U.S. patent application number 10/441757 was filed with the patent office on 2003-10-09 for assay for and uses of peptide hormone receptor agonists.
Invention is credited to Beinborn, Martin, Kopin, Alan S..
Application Number | 20030191114 10/441757 |
Document ID | / |
Family ID | 27075258 |
Filed Date | 2003-10-09 |
United States Patent
Application |
20030191114 |
Kind Code |
A1 |
Kopin, Alan S. ; et
al. |
October 9, 2003 |
Assay for and uses of peptide hormone receptor agonists
Abstract
The invention features a method for determining whether a
candidate compound is a non-peptide agonist of a peptide hormone
receptor. In this method, a candidate compound is exposed to a form
of the peptide hormone receptor, or to a protein that interacts
with a peptide hormone receptor, which has an enhanced ability to
amplify the intrinsic activity of a non-peptide agonist. The second
messenger signaling activity of the enhanced receptor is measured
in the presence of the candidate compound, and compared to the
second messenger signaling activity of the wildtype receptor
measured in the absence of the candidate compound. A change in
second messenger signaling activity indicates that the candidate
compound is an agonist. An increase in second messenger signaling
activity indicates that the compound is either a full or partial
positive agonist; a decrease in second messenger signaling activity
indicates that the compound is an inverse (also termed a
`negative`) agonist. The invention further embraces a method of
using a peptide hormone receptor agonist for the treatment or
prevention of a physiological disease, as well as particular
enhanced receptors and the nucleic acid sequences which code for
them.
Inventors: |
Kopin, Alan S.; (Wellesley,
MA) ; Beinborn, Martin; (Brookline, MA) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
27075258 |
Appl. No.: |
10/441757 |
Filed: |
May 20, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10441757 |
May 20, 2003 |
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09004349 |
Jan 8, 1998 |
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6566080 |
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09004349 |
Jan 8, 1998 |
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08718047 |
Sep 3, 1996 |
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08718047 |
Sep 3, 1996 |
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08570157 |
Dec 11, 1995 |
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5750353 |
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Current U.S.
Class: |
514/221 |
Current CPC
Class: |
A61P 25/24 20180101;
A61P 1/04 20180101; A61K 38/00 20130101; C07K 14/72 20130101; A61P
35/00 20180101; G01N 2333/72 20130101; C07K 2319/00 20130101; A61P
25/18 20180101; G01N 33/74 20130101; C12Q 1/02 20130101; A61P 1/18
20180101; A61P 25/16 20180101; A61P 1/00 20180101 |
Class at
Publication: |
514/221 |
International
Class: |
A61K 031/5513 |
Goverment Interests
[0002] This invention was made in part with Government funding
under National Institute of Health grant #DK46767, and the
Government therefore has certain rights in the invention.
Claims
What is claimed is:
1. A method for treating or preventing a physiological disorder
involving a CCK or gastrin peptide hormone receptor, said method
comprising administering to a mammal an agonist-effective amount of
a compound of formula (I): 6wherein: R.sup.1 represents H,
C.sub.1-6 alkyl optionally substituted by one or more halo groups,
C.sub.3-7 cycloalkyl, cyclopropylmethyl,
(CH.sub.2).sub.rimidazolyl, (CH.sub.2).sub.rtriazolyl,
(CH.sub.2).sub.rtetrazolyl, where r is 1, 2 or 3,
CH.sub.2CO.sub.2R.sup.1- 1, where R.sup.11 is C.sub.1-4alkyl, or
