccatctccaa tcggactgca 420 gctgccatcg tctgtgtcct ctggactttg
gtcatcttgg ggactgtgta tcttctgatg 480 gagagtcacc tgtgtgtgcg
ggggatggtg tcatcttgtg agagcttcat catggagtca 540 gccaacgggt
ggcacgatat catgttccag ctggagttct tcctgcccct gaccatcatc 600
ttgttctgct ccttcaaagt tgtttggagc ctgagacaga ggcaacagct gaccagacag
660 gctcggatga ggagggccac ccggttcatc atggtggtgg cttccgtgtt
catcacgtgt 720 tacctgccca gcgtgttggc gaggctctac ttcctctgga
cggtgccctc cagtgcttgt 780 gacccctctg tccacatagc tctccatgtc
accctgagtc tcacctacct gaacagcatg 840 ctggaccctc ttgtgtacta
cttttcaagc ccctcgttcc ccaaattcta cgccaagctc 900 aaaatccgca
gcttgaaacc cagacgccca ggacgctcgc aggcacggag gtcggaagag 960
atgccaattt cgaatctctg tcgtaagagt tccaccgatg tggtaaatag ttcccagagg
1020 ccgtctgacg ggcagtgggg tctccaagtg tgttga 1056 157 351 PRT Rat
157 Met Leu Phe Leu Ser Pro Ser Ala Met Asp Asn Gly Ser Cys Cys Leu
1 5 10 15 Ile Glu Gly Glu Pro Ile Thr Gln Val Met Pro Pro Leu Leu
Ile Leu 20 25 30 Ala Phe Leu Leu Gly Ala Leu Gly Asn Gly Leu Ala
Leu Cys Gly Phe 35 40 45 Cys Phe His Met Lys Thr Trp Lys Ser Ser
Thr Ile Tyr Leu Phe Asn 50 55 60 Leu Ala Val Ala Asp Phe Leu Leu
Met Ile Cys Leu Pro Leu Arg Thr 65 70 75 80 Asp Tyr Tyr Leu Arg Arg
Arg His Trp Ile Leu Gly Asp Ile Pro Cys 85 90 95 Arg Leu Val Leu
Phe Met Leu Ala Met Asn Arg Ala Gly Ser Ile Val 100 105 110 Phe Leu
Thr Val Val Ala Val Asp Arg Tyr Phe Lys Val Val His Pro 115 120 125
His His Met Val Asn Ala Ile Ser Asn Arg Thr Ala Ala Ala Ile Val 130
135 140 Cys Val Leu Trp Thr Leu Val Ile Leu Gly Thr Val Tyr Leu Leu
Met 145 150 155 160 Glu Ser His Leu Cys Val Arg Gly Met Val Ser Ser
Cys Glu Ser Phe 165 170 175 Ile Met Glu Ser Ala Asn Gly Trp His Asp
Ile Met Phe Gln Leu Glu 180 185 190 Phe Phe Leu Pro Leu Thr Ile Ile
Leu Phe Cys Ser Phe Lys Val Val 195 200 205 Trp Ser Leu Arg Gln Arg
Gln Gln Leu Thr Arg Gln Ala Arg Met Arg 210 215 220 Arg Ala Thr Arg
Phe Ile Met Val Val Ala Ser Val Phe Ile Thr Cys 225 230 235 240 Tyr
Leu Pro Ser Val Leu Ala Arg Leu Tyr Phe Leu Trp Thr Val Pro 245 250
255 Ser Ser Ala Cys Asp Pro Ser Val His Ile Ala Leu His Val Thr Leu
260 265 270 Ser Leu Thr Tyr Leu Asn Ser Met Leu Asp Pro Leu Val Tyr
Tyr Phe 275 280 285 Ser Ser Pro Ser Phe Pro Lys Phe Tyr Ala Lys Leu
Lys Ile Arg Ser 290 295 300 Leu Lys Pro Arg Arg Pro Gly Arg Ser Gln
Ala Arg Arg Ser Glu Glu 305 310 315 320 Met Pro Ile Ser Asn Leu Cys
Arg Lys Ser Ser Thr Asp Val Val Asn 325 330 335 Ser Ser Gln Arg Pro
Ser Asp Gly Gln Trp Gly Leu Gln Val Cys 340 345 350 158 1092 DNA
Homo sapiens 158 atgaatcggc accatctgca ggatcacttt ctggaaatag
acaagaagaa ctgctgtgtg 60 ttccgagatg acttcattgt caaggtgttg
ccgccggtgt tggggctgga gtttatcttc 120 gggcttctgg gcaatggcct
tgccctgtgg attttctgtt tccacctcaa gtcctggaaa 180 tccagccgga
ttttcctgtt caacctggca gtggctgact ttctactgat catctgcctg 240
cccttcctga tggacaacta tgtgaggcgt tgggactgga agtttgggga catcccttgc
