U.S. patent application number 09/788070 was filed with the patent office on 2002-08-01 for modulators of peroxisome proliferator activated receptor-gamma, and methods for the use thereof.
Invention is credited to Evans, Ronald M., Forman, Barry M..
Application Number | 20020103119 09/788070 |
Document ID | / |
Family ID | 22968121 |
Filed Date | 2002-08-01 |
United States Patent
Application |
20020103119 |
Kind Code |
A1 |
Evans, Ronald M. ; et
al. |
August 1, 2002 |
Modulators of peroxisome proliferator activated receptor-gamma, and
methods for the use thereof
Abstract
In accordance with the present invention, there is provided a
class of compounds which are capable of modulating processes
mediated by peroxisome proliferator activated receptor-gamma
(PPAR-.gamma.). The identification of such compounds makes it
possible to intervene in PPAR-.gamma. mediated pathways.
Inventors: |
Evans, Ronald M.; (La Jolla,
CA) ; Forman, Barry M.; (La Jolla, CA) |
Correspondence
Address: |
Stephen E. Reiter
FOLEY & LARDNER
23rd Floor
402 West Broadway
San Diego
CA
92101-3542
US
|
Family ID: |
22968121 |
Appl. No.: |
09/788070 |
Filed: |
February 16, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09788070 |
Feb 16, 2001 |
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09255392 |
Feb 22, 1999 |
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6214850 |
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Current U.S.
Class: |
514/357 ;
514/6.7; 514/6.8 |
Current CPC
Class: |
A61K 31/425 20130101;
A61K 31/44 20130101; A61K 31/53 20130101; A61K 31/4439 20130101;
A61K 31/54 20130101; A61K 31/505 20130101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 038/17 |
Claims
What is claimed is:
1. A method for modulating insulin-sensitivity and blood glucose
levels in a subject, said method comprising administering to a
subject in need of such treatment an amount of a peroxisome
proliferator activated receptor-gamma (PPAR-.gamma.) antagonist or
partial-agonist effective to lower the blood glucose level of said
subject.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods for the modulation
of nuclear receptor mediated processes. In a particular aspect, the
present invention relates to the use of a specific class of
compounds for the modulation of processes mediated by peroxisome
proliferator activated receptor-gamma (PPAR-K).
BACKGROUND OF THE INVENTION
[0002] Peroxisome proliferators are a structurally diverse group of
compounds which, when administered to rodents, elicit dramatic
increases in the size and number of hepatic and renal peroxisomes,
as well as concomitant increases in the capacity of peroxisomes to
metabolize fatty acids via increased expression of the enzymes
required for the .beta.-oxidation cycle (Lazarow and Fujiki, Ann.
Rev. Cell Biol. 1:489-530 (1985); Vamecq and Draye, Essays Biochem.
24:1115-225 (1989); and Nelali et al., Cancer Res. 48:5316-5324
(1988)). Chemicals included in this group are the fibrate class of
hypolipidermic drugs, herbicides, and phthalate plasticizers (Reddy
and Lalwani, Crit. Rev. Toxicol. 12:1-58 (1983)). Peroxisome
proliferation can also be elicited by dietary or physiological
factors such as a high-fat diet and cold acclimatization.
[0003] Insight into the mechanism whereby peroxisome proliferators
exert their pleiotropic effects was provided by the identification
of a member of the nuclear hormone receptor superfamily activated
by these chemicals (Isseman and Green, Nature 347-645-650 (1990)).
This receptor, termed peroxisome proliferator activated receptor
alpha (PPARI), was subsequently shown to be activated by a variety
of medium and long-chain fatty acids and to stimulate expression of
the genes encoding rat acyl-CoA oxidase and hydratase-dehydrogenase
(enzymes required for peroxisomal .beta.-oxidation), as well as
rabbit cytochrome P450 4A6, a fatty acid a-hydroxylase (Gottlicher
et al., Proc. Natl. Acad. Sci. USA 89:4653-4657 (1992); Tugwood et
al., EMBO J. 11:433-439 (1992); Bardot et al., Biochem. Biophys.
Res. Comm. 192:37-45 (1993); Muerhoffet al., J. Biol. Chem.
267:19051-19053 (1992); and Marcus et al., Proc. Natl. Acad. Sci.
USA 90(12):5723-5727 (1993).
[0004] The above-noted references suggest a physiological role for
PPARI in the regulation of lipid metabolism. PPARI activates
transcription by binding to DNA sequence elements, termned
peroxisome proliferator response elements (PPRE), as a heterodimer
with the retinoid X receptor. The retinoid X receptor is activated
by 9-cis retinoic acid (see Kliewer et al., Nature 358:771-774
(1992), Gearing et al., Proc. Natl. Acad. Sci. USA 90:1440-1444
(1993), Keller et al., Proc. Natl. Acad. Sci. USA 90:2160-2164
(1993), Heyman et al., Cell 68:397-406 (1992), and Levin et al.,
Nature 355:359-361 (1992)). Since the PPARI-RXR complex can be
activated by peroxisome proliferators and/or 9-cis retinoic acid,
the retinoid and fatty acid signaling pathways are seen to converge
in modulating lipid metabolism.
[0005] Since the discovery of PPARI, additional isoforns of PPAR
have been identified, e.g., PPARJ, PPARK and PPARL, which are
spatially differentially expressed. Because there are several
isoforms of PPAR, it would be desirable to identify compounds which
are capable of selectively interacting with only one of the PPAR
isoforms. Such compounds would find a wide variety of uses, such
as, for example, in the prevention of obesity, for the treatment of
diabetes, and the like.
BRIEF DESCRIPTION OF THE INVENTION
[0006] In accordance with the present invention, we have identified
a class of compounds which are capable of modulating processes
mediated by peroxisome proliferator activated receptor-gamma
(PPAR-K). The identification of such compounds makes possible
intervention in PPAR-K mediated pathways.
BRIEF DESCRIPTION OF THE FIGURE
[0007] FIG. 1 illustrates the relative reporter activity induced by
two different compounds when added alone or in combination to a
GAL4-PPARK fusion protein. In the figure, BRL 49653 refers to
5-[[4-[2-(methyl-2-pyridinylamino)ethoxy]phenyl]-methyl]-2,4-thiazolidene-
dione) and LY 171883 refers to
2-hydroxy-3-propyl-4-[6-(tetrazole-5-yl)but- oxy]acetophenone.
DETAILED DESCRIPTION OF THE INVENTION
[0008] In accordance with the present invention, there are provided
methods for modulating process(es) mediated by peroxisome
proliferator activated receptor-gamma (PPAR-K), said method
comprising conducting said process(es) in the presence of at least
one antagonist or partial-agonist of PPAR-K.