CH.sub.2CONR.sup.6R.sup.7, where R.sup.6 and R.sup.7 each
independently represent H or C.sub.1-4alkyl, or R.sup.6 and R.sup.7
together form a chain CH.sub.2(p) where p is 4 or 5; R.sup.2
represents NHR.sup.12 or (CH.sub.2).sub.sR.sup.13, where s is 0, 1,
2, or 3; R.sup.3 represents C.sub.1-6alkyl, halo, or
NR.sup.6R.sup.7, where R.sup.6 and R.sup.7 are as previously
defined; R.sup.4 and R.sup.5 each independently represent H,
C.sub.1-12alkyl optionally substituted by NR.sup.9R.sup.9', where
R.sup.9 and R.sup.9, are each independently H or C.sub.1-4alkyl, an
azacyclic or azabicyclic group, C.sub.4-9cycloalkyl optionally
substituted by one or more C.sub.1-4alkyl groups,
C.sub.4-9cycloalkyl, C.sub.1-4alkyl optionally substituted in the
cycloalkyl ring by one or more C.sub.1-4alkyl groups, optionally
substituted aryl, optionally substituted arylC.sub.1-6alkyl or
azacyclic or azabicyclic groups, or R.sup.4 and R.sup.5 together
form the residue of an optionally substituted azacyclic or
azabicyclic ring system; x is 0, 1, 2, or 3; R.sup.12 represents a
phenyl or pyridyl group optionally substituted by one or more
substituents selected form C.sub.1-6alkyl, halo, hydroxy,
C.sub.1-4alkoxy, (CH.sub.2).sub.q-tetrazolyl optionally substituted
in the tetrazole ring by C.sub.1-4alkyl,
(CH.sub.2).sub.q-imidazolyl, (CH.sub.2).sub.q-triazolyl, where q is
0, 1, 2, or 3, 5-hydroxy-4-pyrone, NR.sup.6R.sup.7,
NR.sup.9COR.sup.11, NR.sup.9CONR.sup.9'R.sup.11, where R.sup.6,
R.sup.7, R.sup.9, R.sup.9', and R.sup.11 are each as previously
defined, SO(C.sub.1-6alkyl), SO.sub.2(C.sub.1-6alkyl),
trifluoromethyl, CONHSO.sub.2R.sup.8 or SO.sub.2NHCOR.sup.8, where
R.sup.8 is C.sub.1-6alkyl, optionally substituted aryl,
2,2-difluorocyclopropane or trifluoromethyl, SO.sub.2NHR.sup.10,
where R.sup.10 is a nitrogen containing heterocycle, B(OH).sub.2,
(CH.sub.2).sub.qCO.sub.2H, where q is as previously defined; or
R.sup.12 represents a group: 7where X.sup.1 represents CH or N; W
represents CH.sub.2 or NR.sup.9, where R.sup.9 is as previously
defined, and W.sup.1 represents CH.sub.2, or W and W.sup.1 each
represent O; or R.sup.12 represents phenyl substituted by a group:
8wherein X.sup.2 is O, S, or NR.sup.9, where R.sup.9 is as
previously defined; z is a bond, O, or S; m is 1, 2 or 3; n is 1,
2, or 3; and y is 0, 1, 2, or 3; R.sup.13 represents a group:
9where R.sup.14 represents H or C.sub.1-6alkyl; R.sup.15 represents
H, C.sub.1-6alkyl, halo or NR.sup.6R.sup.7, where R.sup.6 and
R.sup.7 are as previously defined; and the dotted line represents
an optional covalent bond; and pharmaceutically acceptable salts or
prodrugs thereof, with the provision that, when NR.sup.4R.sup.5
represents an unsubstituted azacyclic ring system, R.sup.2 does not
represent NHR.sup.12 where R.sup.12 is optionally substituted
phenyl or: 10where W and W.sup.1 are as previously defined.
2. The method of claim 1, wherein said compound is
3(R,S)-Amino-1,3-dihydr-
o-5-((1S,4S)-5-methyl-2,5-diazabicyclo[2,2,1]heptan-2-vl)-2H-1-propyl-1,4--
benzodiazepin-2-one.
3. The method of claim 1, wherein said compound is
(-)-N-[5-(3-azabicyclo[-
3,2,2]nonan-3-yl)-2,3-dihydro-1-methyl-2-oxo-1H-1,4-benzodiazepin-3-yl]-N'-
-[3-methylphenyl]-urea.
4. The method of claim 1, wherein said compound is
(+)-N-[5-(3-azabicyclo[-
3,2,2]nonan-3-yl)-2,3-dihydro-1-methyl-2-oxo-1H-1,4-benzodiazepin-3-yl]-N'-
-[3-methylphenyl]-urea.
5. The method of claim 1, wherein said peptide hormone receptor is
the CCK-B/gastrin receptor.
6. The method of claim 1, wherein said peptide hormone receptor is
the CCK-A receptor.
7. The method of claim 1, wherein said physiological disorder is a
neoplasm.
8. The method of claim 1, wherein said neoplasm is a primary
tumor.