300 cggctgatgc tcttcatgtt ggctatgaac cgccagggca gcatcatctt
cctcacggtg 360 gtggcggtag acaggtattt ccgggtggtc catccccacc
acgccctgaa caagatctcc 420 aatcggacag cagccatcat ctcttgcctt
ctgtggggca tcactattgg cctgacagtc 480 cacctcctga agaagaagat
gccgatccag aatggcggtg caaatttgtg cagcagcttc 540 agcatctgcc
ataccttcca gtggcacgaa gccatgttcc tcctggagtt cttcctgccc 600
ctgggcatca tcctgttctg ctcagccaga attatctgga gcctgcggca gagacaaatg
660 gaccggcatg ccaagatcaa gagagccatc accttcatca tggtggtggc
catcgtcttt 720 gtcatctgct tccttcccag cgtggttgtg cggatccgca
tcttctggct cctgcacact 780 tcgggcacgc agaattgtga agtgtaccgc
tcggtggacc tggcgttctt tatcactctc 840 agcttcacct acatgaacag
catgctggac cccgtggtgt actacttctc cagcccatcc 900 tttcccaact
tcttctccac tttgatcaac cgctgcctcc agaggaagat gacaggtgag 960
ccagataata accgcagcac gagcgtcgag ctcacagggg accccaacaa aaccagaggc
1020 gctccagagg cgttaatggc caactccggt gagccatgga gcccctctta
tctgggccca 1080 acctctcctt aa 1092 159 363 PRT Homo sapiens 159 Met
Asn Arg His His Leu Gln Asp His Phe Leu Glu Ile Asp Lys Lys 1 5 10
15 Asn Cys Cys Val Phe Arg Asp Asp Phe Ile Val Lys Val Leu Pro Pro
20 25 30 Val Leu Gly Leu Glu Phe Ile Phe Gly Leu Leu Gly Asn Gly
Leu Ala 35 40 45 Leu Trp Ile Phe Cys Phe His Leu Lys Ser Trp Lys
Ser Ser Arg Ile 50 55 60 Phe Leu Phe Asn Leu Ala Val Ala Asp Phe
Leu Leu Ile Ile Cys Leu 65 70 75 80 Pro Phe Leu Met Asp Asn Tyr Val
Arg Arg Ser Asp Trp Lys Phe Gly 85 90 95 Asp Ile Pro Cys Arg Leu
Met Leu Phe Met Leu Ala Met Asn Arg Gln 100 105 110 Gly Ser Ile Ile
Phe Leu Thr Val Val Ala Val Asp Arg Tyr Phe Arg 115 120 125 Val Val
His Pro His His Ala Leu Asn Lys Ile Ser Asn Arg Thr Ala 130 135 140
Ala Ile Ile Ser Cys Leu Leu Trp Gly Ile Thr Ile Gly Leu Thr Val 145
150 155 160 His Leu Leu Lys Lys Lys Met Pro Ile Gln Asn Gly Gly Ala
Asn Leu 165 170 175 Cys Ser Ser Phe Ser Ile Cys His Thr Phe Gln Trp
His Glu Ala Met 180 185 190 Phe Leu Leu Glu Phe Phe Leu Pro Leu Gly
Ile Ile Leu Phe Cys Ser 195 200 205 Ala Arg Ile Ile Trp Ser Leu Arg
Gln Arg Gln Met Asp Arg His Ala 210 215 220 Lys Ile Lys Arg Ala Ile
Thr Phe Ile Met Val Val Ala Ile Val Phe 225 230 235 240 Val Ile Cys
Phe Leu Pro Ser Val Val Val Arg Ile Arg Ile Phe Trp 245 250 255 Leu
Leu His Thr Ser Gly Thr Gln Asn Cys Glu Val Tyr Arg Ser Val 260 265
270 Asp Leu Ala Phe Phe Ile Thr Leu Ser Phe Thr Tyr Met Asn Ser Met
275 280 285 Leu Asp Pro Val Val Tyr Tyr Phe Ser Ser Pro Ser Phe Pro
Asn Phe 290 295 300 Phe Ser Thr Leu Ile Asn Arg Cys Leu Gln Arg Lys
Met Thr Gly Glu 305 310 315 320 Pro Asp Asn Asn Arg Ser Thr Ser Val
Glu Leu Thr Gly Asp Pro Asn 325 330 335 Lys Thr Arg Gly Ala Pro Glu
Ala Leu Met Ala Asn Ser Gly Glu Pro 340 345 350 Trp Ser Pro Ser Tyr
Leu Gly Pro Thr Ser Pro 355 360 160 29 DNA Artificial Novel
Sequence 160 ctatgtgagg cgttcagact ggaagtttg 29 161 29 DNA
Artificial Novel Sequence 161 caaacttcca gtctgaacgc ctcacatag
29
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