[0009] Antagonists and partial-agonists of PPAR contemplated for
use in the practice of the present invention can be described
broadly with reference to the general structure I: 1
[0010] wherein:
[0011] each of X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5 and
X.sub.6 is independently selected from carbon, nitrogen, oxygen or
sulfur, with the proviso that at least three of the atoms forming
the ring are carbon,
[0012] R.sub.1 is selected from alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, aryl,
substituted aryl, alkylaryl, substituted alkylaryl, alkenylaryl,
substituted alkenylaryl, alkynylaryl, substituted alkynylaryl,
arylalkyl, substituted arylalkyl, arylalkenyl, substituted
arylalkenyl, arylalkynyl, substituted arylalkynyl, poly(alkylene
oxide), substituted poly(alkylene oxide), poly(alkylene sulfide),
substituted poly(alkylene sulfide), poly(alkylene amine),
substituted poly(alkylene amine), --OR, --SR or --NR.sub.2, wherein
each R is independently selected from alkyl, substituted alkyl,
alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,
substituted aryl, alkylaryl, substituted alkylaryl, arylalkyl,
substituted arylalkyl, poly(alkylene oxide), substituted
poly(alkylene oxide), poly(alkylene sulfide), substituted
poly(alkylene sulfide), poly(alkylene amine) or substituted
poly(alkylene amine); with R.sub.1 having in the range of 2 up to
15 carbon atoms being preferred;
[0013] R.sub.2 is selected from hydrogen, alkyl, substituted alkyl,
alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,
substituted aryl, alkylaryl, substituted alkylaryl, alkenylaryl,
substituted alkenylaryl, alkynylaryl, substituted alkynylaryl,
arylalkyl, substituted arylalkyl, arylalkenyl, substituted
arylalkenyl, arylalkynyl, substituted arylalkynyl, oxyalkyl,
poly(alkylene oxide) or substituted poly(alkylene oxide); with
R.sub.2 having in the range of 1 up to about 15 carbon atoms being
preferred;
[0014] R.sub.3 is selected from hydrogen, hydroxy, halogen, alkoxy,
lower alkyl, substituted lower alkyl, alkenyl, substituted alkenyl,
alkynyl or substituted alkynyl; with R.sub.3 having in the range of
0 up to about 6 carbon atoms being preferred;
[0015] R4 is selected from hydrogen, formyl, acyl, lower alkyl or
substituted lower alkyl; with R.sub.4 having in the range of 0 up
to about 4 carbon atoms being preferred;
[0016] R.sub.5 is selected from hydrogen, hydroxy, lower alkoxy,
lower alkyl, substituted lower alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl or halogen; with R.sub.5 having in the
range of 0 up to about 6 carbon atoms being preferred; and
[0017] R.sub.6 is selected from hydrogen, hydroxy, lower alkoxy,
lower alkyl, substituted lower alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl or halogen; with R.sub.6 having in the
range of 0 up to about 6 carbon atoms being preferred.
[0018] Those of skill in the art recognize that the core ring of
structure I can be any one of a number of different aromatic or
pseudo-aromatic structures, e.g., a benzene ring, a pyridine ring,
a pyrazine, an oxazine, and the like.
[0019] As employed herein, "lower alkyl" refers to straight or
branched chain alkyl groups having in the range of about 1 up to 4
carbon atoms; "alkyl" refers to straight or branched chain alkyl
groups having in the range of about 1 up to 12 carbon atoms;
"substituted alkyl" refers to alkyl groups further bearing one or
more substituents such as hydroxy, alkoxy (of a lower alkyl group),
mercapto (of a lower alkyl group), halogen, trifluoromethyl, cyano,
nitro, amino, carboxyl, carbamate, sulfonyl, sulfonamide,
heteroatom-containing cyclic moieties, substituted
heteroatom-containing cyclic moieties, and the like.
[0020] As employed herein, "alkenyl" refers to straight or branched
chain hydrocarbyl groups having at least one carbon-carbon double
bond, and having in the range of about 2 up to 12 carbon atoms and
"substituted alkenyl" refers to alkenyl groups further bearing one
or more substituents as set forth above.
[0021] As employed herein, "alkynyl" refers to straight or branched
chain hydrocarbyl groups having at least one carbon-carbon triple
bond, and having in the range of about 2 up to 12 carbon atoms, and
"substituted alkynyl" refers to alkynyl groups further bearing one
or more substituents as set forth above.
[0022] As employed herein, "aryl" refers to aromatic groups having
in the range of 6 up to 14 carbon atoms and "substituted aryl"
refers to aryl groups further bearing one or more substituents as
set forth above.
[0023] As employed herein, "alkylaryl" refers to alkyl-substituted
aryl groups and "substituted alkylaryl" refers to alkylaryl groups
further bearing one or more substituents as set forth above.
[0024] As employed herein, "alkenylaryl" refers to
alkenyl-substituted aryl groups and "substituted alkenylaryl"
refers to alkenylaryl groups further bearing one or more
substituents as set forth above.
[0025] As employed herein, "alkynylaryl" refers to
alkynyl-substituted aryl groups and "substituted alkynylaryl"
refers to alkynylaryl groups further bearing one or more
substituents as set forth above.
[0026] As employed herein, "arylalkyl" refers to aryl-substituted
alkyl groups and "substituted arylalkyl" refers to arylalkyl groups
further bearing one or more substituents as set forth above.
[0027] As employed herein, "arylalkenyl" refers to aryl-substituted
alkenyl groups and "substituted arylalkenyl" refers to arylalkenyl
groups further bearing one or more substituents as set forth
above.
[0028] As employed herein, "arylalkynyl" refers to aryl-substituted
alkynyl groups and "substituted arylalkynyl" refers to arylalkynyl
groups further bearing one or more substituents as set forth
above.
[0029] As employed herein, "poly(alkylene oxide)" refers to
compounds having the general structure:
--[(CR'.sub.2).sub.x--O].sub.y--H,
[0030] wherein each R' is independently selected from hydrogen or
lower alkyl, x falls in the range of 1 up to about 4 and y falls in
the range of 2 up to about 8; "substituted poly(alkylene oxide)"
refers to poly(alkylene oxide) groups further bearing one or more
substituents as set forth above.
[0031] As employed herein, "poly(alkylene sulfide)" refers to
compounds having the general structure:
--[(CR'.sub.2).sub.x--S].sub.y--H,
[0032] wherein R', x and y are as defined above; "substituted
poly(alkylene sulfide)" refers to poly(alkylene sulfide) groups
further bearing one or more substituents as set forth above.
[0033] As employed herein, "poly(alkylene amine)" refers to
compounds having the general structure:
--[(CR'.sub.2).sub.x--N(R')].sub.y--H,
[0034] wherein R', x and y are as defined above; "substituted
poly(alkylene amine)" refers to poly(alkylene amine) groups firther
bearing one or more substituents as set forth above.
[0035] As employed herein, "heteroatom-containing cyclic moiety"
refers to cyclic (i.e., 5-, 6- or 7-membered ring-containing)
groups containing one or more heteroatoms (e.g., N, O, S, or the
like) as part of the ring structure, and having in the range of 1
up to about 14 carbon atoms; and "substituted heteroatom-containing
cyclic moiety" refers to heterocyclic groups further bearing one or
more substituents as set forth above. Examples of
heteroatom-containing cyclic moieties include furans, thiophenes,
pyrroles, pyrazoles, diazoles, triazoles, tetrazoles, dithioles,
oxathioles, oxazoles, isoxazoles, thiazoles, isothiazoles,
oxadiazoles, oxatriazoles, dioxazoles, oxathiazoles, pyrans,
pyrones, dioxins, pyridines, pyrimidines, pyrazines, pyridazines,
piperazines, diazines, triazines, oxazines, isoxazines,
oxathiazines, oxadiazines, morpholines, azepins, oxepins, thiopins,
diazepins, benzothiazoles, thiazolidinediones, and the like.
[0036] As employed herein, "acyl" refers to alkyl-carbonyl
species.
[0037] As employed herein, "halogen" or "halo" refers to fluoro
substituents, chloro substituents, bromo substituents or iodo
substituents.