9. A compound of formula (I): 11wherein: R.sup.1 represents H,
C.sub.1-6 alkyl optionally substituted by one or more halo groups,
C.sub.3-7 cycloalkyl, cyclopropylmethyl,
(CH.sub.2).sub.rimidazolyl, (CH.sub.2).sub.rtriazolyl,
(CH.sub.2).sub.rtetrazolyl, where r is 1, 2 or 3,
CH.sub.2CO.sub.2R.sup.11, where R.sup.11 is C.sub.1-4alkyl, or
CH.sub.2CONR.sup.6R.sup.7, where R.sup.6 and R.sup.7 each
independently represent H or C.sub.1-4alkyl, or R.sup.6 and R.sup.7
together form a chain CH.sub.2(p) where p is 4 or 5; R.sup.2
represents NHR.sup.12 or (CH.sub.2).sub.sR.sup.13, where s is 0, 1,
2, or 3; R.sup.3 represents C.sub.1-6alkyl, halo, or
NR.sup.6R.sup.7, where R.sup.6 and R.sup.7 are as previously
defined; R.sup.4 and R.sup.5 each independently represent H,
C.sub.1-12alkyl optionally substituted by NR.sup.9R.sup.9', where
R.sup.9 and R.sup.9, are each independently H or C.sub.1-4alkyl, an
azacyclic or azabicyclic group, C.sub.4-9cycloalkyl optionally
substituted by one or more C.sub.1-4alkyl groups,
C.sub.4-9cycloalkyl, C.sub.1-4alkyl optionally substituted in the
cycloalkyl ring by one or more C.sub.1-4alkyl groups, optionally
substituted aryl, optionally substituted arylC.sub.1-6alkyl or
azacyclic or azabicyclic groups, or R.sup.4 and R.sup.5 together
form the residue of an optionally substituted azacyclic or
azabicyclic ring system; x is 0, 1, 2, or 3; R.sup.12 represents a
phenyl or pyridyl group optionally substituted by one or more
substituents selected form C.sub.1-6alkyl, halo, hydroxy,
C.sub.1-4alkoxy, (CH.sub.2).sub.q-tetrazolyl optionally substituted
in the tetrazole ring by C.sub.1-4alkyl,
(CH.sub.2).sub.q-imidazolyl, (CH.sub.2).sub.q-triazolyl, where q is
0, 1, 2, or 3, 5-hydroxy-4-pyrone, NR.sup.6R.sup.7,
NR.sup.9COR.sup.11, NR.sup.9CONR.sup.9'R.sup.11, where R.sup.6,
R.sup.7, R.sup.9, R.sup.9', and R.sup.11 are each as previously
defined, SO(C.sub.1-6alkyl), SO.sub.2(C.sub.1-6alkyl),
trifluoromethyl, CONHSO.sub.2R.sup.8 or SO.sub.2NHCOR.sup.8, where
R.sup.8 is C.sub.1-6alkyl, optionally substituted aryl,
2,2-difluorocyclopropane or trifluoromethyl, SO.sub.2NHR.sup.10,
where R.sup.10 is a nitrogen containing heterocycle, B(OH).sub.2,
(CH.sub.2).sub.qCO.sub.2H, where q is as previously defined; or
R.sup.12 represents a group: 12where X.sup.1 represents CH or N; W
represents CH.sub.2 or NR.sup.9, where R.sup.9 is as previously
defined, and W.sup.1 represents CH.sub.2, or W and W.sup.1 each
represent O; or R.sup.12 represents phenyl substituted by a group:
13wherein X.sup.2 is O, S, or NR.sup.9, where R.sup.9 is as
previously defined; z is a bond, O, or S; m is 1, 2 or 3; n is 1,
2, or 3; and y is 0, 1, 2, or 3; R.sup.13 represents a group:
14where R.sup.14 represents H or C.sub.1-6alkyl; R.sup.15
represents H, C.sub.1-6alkyl, halo or NR.sup.6R.sup.7, where
R.sup.6 and R.sup.7 are as previously defined; and the dotted line
represents an optional covalent bond; and pharmaceutically
acceptable salts or prodrugs thereof, with the provision that, when
NR.sup.4R.sup.5 represents an unsubstituted azacyclic ring system,
R.sup.2 does not represent NHR.sup.12 where R.sup.12 is optionally
substituted phenyl or: 15where W and W.sup.1 are as previously
defined.
10. The method of claim 9, wherein said compound is
3(R,S)-Amino-1,3-dihydro-5-((1S,4S)-5-methyl-2,5-diazabicyclo[2,2,1]hepta-
n-2-vl)-2H-1-propyl-1,4-benzodiazepin-2-one.
11. The method of claim 9, wherein said compound is
(-)-N-[5-(3-azabicyclo[3,2,2]nonan-3-yl)-2,3-dihydro-1-methyl-2-oxo-1H-1,-
4-benzodiazepin-3-yl]-N'-[3-methylphenyl]-urea.
12. The method of claim 9, wherein said compound is
(+)-N-[5-(3-azabicyclo[3,2,2]nonan-3-yl)-2,3-dihydro-1-methyl-2-oxo-1H-1,-
4-benzodiazepin-3-yl]-N'-[3-methylphenyl]-urea.
Description
BACKGROUND OF THE INVENTION
[0001] This application is a divisional of U.S. application Ser.
No. 09/004,349, filed Jan. 8, 1998 (now U.S. Pat. No. 6,566,080)
which is a continuation of U.S. application Ser. No. 08/718,047,
filed Sep. 3, 1996 (now abandoned) which is a continuation-in-part
of U.S. application Ser. No. 08/570,157, filed Dec. 11, 1995 (now
U.S. Pat. No. 5,750,353).
[0003] The invention relates to methods of identifying and using a
compound that is an agonist to a peptide hormone receptor.
[0004] Peptide hormone receptors are important targets for drug
research because a considerable number of diseases and other
adverse effects result from abnormal receptor activity. One peptide
hormone of interest, cholecystokinin (CCK), is a neuropeptide with
two distinct receptors: CCK-A and CCK-B/gastrin (Vanderhaeghen et
al., Nature, 257:604-605, 1975; Dockray, Nature, 264:568-570, 1976;
Rehfeld, J. Biol. Chem., 253:4022-4030, 1978; Hill et al., Brain
Res., 526:276-283, 1990; Hill et al., J. Neurosci., 10:1070-1081,
1990; Woodruff et al., Neuropeptides, (Suppl.) 19:57-64, 1991). The
peripheral type receptor, the CCK-A receptor, is located in
discrete brain nuclei and, in certain species, the spinal cord, and
is also involved in gallbladder contraction and pancreatic enzyme
secretion. The CCK-B/gastrin receptor is most abundant in the
cerebral cortex, cerebellum, basal ganglia, and amygdala of the
brain, in parietal cells of the gastrointestinal tract, in ECL
cells, as well as in kidney cells. CCK-B receptor antagonists have
been postulated to modulate anxiety, panic attacks, analgesia, and
satiety (Ravard et al., Trends Pharmacol. Sci., 11:271-273, 1990;
Singh et al., Proc. Natl. Acad. Sci. U.S.A., 88:1130-1133, 1991;
Faris et al., Science, 219:310-312, 1983; Dourish et al., Eur. J.
Pharmacol., 176:35-44, 1990; Wiertelak et al., Science,
256:830-833, 1992; Dourish et al., Science, 245:1509-1511,
1989).
SUMMARY OF THE INVENTION
[0005] Applicants have developed a systematic screening assay for
identifying an agonist specific for a peptide hormone receptor,
e.g., a peptide, peptoid, or non-peptide agonist. The assay is
based on applicants' recognition that a peptide hormone receptor
able to amplify the intrinsic activity of a ligand, e.g., a
constitutively active peptide hormone receptor, is useful as a
screening vehicle for identifying a receptor-specific agonist. A
receptor with a signaling activity higher than the corresponding
human wild-type basal level of signaling activity is further useful
for detecting the reduction in signaling activity induced by an
inverse agonist. In both cases, the receptor amplifies the signal
generated when the ligand interacts with its receptor, relative to
the signal generated when the ligand interacts with a human
wild-type receptor. Thus, forms of a receptor with the ability to
amplify receptor signaling are useful for efficiently screening
positive and inverse agonists to the corresponding human wild-type
form of the receptor.
[0006] Accordingly, the invention features a method for determining
whether a candidate compound is an agonist of a peptide hormone
receptor. In this method, a candidate compound is exposed to a form
of the peptide hormone receptor which has a greater, or an
enhanced, ability to amplify the intrinsic activity of an agonist
(hereafter,an `enhanced receptor`). The second messenger signaling
activity of the enhanced receptor is measured in the presence of
the candidate compound, and compared to the second messenger
signaling activity of the enhanced receptor measured in the absence
of the candidate compound. A change in second messenger signaling
activity indicates that the candidate compound is an agonist. For
example, an increase in second messenger signaling activity
indicates that the compound is either a full or partial positive
agonist; a decrease in second messenger signaling activity
indicates that the compound is an inverse (also termed a
`negative`) agonist. The method can further comprise using the
agonist to treat or to prevent a physiological disorder involving a
peptide hormone receptor by administering to a mammal the
identified agonist in an agonist-effective amount.