[0038] In a presently preferred aspect of the present invention,
"R.sub.1" of Formula I is selected from:
--Y.sub.n--(CR"R").sub.m--Z,
--Y.sub.n--(CR"R").sub.m'--O--(CR"R").sub.m'--Z, or
--Y.sub.n--(CR"R").sub.m"--N(R'")--(CR"R").sub.m"--Z,
[0039] wherein:
[0040] Y is --O-- or --S--,
[0041] n is 0 or 1,
[0042] each R" is independently selected from hydrogen, lower
alkyl, substituted lower alkyl, hydroxy, lower alkoxy, thioalkyl,
halogen, trifluoromethyl, cyano, nitro, amino, carboxyl, carbamate,
sulfonyl or sulfonamide,
[0043] R'" is selected from hydrogen, lower alkyl or substituted
allkyl,
[0044] m falls in the range of 1 up to 15,
[0045] each m' falls independently in the range of 1 up to 8,
[0046] each m" falls independently in the range of 0 up to 12,
and
[0047] Z is selected from a heteroatom-containing cyclic moiety, a
substituted heteroatom-containing cyclic moiety, cyano, nitro,
amino, carbamate, --OR.sup.a, wherein R.sup.a is selected from H,
alkyl, alkenyl, alkynyl, acyl or aryl; --C(O)R.sup.b, wherein
R.sup.b is selected from H, alkyl, substituted alkyl, alkoxy,
alkylamino, alkenyl, substituted alkenyl, alkynyl, substituted
alkynyl, aryl, substituted aryl, aryloxy, arylamino, alkylaryl,
substituted alkylaryl, alkenylaryl, substituted alkenylaryl,
alkynylaryl, substituted alkynylaryl, arylalkyl, substituted
arylalkyl, arylalkenyl, substituted arylalkenyl, arylalkynyl,
substituted arylalkynyl, heterocyclic, substituted heterocyclic or
trifluoromethyl; --CO.sub.2R.sup.c, wherein R.sup.c is selected
from H, alkyl, alkenyl, alkynyl or aryl; --SR.sup.a, --S(O)R.sup.a,
--S(O).sub.2R.sup.a or --S(O).sub.2NHR.sup.a, wherein each R.sup.a
is as defined above, and the like.
[0048] It is presently preferred that Z be selected from
heteroatom-containing cyclic moieties, with
polyheteroatom-containing cyclic moieties being especially
preferred. Those of skill in the art can readily identify numerous
groups which fall within the definition of "heteroatom-containing
cyclic moieties", as set forth herein. Especially preferred are
polyheteroatom-containing cyclic moieties, e.g., pyrazoles,
diazoles, triazoles, tetrazoles, dithioles, oxathioles, oxazoles,
isoxazoles, thiazoles, isothiazoles, oxadiazoles, oxatriazoles,
dioxazoles, oxathiazoles, pyridazines, piperazines, diazines,
triazines, oxazines, isoxazines, oxathiazines, oxadiazines,
morpholines, diazepins, thiazolidinediones, and the like.
[0049] Especially preferred compounds employed in the practice of
the present invention are those wherein "R.sub.1" of Formula I
is
--Y.sub.n--(CH.sub.2).sub.x--Z
[0050] wherein:
[0051] Y is --O-- or --S--,
[0052] n is 0 or 1,
[0053] x falls in the range of 2 up to 12; and
[0054] Z is a triazole, a tetrazole, an oxadiazole, an oxatriazole,
a dioxazole, an oxathiazole, a triazine, an isoxazine, an
oxathiazine, an oxadiazine, a thiazolidinedione, and the like.
[0055] A presently preferred species of R.sub.1 is
--O--(CH.sub.2).sub.4--- [tetrazoline].
[0056] In another preferred aspect of the present invention,
"R.sub.2" of Formula I is selected from methyl, ethyl, propyl,
butyl, methoxy, ethoxy, propoxy, butoxy, and the like.
[0057] In yet another preferred aspect of the present invention,
"R.sub.3" of Formula I is selected from hydrogen, hydroxy, alkoxy,
and the like.
[0058] In still another preferred aspect of the present invention,
"R.sub.4" of Formula I is selected from formyl, acyl,
thiazolidenedione, alkyl-substituted thiazolidenedione, and the
like.
[0059] In a further preferred aspect of the present invention,
"R.sub.5" of Formula I is hydrogen.
[0060] In a still further preferred aspect of the present
invention, "R.sub.6" of Formula I is hydrogen.
[0061] In yet another preferred aspect of the present invention, at
least one of R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 (in
addition to R.sub.1) are not hydrogen. It is especially preferred
that at least two of R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6
(in addition to R.sub.1) are not hydrogen. A plurality of
substituents on the ring of structure I is especially preferred
when the backbone of R.sub.1 contains no greater than 6 atoms.
[0062] Presently preferred species contemplated for use in the
practice of the present invention include compounds wherein:
[0063] R.sub.1 is --O--(CH.sub.2).sub.4--[tetrazoline],
[0064] R.sub.2 is hydrogen or lower alkyl,
[0065] R.sub.3 is hydroxy or alkoxy,
[0066] R.sub.4 is acyl or thiazolidenedione; and
[0067] R.sub.5 and R.sub.6 are each hydrogen, as well as compounds
wherein:
[0068] R.sub.1 is --O--(CH.sub.2).sub.y--thiazolidenedione, wherein
y falls in the range of about 2 up to 8;
[0069] R.sub.2 is hydrogen or lower alkyl,
[0070] R.sub.3 is hydroxy or alkoxy,
[0071] R.sub.4 is acyl or thiazolidenedione; and
[0072] R.sub.5 and R.sub.6 are each hydrogen.
[0073] The above-described compounds can be readily prepared using
a variety of synthetic methods, as are well known by those of skill
in the art. For example, many of the above-described compounds can
be prepared chemically or enzymatically.
[0074] As employed herein, the term "modulate" refers to the
ability of a modulator for a member of the steroid/thyroid
superfamily to either directly (by binding to the receptor as a
ligand) or indirectly (as a precursor for a ligand or an inducer
which promotes production of ligand from a precursor) induce
expression of gene(s) maintained under hormone expression control,
or to repress expression of gene(s) maintained under such
control.
[0075] As employed herein, the phrase "processes mediated by PPARK"
refers to biological, physiological, endocrinological, and other
bodily processes which are mediated by receptor or receptor
combinations which are responsive to the PPAR-K antagonists and
partial-agonists described herein (e.g., cell differentiation to
produce lipid-accumulating cells, regulation of insulin-sensitivity
and blood glucose levels, especially as related to
hypoglycemia/hyperinsulinism (resulting, for example, from abnormal
pancreatic beta-cell function, insulin-secreting tumors and/or
autoimmune hypoglycemia due to autoantibodies to insulin, the
insulin receptor or autoantibodies that are stimulatory to
pancreatic beta-cells), the formation of macrophages which lead to
the development of atherosclerotic plaques, and the like).
Modulation of such processes can be accomplished in vitro or in
vivo. In vivo modulation can be carried out in a wide range of
subjects, such as, for example, humans, rodents, sheep, pigs, cows,
and the like.
[0076] PPAR-K-selective antagonists or partial-agonists
contemplated for use in the practice of the present invention can
be employed for both in vitro and in vivo applications. For in vivo
applications, the invention compounds can be incorporated into a
pharmaceutically acceptable formulation for administration. Those
of skill in the art can readily determine suitable dosage levels
when compounds contemplated for use in the practice of the present
invention are so used.