[0007] By "intrinsic activity" is meant the ability of a ligand to
activate a receptor, i.e., to act as an agonist. By `amplify` is
meant that the signal generated when the ligand interacts with the
enhanced receptor is either higher for a positive agonist, or lower
for an inverse agonist, than the signal produced when the same
ligand interacts with a corresponding non-enhanced receptor, e.g.,
a wild-type human receptor. A `non-enhanced receptor,` for the
purposes of this invention, is a wild-type human receptor for the
peptide hormone of interest. By "corresponding" is meant the same
type of peptide hormone receptor albeit in another form, e.g., a
constitutively active mutant receptor. By way of example, the
corresponding wild-type form of a constitutively active mutant
CCK-B/gastrin receptor would be a wild-type CCK-B/gastrin receptor;
the human CCK-B/gastrin receptor is the corresponding human form of
the rat CCK-B/gastrin receptor.
[0008] An "agonist," as used herein, includes a positive agonist,
e.g., a full or a partial positive agonist, or a negative agonist,
i.e., an inverse agonist. An agonist is a chemical substance that
combines with a receptor so as to initiate an activity of the
receptor; for a peptide hormone receptor, the agonist preferably
alters a second messenger signaling activity. A positive agonist is
a compound that enhances or increases an activity, e.g., a second
messenger signaling activity, of a receptor. A "full agonist"
refers to an agonist capable of activating the receptor to the
maximum level of activity, e.g., a level of activity which is
induced by a natural, i.e., an endogenous, peptide hormone. A
"partial agonist" refers to a positive agonist with reduced
intrinsic activity relative to a full agonist. As used herein, a
"peptoid" is a peptide-derived partial or full agonist (Horwell et
al., Eur. J. Med. Chem., 30 Suppl.:537S-550S, 1995; Horwell et al.,
J. Med. Chem., 34:404-14, 1991).
[0009] An "inverse agonist," as used herein, has a negative
intrinsic activity, and reduces the receptor's signaling activity
relative to the signaling activity of the wild-type receptor
measured in the absence of the inverse agonist. In contrast, an
"antagonist," as used herein, refers to a chemical substance that
inhibits the ability of an agonist to increase or decrease receptor
activity. A `full,` or `perfect` antagonist has no intrinsic
activity, and no effect on the receptor's basal activity (FIG. 1).
Peptide-derived antagonists are, for the purposes herein,
considered to be non-peptide ligands.
[0010] A diagram explaining the difference between full and partial
agonists, inverse agonists, and antagonists is shown in FIG. 1 (see
also Milligan et al., TIPS, 16:10-13, 1995). In FIG. 1, the
position of the equilibrium between an inactive state R and an
active state R* varies with individual receptors and is altered by
the presence of receptor ligands. Agonists function by stabilizing
R* while inverse agonists preferentially stabilize R. A continuum
of ligands between full agonists (at the extreme right-hand side of
the see-saw as these move the equilibrium furthest to the right)
and full inverse agonists (at the extreme left-hand side of the
see-saw) is expected to exist. Antagonists, which do not alter the
position of the equilibrium, define the position of the fulcrum. An
antagonist is, e.g., a competitive or a non-competitive
inhibitor.
[0011] Examples of peptide hormone receptor specific peptide and
non-peptide agonists useful in the screening assay of the invention
are described below. Non-peptide ligands include, but are not
limited to, benzodiazepines and derivatives thereof, e.g.,
azabicyclo[3.2.2]nonane benzodiazepine ("L-740,093"; see Castro
Pineiro et al., WO 94/03437; Castro Pineiro et al., U.S. Pat. No.
5,521,175). L-740,093 S and L-740,093 R refer to the S-enantiomer
and the R-enantiomer of L-740,093, respectively. Where the peptide
hormone receptor is a CCK-A receptor or a CCK-B/gastrin receptor,
useful peptide agonists include, but are not limited to, gastrin
(e.g., sulphated ("gastrin II") or unsulphated ("gastrin I") forms
of gastrin-17, or sulphated or unsulphated forms of gastrin-34), or
cholecystokinin (CCK) (e.g., sulfated CCK-8 (CCK-8s), unsulphated
CCK-8 (CCK-8d), CCK-4, or pentagastrin). Full agonists of the
CCK-B/gastrin receptor include, but are not limited to, CCK-8s, and
more preferably gastrin (gastrin I).
[0012] An enhanced receptor may, but does not always, have a higher
basal activity than the basal activity of a corresponding human
wild-type receptor. Methods for measuring the activity of an
enhanced receptor relative to the activity of a corresponding
wild-type receptor are described and demonstrated below. Examples
of enhanced receptors include synthetic mutant receptors, e.g.,
constitutively active mutant receptors; other mutant receptors with
normal basal activity which amplify the intrinsic activity of a
compound; naturally-occurring mutant receptors, e.g., those which
cause a disease phenotype by virtue of their enhanced receptor
activity, e.g., a naturally-occurring constitutively active
receptor; and either constitutively active or wild-type non-human
receptors, e.g., rat, mouse, mastomys, Xenopus, or canine receptors
or hybrid variants thereof, which amplify an agonist signal to a
greater extent than does the corresponding wild-type human
receptor.
[0013] Further examples of peptide hormone receptors useful in the
screening assay of the invention include, but are not limited to,
receptors specific for the following peptide hormones: amylin,
angiotensin, bombesin, bradykinin, C5a anaphylatoxin, calcitonin,
calcitonin-gene related peptide (CGRP), corticotropin releasing
hormone (CRH), chemokines, cholecystokinin (CCK), endothelin,
erythropoietin (EPO), follicle stimulating hormone (FSH),
formyl-methionyl peptides, galanin, gastrin, gastrin releasing
peptide, glucagon, glucagon-like peptide 1, glycoprotein hormones,
gonadotrophin-releasing hormone, leptin, luteinizing hormone (LH),
melanocortins, neuropeptide Y, neurotensin, opioid, oxytocin,
parathyroid hormone, secretin, somatostatin, tachykinins, thrombin,
thyrotrophin, thyrotrophin releasing hormone, vasoactive intestinal
polypeptide (VIP), and vasopressin. An enhanced receptor can
further embrace a single transmembrane domain peptide hormone
receptor, e.g., an insulin receptor.
[0014] The invention also features a method of isolating a form,
e.g., a mutant form, of a peptide hormone receptor that is suitable
for detecting agonist activity in a ligand, e.g., a peptide,
peptoid, or non-peptide ligand. The method involves (a) exchanging
a region of a functional domain of a first peptide hormone receptor
with a corresponding region of a functional domain of a second
peptide hormone receptor; and (b) measuring the ability of the
first peptide hormone receptor to amplify an agonist signal
relative to a corresponding wild-type human receptor. The
functional domain can be an intracellular loop, parts of a
transmembrane domain adjacent to an intracellular loop, a
transmembrane domain, a region of a transmembrane domain distal to
an intracellular loop, or an extracellular loop. A level of
amplification by the first peptide hormone receptor that is greater
than the level of amplification by the wild-type human receptor
indicates that the first peptide hormone receptor is suitable for
detecting agonist activity in a non-peptide ligand. The
corresponding region can be between one and ten amino acids, e.g.,
a block of five to ten amino acids, or up to thirty or a hundred
amino acids in length. The first and second peptide hormone
receptors are preferably linked to different second messenger
pathways. Those skilled in the art know which particular amino
acids of the peptide hormone receptors are considered to be within
extracellular, intracellular (cytoplasmic), or transmembrane
regions of the receptor. For example, extracellular, intracellular,
and transmembrane regions of the CCK-B/gastrin receptor are
determined by sequence alignment with other receptors (FIG. 2), or
by hydropathy analysis (Baldwin, EMBO J., 12:1693-1703, 1993).
Conformation receptor modelling is described further below.