[0077] In accordance with another embodiment of the present
invention, there is provided a method of screening for antagonists
of PPARK receptor proteins, said method comprising
[0078] culturing test cells containing
[0079] (i) increasing concentrations of at least one compound whose
ability to inhibit the transcription activation activity of PPARK
agonists is sought to be determined, and
[0080] (ii) optionally, at least one PPARK agonist,
[0081] wherein said test cells contain
[0082] (i) exogenous DNA which expresses intact PPARK or a modified
form of PPARK, wherein the modified form of PPARK contains the DNA
binding domain of GAL4, and
[0083] (ii) a PPRE or GAL4 response element, respectively,
operatively linked to a reporter gene; and thereafter
[0084] assaying for evidence of transcription of said reporter gene
in said cells as a function of the concentration of said compound
in said culture medium, thereby indicating the ability of said
compound to inhibit activation of transcription by PPARK
agonists.
[0085] Media employed for such culturing may include agonist for
the receptor being tested, or the receptor may be constitutive
(i.e., not require the presence of agonist for activation), or a
fixed concentration of agonist can be added to the media employed
for such testing.
[0086] The above-described assays of the present invention have low
background and a broad dynamic range.
[0087] Thus, in accordance with the present invention, compound(s)
which fall within the structure of Formula I can readily be tested
for the ability to regulate the transcription-activating effects of
peroxisome proliferator activated receptor-gamma (PPAR-K). This can
be carried out by assaying for changes in the level of reporter
protein present as a result of contacting cells containing the
receptor and reporter vector with test compound;
[0088] wherein the reporter vector comprises:
[0089] (a) a promoter that is operable in the cell,
[0090] (b) a hormone response element, and
[0091] (c) a DNA segment encoding a reporter protein,
[0092] wherein the reporter protein-encoding DNA segment is
operatively linked to the promoter for transcription of the DNA
segment, and
[0093] wherein the hormone response element is operatively linked
to the promoter for activation thereof.
[0094] Hormone response elements contemplated for use in the
practice of the present invention are composed of at least one
direct repeat of two or more half sites separated by a spacer of
one nucleotide. The spacer nucleotide can be selected from any one
of A, C, G or T. Each half site of response elements contemplated
for use in the practice of the invention comprises the sequence
--RGBNNM--,
[0095] wherein
[0096] R is selected from A or G;
[0097] B is selected from G, C, or T;
[0098] each N is independently selected from A, T, C, or G; and
[0099] M is selected from A or C;
[0100] with the proviso that at least 4 nucleotides of said
--RGBNNM-- sequence are identical with the nucleotides at
corresponding positions of the sequence --AGGTCA--. Response
elements employed in the practice of the present invention can
optionally be preceded by N.sub.x, wherein x falls in the range of
0 up to 5.
[0101] Presently preferred response elements contain at least one
copy (with one, two or three copies most common) of the minimal
sequence:
1 AGGACA A AGGTCA (SEQ ID NO:4).
[0102] As noted above, the minimal sequence can optionally be
flanked by additional residues, for example, as in the
sequence:
2 GGACC AGGACA A AGGTCA CGTTC (SEQ ID NO:5).
[0103] In a preferred embodiment of the present invention, only the
ligand binding domain of PPARK is utilized, in combination with the
DNA binding domain of GAL4 protein, for the identification of PPARK
ligands or ligand-precursors. This allows one to avoid possible
background signal caused by the potential presence of endogenous
PPAR isoforms in the host cells used for the assay.
[0104] The DNA binding domain of the yeast GAL4 protein comprises
at least the first 74 amino acids thereof (see, for example, Keegan
et al., Science 231:699-704 (1986)). Preferably, the first 90 or
more amino acids of the GAL4 protein will be used, with the first
147 amino acid residues of yeast GAL4 being presently most
preferred.
[0105] The GAL4 fragment employed in the practice of the present
invention can be incorporated into any of a number of sites within
the PPARK receptor protein. For example, the GAL4 DNA binding
domain can be introduced at the amino terminus of the PPARK
receptor protein, or the GAL4 DNA binding domain can be substituted
for the native DNA binding domain of the PPARK receptor, or the
GAL4 DNA binding domain can be introduced at the carboxy terminus
of the PPARK receptor protein, or at other positions as can readily
be determined by those of skill in the art. Thus, for example, a
modified receptor protein can be prepared which consists
essentially of amino acid residues 1-147 of GAL4, plus the ligand
binding domain of PPARK (i.e., containing the ligand binding domain
only of said receptor (i.e., residues 163-475 of SEQ ID NO:1),
substantially absent the DNA binding domain and amino terminal
domain thereof).
[0106] Identification methods according to the present invention
involve the use of a functional bioassay system, wherein the
modified receptor and a reporter plasmid are cultured in suitable
host cells in the presence of test compound. Evidence of
transcription (e.g., expression) of reporter gene is then monitored
to determine the presence of an activated receptor-ligand complex.
Accordingly, the functional bioassay system utilizes two plasmids:
an "expression" plasmid and a "reporter" plasmid. The expression
plasmid can be any plasmid which contains and is capable of
expressing DNA encoding the desired form of PPARK receptor protein
(i.e., intact receptor or GAL4 chimeric receptor as described
hereinabove), in a suitable host cell. The reporter plasmid can be
any plasmid which contains an operative PPRE or GAL4 response
element, as appropriate, functionally linked to an operative
reporter gene.
[0107] Exemplary PPREs have been described in detail hereinabove.
Exemplary GAL4 response elements are those containing the
palindromic 17-mer:
5'-CGGAGGACTGTCCTCCG-3' (SEQ ID NO:6),
[0108] such as, for example, 17MX, as described by Webster et al.,
in Cell 52:169-178 (1988), as well as derivatives thereof.
Additional examples of suitable response elements include those
described by Hollenberg and Evans in Cell 55:899-906 (1988); or
Webster et al. in Cell 54:199-207 (1988).
[0109] Exemplary reporter genes include chloramphenicol transferase
(CAT), luciferase (LUC), beta-galactosidase (J-gal), and the like.
Exemplary promoters include the simian virus (SV) promoter or
modified form thereof (e.g., -SV), the thymidine kinase (TK)
promoter, the mammary tumor virus (MTV) promoter or modified form
thereof (e.g., -MTV), and the like [see, for example, Mangelsdorf
et al., in Nature 345:224-229 (1990), Mangelsdorf et al., in Cell
66:555-561 (1991), and Berger et al., in J. Steroid Biochem. Molec.
Biol. 41:733-738 (1992)]. The plasmids pGMCAT, pGHCAT,
pTK-GAL.sub.p3-LUC, -MTV-GAL.sub.p3-LUC, -MTV-GAL.sub.p3-CAT, and
the like, are examples of reporter plasmids which contain an
operative hormone responsive promoter/enhancer element functionally
linked to an operative reporter gene, and can therefore be used in
the above-described functional bioassay (see Example 2 for details
on the preparation of these plasmids). In pGMCAT, the operative
hormone responsive promoter/enhancer element is the MTV LTR; in
pGHCAT it is the functional portion of the growth hormone promoter.
In both pGMCAT and GHCAT the operative reporter gene is the
bacterial gene for chloramphenicol acetyltransferase (CAT).
[0110] As used herein in the phrase "operative response element
functionally linked to an operative reporter gene", the word
"operative" means that the respective DNA sequences (represented by
the terms "PPRE," "GAL4 response element" and "reporter gene") are
operational, i.e., work for their intended purposes; the word
"functionally" means that after the two segments are linked, upon
appropriate activation by a ligand-receptor complex, the reporter
gene will be expressed as the result of the fact that the "PPRE" or
"GAL4 response element" was "turned on" or otherwise activated.
[0111] In practicing the above-described functional bioassay, the
expression plasmid and the reporter plasmid are co-transfected into
suitable host cells. The transfected host cells are then cultured
in the presence and absence of a test compound to determine if the
test compound is able to produce activation of the promoter
operatively linked to the PPRE or GAL4 response element of the
reporter plasmid. Thereafter, the transfected and cultured host
cells are monitored for induction (i.e., the presence) of the
product of the reporter gene sequence.
[0112] Any cell line can be used as a suitable "host" for the
functional bioassay contemplated for use in the practice of the
present invention. Thus, in contrast to the requirements of prior
art assay systems, when GAL4 chimerics are employed, there is no
need to use receptor-negative cells in carrying out the invention
process. Since the modified receptor employed in the practice of
the present invention is the only species in the test cell which is
capable of initiating transcription from a GAL4 response element,
the expression of native receptor by the test cell does not
contribute to background levels. Thus, the invention bioassay can
be made to be very selective.
[0113] Cells contemplated for use in the practice of the present
invention include transformed cells, non-transformed cells,
neoplastic cells, primary cultures of different cell types, and the
like. Exemplary cells which can be employed in the practice of the
present invention include Schneider cells, CV-1 cells, HuTu80
cells, F9 cells, NTERA2 cells, NB4 cells, HL-60 cells, 293 cells,
Hela cells, yeast cells, and the like. Preferred host cells for use
in the finctional bioassay system are COS cells and CV-1 cells.
COS-1 (referred to as COS) cells are monkey kidney cells that
express SV40 T antigen (Tag); while CV-1 cells do not express SV40
Tag. The presence of Tag in the COS-1 derivative lines allows the
introduced expression plasmid to replicate and provides a relative
increase in the amount of receptor produced during the assay
period. CV-1 cells are presently preferred because they are
particularly convenient for gene transfer studies and provide a
sensitive and well-described host cell system.
[0114] The above-described cells (or fractions thereof) are
maintained under physiological conditions when contacted with
physiologically active compound. "Physiological conditions" are
readily understood by those of skill in the art to comprise an
isotonic, aqueous nutrient medium at a temperature of about
37EC.
[0115] In accordance with yet another embodiment of the present
invention, there is provided a method for treating obesity, said
method comprising administering to a subject in need thereof an
amount of a peroxisome proliferator activated receptor-gamma
(PPAR-K) antagonist effective to block cell differentiation to
produce lipid-accumulating cells. As employed herein "treating"
obesity embraces preventing as well as reversing obesity.
[0116] As employed here, "obesity" refers generally to individuals
who are at least about 20-30% over the average weight for his/her
age, sex and height. Technically, "obese" is defined, for males, as
individuals whose body mass index is greater than 27.8 kg/m.sup.2,
and for females, as individuals whose body mass index is greater
than 27.3 kg/m.sup.2. Those of skill in the art readily recognize
that the invention method is not limited to those who fall within
the above criteria. Indeed, the invention method can also be
advantageously practiced by individuals who fall outside of these
traditional criteria, for example, by those who may be prone to
obesity.
[0117] Those of skill in the art recognize that there are numerous
cell types which are capable of differentiation to produce
"lipid-accumulating cells," such as, for example, mesenchymal cells
(e.g., fibroblasts).
[0118] As employed herein, the phrase "amount . . . effective to
block cell differentiation" refers to levels of compound sufficient
to provide circulating concentrations high enough to effect
activation of PPARK. Such a concentration typically falls in the
range of about 10 nM up to 2 TM; with concentrations in the range
of about 100 nM up to 500 nM being preferred. Since the activity of
different compounds which fall within the definition of structure I
as set forth above may vary considerably, and since individual
subjects may present a wide variation in severity of symptoms, it
is up to the practitioner to determine a subject's response to
treatment and vary the dosages accordingly.
[0119] In accordance with a particular embodiment of the present
invention, compositions comprising at least one antagonist or
partial-agonist of PPAR-K (as described herein), and a
pharmaceutically acceptable carrier are contemplated. Exemplary
pharmaceutically acceptable carriers include carriers suitable for
oral, intravenous, subcutaneous, intramuscular, intracutaneous, and
the like administration. Administration in the form of creams,
lotions, tablets, dispersible powders, granules, syrups, elixirs,
sterile aqueous or non-aqueous solutions, suspensions or emulsions,
and the like, is contemplated.
[0120] For the preparation of oral liquids, suitable carriers
include emulsions, solutions, suspensions, syrups, and the like,
optionally containing additives such as wetting agents, emulsifying
and suspending agents, sweetening, flavoring and perfuming agents,
and the like.
[0121] For the preparation of fluids for parenteral administration,
suitable carriers include sterile aqueous or non-aqueous solutions,
suspensions, or emulsions. Examples of non-aqueous solvents or
vehicles are propylene glycol, polyethylene glycol, vegetable oils,
such as olive oil and corn oil, gelatin, and injectable organic
esters such as ethyl oleate. Such dosage forms may also contain
adjuvants such as preserving, wetting, emulsifying, and dispersing
agents. They may be sterilized, for example, by filtration through
a bacteria-retaining filter, by incorporating sterilizing agents
into the compositions, by irradiating the compositions, or by
heating the compositions. They can also be manufactured in the form
of sterile water, or some other sterile injectable medium
immediately before use.
[0122] In accordance with still another embodiment of the present
invention, there is provided a method for modulating
insulin-sensitivity and blood glucose levels in a subject, said
method comprising administering to a subject in need of such
treatment an amount of a peroxisome proliferator activated
receptor-gamma (PPAR-K) antagonist or partial-agonist effective to
lower the blood glucose level of said subject.
[0123] As employed herein, the phrase "amount . . . effective to
lower blood glucose levels" refers to levels of compound sufficient
to provide circulating concentrations high enough to accomplish the
desired effect. Such a concentration typically falls in the range
of about 10 nM up to 2 TM; with concentrations in the range of
about 100 nM up to 500 nM being preferred. As noted previously,
since the activity of different compounds which fall within the
definition of structure I as set forth above may vary considerably,
and since individual subjects may present a wide variation in
severity of symptoms, it is up to the practitioner to determine a
subject's response to treatment and vary the dosages
accordingly.
[0124] The invention will now be described in greater detail by
reference to the following non-limiting examples.
EXAMPLE 1
Preparation of GAL4-receptor Fusion Proteins
[0125] A basic vector useful for the generation of GAL4-receptor
fusion proteins is called pCMX-GAL4 (see SEQ ID NO:2). This vector
encodes GAL4 DNA binding domain, followed by a polylinker sequence
useful in the cloning. The parental expression vector pCMX has been
described by Umesono et al., in Cell 65:1255-1266 (1991), and the
GAL4 portion of pCMX-GAL4 is derived from plasmid pSG424, described
by Sadowski and Ptasbne, in Nucleic Acids Res. 17:7539 (1989).
[0126] In general, GAL4-receptor ligand binding domain fusions are
prepared by talking advantage of mutant receptor cDNA clones, such
as GR-RAR chimera [see, for example, Giguere et al., in Nature
330:624-629 (1987)]. These mutant receptor cDNAs encode common XhoI
sites at the end of the DNA binding domain, as described by Giguere
et al., supra. To do so, a new pCMX-GAL4 vector was prepared which
encodes a compatible SalI site in the polylinker sequence (there is
an XhoI site in the GAL4 sequence):
SalI site: G'TCGAC
XhoI site: C'TCGAG
[0127] This allows efficient transfer of the receptor ligand
binding domain to GAL4 DNA binding domain. Through this method, a
number of chimeric species have been generated, including
GAL4-PPARK, containing residues 163-475 of PPARK (see SEQ ID
NO:1).
[0128] If mutants of the type referred to above are not available
for the construction of GAL4-containing chimerics, one may simply
look for any convenient restriction enzyme site within or
downstream of the DNA binding domain of the receptor of interest
(i.e., within about the first 30 amino acid residues downstream of
the conserved Gly-Met residues of the DNA binding domain, i.e.,
within 30 residues of the last two residues shown in SEQ ID NO:1),
and utilize the carboxy terminal sequences therefrom.
EXAMPLE 2
Preparation of Reporter Constructs
[0129] Various reporter constructs are used in the examples which
follow. They are prepared as follows:
[0130] TK-LUC: The MTV-LTR promoter sequence was removed from the
MTV-LUC plasmid described by Hollenberg and Evans in Cell
55:899-906 (1988) by HindIII and XhoI digest, and cloned with the
HindIII-XhoI fragment of the Herpes simplex virus thymidine kinase
gene promoter (-105 to +51 with respect to the transcription start
site, m, isolated from plasmid pBLCAT2, described by Luckow &
Schutz in Nucleic Acids Res. 15:5490 (1987)) to generate parental
construct TK-LUC.
[0131] pTK-PPRE3-LUC: Three copies of double-stranded peroxisome
proliferator response element (PPRE) oligonucleotides (see SEQ ID
NO:5) were cloned upstream of the TK promoter of TK-LUC at the SalI
site.
[0132] pTK-MH100x4-LUC: Four copies of double-stranded MH100
oligonucleotides, encoding a GAL4 binding site, were cloned
upstream of the TK promoter of TK-LUC at the HindIII site.
[0133] CMX-JGAL: The coding sequence for the E. coli
J-galactosidase gene was isolated from plasmid pCH110 [see Hall et
al., J. Mol. Appl. Genet. 2:101-109 (1983)] by HindIII and BamHI
digest, and cloned into pCMX eucaryotic expression vector [see
Umesono et al., supra].
EXAMPLE 3
Screening Assay For PPAR-K Antagonists
[0134] Effector plasmid, reporter plasmid, and J-galactosidase
control plasmid are co-transfected into CV-1 cells at a ratio of
about 1:3:5, using a liposome-mediated method, employing
N-{2-(2,3)-dioleoyloxy)propyl- -N,N,N-trimethyl ammonium methyl
sulfate} (i.e., DOTAP, Boehringer Mannheim) according to the
manufacturer's instructions in Dulbecco's modified Eagle's medium
(DMEM) with 10% delipidated hormone-depleted fetal calf serum.
After about 2-3 hours, the cells are washed with DMEM and agonist
(200 nM BRL 49653) and/or an appropriate test compound (LY 171883;
see FIG. 1) is added to the media. After 24-48 hours of incubation,
the cells are rinsed with phosphate buffered saline (pH 7.2) and
lysed. Aliquots are assayed for luciferase and J-galactosidase
activity. Luciferase activity is normalized to optical density
units of J-galactosidase per minute of incubation.
[0135] Thus, CV-1 cells are co-transfected with CMX-GAL-PPARK and
pTK-MH100x4-LUC at a ratio of about 100 ng of receptor-encoding DNA
per 10.sup.5 cells. The usual amounts of DNA per 10.sup.5 cells are
100 ng of CMX-GAL-PPARK, 300 ng of pTK-MH100x4-LUC, and 500 ng of
CMX-JGAL. Typically, transfections are performed in triplicate. The
plates are then incubated for 2-3 hours at 37EC.
[0136] The cells are washed with fresh medium. Fresh medium
containing agonist (200 nM BRL 49653) and/or an appropriate test
compound (LY 171883; see FIG. 1) is added to the media. A solvent
control is also performed. The cells are incubated at 37EC for 1-2
days.
[0137] The cells are rinsed twice with buffered saline solution.
Subsequently, cells are lysed, in situ, by adding 200 Tl of lysis
buffer. After 30 minutes incubation at room temperature, 40 Tl
aliquots of cell lysate are transferred to 96-well plates for
luciferase reporter gene assays and J-galactosidase transfection
controls [see Heyman et al., Cell 68:397-406 (1992)].
[0138] The data are expressed as relative light units (RLUs) per
O.D. unit of J-galactosidase per minute. The triplicates are
averaged and plotted (see FIG. 1) as relative reporter activity
induced by agonist alone, antagonist alone, or combinations
thereof. Review of the data presented in FIG. 1 reveals that
2-hydroxy-3-propyl-4-[6-(tetrazole-5-yl)butoxy]ace- tophenone
(i.e., LY 171883) is a functional antagonist of PPARK.
[0139] While the invention has been described in detail with
reference to certain preferred embodiments thereof, it will be
understood that modifications and variations are within the spirit
and scope of that which is described and claimed.
Sequence CWU 1
1
7 1 2005 DNA Mus Musculus CDS (352)..(1776) 1 atcgaatccc gcgccccagg
cgctgccgct ctgagtgcga cgggccccgc ctggccggcc 60 ggaggacgcg
gaagaagaga cctggggcgc tgcctggggt attgggtcgc gcgcagtgag 120
gggaccgagt gtgacgacaa ggtgaccggg ctgaggggac gggctgagga gaagtcacac
180 tctgacagga gcctgtgaga ccaacagcct gacggggtct cggttgaggg
gacgcgggct 240 gagaagtcac gttctgacag gactgtgtga cagacaagat
ttgaaagaag cggtgaacca 300 ctgatattca ggacattttt aaaaacaaga
ctacccttta ctgaaattac c atg gtt 357 Met Val 1 gac aca gag atg cca
ttc tgg ccc acc aac ttc gga atc agc tct gtg 405 Asp Thr Glu Met Pro
Phe Trp Pro Thr Asn Phe Gly Ile Ser Ser Val 5 10 15 gac ctc tcc gtg
atg gaa gac cac tcg cat tcc ttt gac atc aag ccc 453 Asp Leu Ser Val
Met Glu Asp His Ser His Ser Phe Asp Ile Lys Pro 20 25 30 ttt acc
aca gtt gat ttc tcc agc att tct gct cca cac tat gaa gac 501 Phe Thr
Thr Val Asp Phe Ser Ser Ile Ser Ala Pro His Tyr Glu Asp 35 40 45 50
att cca ttc aca aga gct gac cca atg gtt gct gat tac aaa tat gac 549
Ile Pro Phe Thr Arg Ala Asp Pro Met Val Ala Asp Tyr Lys Tyr Asp 55
60 65 ctg aag ctc caa gaa tac caa agt gcg atc aaa gta gaa cct gca
tct 597 Leu Lys Leu Gln Glu Tyr Gln Ser Ala Ile Lys Val Glu Pro Ala
Ser 70 75 80 cca cct tat tat tct gaa aag acc cag ctc tac aac agg
cct cat gaa 645 Pro Pro Tyr Tyr Ser Glu Lys Thr Gln Leu Tyr Asn Arg
Pro His Glu 85 90 95 gaa cct tct aac tcc ctc atg gcc att gag tgc
cga gtc tgt ggg gat 693 Glu Pro Ser Asn Ser Leu Met Ala Ile Glu Cys
Arg Val Cys Gly Asp 100 105 110 aaa gca tca ggc ttc cac tat gga gtt
cat gct tgt gaa gga tgc aag 741 Lys Ala Ser Gly Phe His Tyr Gly Val
His Ala Cys Glu Gly Cys Lys 115 120 125 130 ggt ttt ttc cga aga acc
atc cga ttg aag ctt att tat gat agg tgt 789 Gly Phe Phe Arg Arg Thr
Ile Arg Leu Lys Leu Ile Tyr Asp Arg Cys 135 140 145 gat ctt aac tgc
cgg atc cac aaa aaa agt aga aat aaa tgt cag tac 837 Asp Leu Asn Cys
Arg Ile His Lys Lys Ser Arg Asn Lys Cys Gln Tyr 150 155 160 tgt cgg
ttt cag aag tgc ctt gct gtg ggg atg tct cac aat gcc atc 885 Cys Arg
Phe Gln Lys Cys Leu Ala Val Gly Met Ser His Asn Ala Ile 165 170 175
agg ttt ggg cgg atg cca cag gcc gag aag gag aag ctg ttg gcg gag 933
Arg Phe Gly Arg Met Pro Gln Ala Glu Lys Glu Lys Leu Leu Ala Glu 180
185 190 atc tcc agt gat atc gac cag ctg aac cca gag tct gct gat ctg
cga 981 Ile Ser Ser Asp Ile Asp Gln Leu Asn Pro Glu Ser Ala Asp Leu
Arg 195 200 205 210 gcc ctg gca aag cat ttg tat gac tca tac ata aag
tcc ttc ccg ctg 1029 Ala Leu Ala Lys His Leu Tyr Asp Ser Tyr Ile
Lys Ser Phe Pro Leu 215 220 225 acc aaa gcc aag gcg agg gcg atc ttg
aca gga aag aca acg gac aaa 1077 Thr Lys Ala Lys Ala Arg Ala Ile
Leu Thr Gly Lys Thr Thr Asp Lys 230 235 240 tca cca ttt gtc atc tac
gac atg aat tcc tta atg atg gga gaa gat 1125 Ser Pro Phe Val Ile
Tyr Asp Met Asn Ser Leu Met Met Gly Glu Asp 245 250 255 aaa atc aag
ttc aaa cat atc acc ccc ctg cag gag cag agc aaa gag 1173 Lys Ile
Lys Phe Lys His Ile Thr Pro Leu Gln Glu Gln Ser Lys Glu 260 265 270
gtg gcc atc cga att ttt caa ggg tgc cag ttt cga tcc gta gaa gcc
1221 Val Ala Ile Arg Ile Phe Gln Gly Cys Gln Phe Arg Ser Val Glu
Ala 275 280 285 290 gtg caa gag atc aca gag tat gcc aaa aat atc cct
ggt ttc att aac 1269 Val Gln Glu Ile Thr Glu Tyr Ala Lys Asn Ile
Pro Gly Phe Ile Asn 295 300 305 ctt gat ttg aat gac caa gtg act ctg
ctc aag tat ggt gtc cat gag 1317 Leu Asp Leu Asn Asp Gln Val Thr
Leu Leu Lys Tyr Gly Val His Glu 310 315 320 atc atc tac acg atg ctg
gcc tcc ctg atg aat aaa gat gga gtc ctc 1365 Ile Ile Tyr Thr Met
Leu Ala Ser Leu Met Asn Lys Asp Gly Val Leu 325 330 335 atc tca gag
ggc caa gga ttc atg acc agg gag ttc ctc aaa agc ctg 1413 Ile Ser
Glu Gly Gln Gly Phe Met Thr Arg Glu Phe Leu Lys Ser Leu 340 345 350
cgg aag ccc ttt ggt gac ttt atg gag cct aag ttt gag ttt gct gtg
1461 Arg Lys Pro Phe Gly Asp Phe Met Glu Pro Lys Phe Glu Phe Ala
Val 355 360 365 370 aag ttc aat gca ctg gaa tta gat gac agt gac ttg
gct ata ttt ata 1509 Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp
Leu Ala Ile Phe Ile 375 380 385 gct gtc att att ctc agt gga gac cgc
cca ggc ttg ctg aac gtg aag 1557 Ala Val Ile Ile Leu Ser Gly Asp
Arg Pro Gly Leu Leu Asn Val Lys 390 395 400 ccc atc gag gac atc caa
gac aac ctg ctg cag gcc ctg gaa ctg cag 1605 Pro Ile Glu Asp Ile
Gln Asp Asn Leu Leu Gln Ala Leu Glu Leu Gln 405 410 415 ctc aag ctg
aat cac cca gag tcc tct cag ctg ttc gcc aag gtg ctc 1653 Leu Lys
Leu Asn His Pro Glu Ser Ser Gln Leu Phe Ala Lys Val Leu 420 425 430
cag aag atg aca gac ctc agg cag atc gtc aca gag cac gtg cag cta
1701 Gln Lys Met Thr Asp Leu Arg Gln Ile Val Thr Glu His Val Gln
Leu 435 440 445 450 ctg cat gtg atc aag aag aca gag aca gac atg agc
ctt cac ccc ctg 1749 Leu His Val Ile Lys Lys Thr Glu Thr Asp Met
Ser Leu His Pro Leu 455 460 465 ctc cag gag atc tac aag gac ttg tat
tagcaggaaa gtcccacccg 1796 Leu Gln Glu Ile Tyr Lys Asp Leu Tyr 470
475 ctgacaacgt gttccttcta ttgattgcac tattattttg agggaaaaaa
atctgacacc 1856 taagaaattt actgtgaaaa agcatttaaa aacaaaaagt
tttagaacat gatctatttt 1916 atgcatattg tttataaaga tacatttaca
atttactttt aatattaaaa attaccacat 1976 tataaaaaaa aaaaaaaaaa
aggaattcc 2005 2 475 PRT Mus Musculus 2 Met Val Asp Thr Glu Met Pro
Phe Trp Pro Thr Asn Phe Gly Ile Ser 1 5 10 15 Ser Val Asp Leu Ser
Val Met Glu Asp His Ser His Ser Phe Asp Ile 20 25 30 Lys Pro Phe
Thr Thr Val Asp Phe Ser Ser Ile Ser Ala Pro His Tyr 35 40 45 Glu
Asp Ile Pro Phe Thr Arg Ala Asp Pro Met Val Ala Asp Tyr Lys 50 55
60 Tyr Asp Leu Lys Leu Gln Glu Tyr Gln Ser Ala Ile Lys Val Glu Pro
65 70 75 80 Ala Ser Pro Pro Tyr Tyr Ser Glu Lys Thr Gln Leu Tyr Asn
Arg Pro 85 90 95 His Glu Glu Pro Ser Asn Ser Leu Met Ala Ile Glu
Cys Arg Val Cys 100 105 110 Gly Asp Lys Ala Ser Gly Phe His Tyr Gly
Val His Ala Cys Glu Gly 115 120 125 Cys Lys Gly Phe Phe Arg Arg Thr
Ile Arg Leu Lys Leu Ile Tyr Asp 130 135 140 Arg Cys Asp Leu Asn Cys
Arg Ile His Lys Lys Ser Arg Asn Lys Cys 145 150 155 160 Gln Tyr Cys
Arg Phe Gln Lys Cys Leu Ala Val Gly Met Ser His Asn 165 170 175 Ala
Ile Arg Phe Gly Arg Met Pro Gln Ala Glu Lys Glu Lys Leu Leu 180 185
190 Ala Glu Ile Ser Ser Asp Ile Asp Gln Leu Asn Pro Glu Ser Ala Asp
195 200 205 Leu Arg Ala Leu Ala Lys His Leu Tyr Asp Ser Tyr Ile Lys
Ser Phe 210 215 220 Pro Leu Thr Lys Ala Lys Ala Arg Ala Ile Leu Thr
Gly Lys Thr Thr 225 230 235 240 Asp Lys Ser Pro Phe Val Ile Tyr Asp
Met Asn Ser Leu Met Met Gly 245 250 255 Glu Asp Lys Ile Lys Phe Lys
His Ile Thr Pro Leu Gln Glu Gln Ser 260 265 270 Lys Glu Val Ala Ile
Arg Ile Phe Gln Gly Cys Gln Phe Arg Ser Val 275 280 285 Glu Ala Val
Gln Glu Ile Thr Glu Tyr Ala Lys Asn Ile Pro Gly Phe 290 295 300 Ile
Asn Leu Asp Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly Val 305 310
315 320 His Glu Ile Ile Tyr Thr Met Leu Ala Ser Leu Met Asn Lys Asp
Gly 325 330 335 Val Leu Ile Ser Glu Gly Gln Gly Phe Met Thr Arg Glu
Phe Leu Lys 340 345 350 Ser Leu Arg Lys Pro Phe Gly Asp Phe Met Glu
Pro Lys Phe Glu Phe 355 360 365 Ala Val Lys Phe Asn Ala Leu Glu Leu
Asp Asp Ser Asp Leu Ala Ile 370 375 380 Phe Ile Ala Val Ile Ile Leu
Ser Gly Asp Arg Pro Gly Leu Leu Asn 385 390 395 400 Val Lys Pro Ile
Glu Asp Ile Gln Asp Asn Leu Leu Gln Ala Leu Glu 405 410 415 Leu Gln
Leu Lys Leu Asn His Pro Glu Ser Ser Gln Leu Phe Ala Lys 420 425 430
Val Leu Gln Lys Met Thr Asp Leu Arg Gln Ile Val Thr Glu His Val 435
440 445 Gln Leu Leu His Val Ile Lys Lys Thr Glu Thr Asp Met Ser Leu
His 450 455 460 Pro Leu Leu Gln Glu Ile Tyr Lys Asp Leu Tyr 465 470
475 3 546 DNA Saccharomyces cerevisiae CDS (35)..(544) 3 gggagaccca
agcttgaagc aagcctcctg aaag atg aag cta ctg tct tct atc 55 Met Lys
Leu Leu Ser Ser Ile 1 5 gaa caa gca tgc gat att tgc cga ctt aaa aag
ctc aag tgc tcc aaa 103 Glu Gln Ala Cys Asp Ile Cys Arg Leu Lys Lys
Leu Lys Cys Ser Lys 10 15 20 gaa aaa ccg aag tgc gcc aag tgt ctg
aag aac aac tgg gag tgt cgc 151 Glu Lys Pro Lys Cys Ala Lys Cys Leu
Lys Asn Asn Trp Glu Cys Arg 25 30 35 tac tct ccc aaa acc aaa agg
tct ccg ctg act agg gca cat ctg aca 199 Tyr Ser Pro Lys Thr Lys Arg
Ser Pro Leu Thr Arg Ala His Leu Thr 40 45 50 55 gaa gtg gaa tca agg
cta gaa aga ctg gaa cag cta ttt cta ctg att 247 Glu Val Glu Ser Arg
Leu Glu Arg Leu Glu Gln Leu Phe Leu Leu Ile 60 65 70 ttt cct cga
gaa gac ctt gac atg att ttg aaa atg gat tct tta cag 295 Phe Pro Arg
Glu Asp Leu Asp Met Ile Leu Lys Met Asp Ser Leu Gln 75 80 85 gat
ata aaa gca ttg tta aca gga tta ttt gta caa gat aat gtg aat 343 Asp
Ile Lys Ala Leu Leu Thr Gly Leu Phe Val Gln Asp Asn Val Asn 90 95
100 aaa gat gcc gtc aca gat aga ttg gct tca gtg gag act gat atg cct
391 Lys Asp Ala Val Thr Asp Arg Leu Ala Ser Val Glu Thr Asp Met Pro
105 110 115 cta aca ttg aga cag cat aga ata agt gcg aca tca tca tcg
gaa gag 439 Leu Thr Leu Arg Gln His Arg Ile Ser Ala Thr Ser Ser Ser
Glu Glu 120 125 130 135 agt agt aac aaa ggt caa aga cag ttg act gta
tcg ccg gaa ttc ccg 487 Ser Ser Asn Lys Gly Gln Arg Gln Leu Thr Val
Ser Pro Glu Phe Pro 140 145 150 ggg atc cgt cga cgg tac cag ata tca
gga tcc tgg cca gct agc tag 535 Gly Ile Arg Arg Arg Tyr Gln Ile Ser
Gly Ser Trp Pro Ala Ser 155 160 165 gta gct aga gg 546 Val Ala Arg
4 166 PRT Saccharomyces cerevisiae 4 Met Lys Leu Leu Ser Ser Ile
Glu Gln Ala Cys Asp Ile Cys Arg Leu 1 5 10 15 Lys Lys Leu Lys Cys
Ser Lys Glu Lys Pro Lys Cys Ala Lys Cys Leu 20 25 30 Lys Asn Asn
Trp Glu Cys Arg Tyr Ser Pro Lys Thr Lys Arg Ser Pro 35 40 45 Leu
Thr Arg Ala His Leu Thr Glu Val Glu Ser Arg Leu Glu Arg Leu 50 55
60 Glu Gln Leu Phe Leu Leu Ile Phe Pro Arg Glu Asp Leu Asp Met Ile
65 70 75 80 Leu Lys Met Asp Ser Leu Gln Asp Ile Lys Ala Leu Leu Thr
Gly Leu 85 90 95 Phe Val Gln Asp Asn Val Asn Lys Asp Ala Val Thr
Asp Arg Leu Ala 100 105 110 Ser Val Glu Thr Asp Met Pro Leu Thr Leu
Arg Gln His Arg Ile Ser 115 120 125 Ala Thr Ser Ser Ser Glu Glu Ser
Ser Asn Lys Gly Gln Arg Gln Leu 130 135 140 Thr Val Ser Pro Glu Phe
Pro Gly Ile Arg Arg Arg Tyr Gln Ile Ser 145 150 155 160 Gly Ser Trp
Pro Ala Ser 165 5 13 DNA Mus musculus 5 aggacaaagg tca 13 6 23 DNA
Mus musculus 6 ggaccaggac aaaggtcacg ttc 23 7 17 DNA Saccharomyces
cerevisiae 7 cggaggactg tcctccg 17
* * * * *