[0015] Another method of isolating a form of a peptide hormone
receptor suitable for detecting agonist activity in a non-peptide
ligand involves (a) constructing a series of mutant forms of the
receptor by replacing an original amino acid with another amino
acid, i.e., a replacement amino acid; and (b) measuring the ability
of the resulting peptide hormone receptor mutant form to amplify an
agonist signal relative to the level of amplification by a
corresponding wild-type human receptor. An amplification in the
peptide hormone receptor mutant form that is greater than the level
of amplification of the corresponding wild-type human receptor
indicates that the mutant form is suitable for detecting agonist
activity in a non-peptide ligand. The replaced amino acid can lie
in an intracellular domain of the receptor or in a region of a
transmembrane domain flanking an intracellular portion of the
receptor, e.g., the intracellular domain-proximal half of the
transmembrane domain, or within, e.g., 8 or 10 amino acids of the
intracellular domain. The replacement amino acid can be of the same
type in each of the mutant constructs; alternatively, various types
of amino acids can be substituted at random. The replacement amino
acid can be of the same charge, or of a different charge, than the
original amino acid, e.g., a negative amino acid can be exchanged
for a positive amino acid, a positive amino acid can be exchanged
for a negative amino acid, or a positive or negative amino acid can
be exchanged for a neutral amino acid. Preferably, the replacement
amino acid is glutamine, glutamic acid, aspartic acid, or
serine.
[0016] Also embraced are the various mutant peptide hormone
receptors disclosed herein, and their respective nucleic acid
coding sequences. Mutant peptide hormone receptors of the invention
include, but are not limited to, the CCK-A receptor MHA21/35, and
the mutant CCK-B/gastrin receptors MH40, MH128, MH156, MH162, MH31,
MH131, MH13, MH130, MH129, and MH72. Plasmid manipulation, storage,
and cell transformation can be performed by methods known to those
of ordinary skill in the art. See, e.g., Ausubel et al. (eds.),
Current Protocols in Molecular Biology, John Wiley & Sons,
Inc., NY, 1988, 1995.
[0017] With an efficient and rapid assay for identifying an agonist
specific for a given peptide hormone receptor, those skilled in the
art can identify agonists to serve as lead compounds for further
pharmaceutical research. In particular, systematic chemical
modifications can be made, and their effects can be further
assessed using an enhanced receptors according to the method of the
invention. By following such a development strategy the intrinsic
activity of new agonists can be optimized so as to be useful
therapeutically against a disease involving a peptide hormone
receptor.
[0018] Knowing that a particular ligand functions as a positive or
inverse agonist, as opposed to an antagonist, facilitates
identifying which ligand species is most likely to achieve a given
physiological effect, or to achieve a physiological effect absent
an unwanted side effect. Thus, the invention further features a
method for the treatment or prevention of a physiological disorder
involving a peptide hormone receptor that includes administering to
a mammal, e.g., a human, a ligand which acts as an agonist on a
peptide hormone receptor. The ligand is administered in an
agonist-effective amount, i.e., in an amount that has a full or a
partial inverse, or a full or a partial positive, agonist effect on
a peptide hormone receptor. The peptide hormone receptor can be,
but need not be limited to, a receptor specific for one of the
following peptide hormones: amylin, angiotensin, bombesin,
bradykinin, C5a anaphylatoxin, calcitonin, calcitonin-gene related
peptide (CGRP), corticotropin releasing hormone (CRH), chemokines,
cholecystokinin (CCK) (e.g., the CCK-A or the CCK-B/gastrin
receptor), endothelin, erythropoietin (EPO), follicle stimulating
hormone (FSH), formyl-methionyl peptides, galanin, gastrin, gastrin
releasing peptide, glucagon, glucagon-like peptide 1, glycoprotein
hormones, gonadotrophin-releasing hormone, insulin, leptin,
luteinizing hormone (LH), melanocortins, neuropeptide Y,
neurotensin, opioid, oxytocin, parathyroid hormone, secretin,
somatostatin, tachykinins, thrombin, thyrotrophin, thyrotrophin
releasing hormone, vasoactive intestinal polypeptide (VIP), and
vasopressin.
[0019] An inverse agonist is particularly useful for treating or
preventing a physiological disorder that results from enhanced
basal activity of a peptide hormone receptor, e.g., a
constitutively active receptor. For example, an inverse agonist is
useful for treating a neoplasm that results from, or is sustained
or aggravated by, enhanced activity of a peptide hormone receptor,
e.g., a naturally-occurring peptide hormone receptor. Examples
include, but are not limited to, a CCK-B/gastrin related tumor,
e.g., a neuroendocrine tumor, Zollinger Ellinger Syndrome, or a
gastric carcinoid tumor; a TSH-related tumor; multiple endocrine
neoplasia type I; a lung tumor, e.g., a small-cell carcinoma; a
brain tumor, e.g., a brain tumor involving CCK; a kidney tumor,
e.g., hypernephroma or renal cell carcinoma. Agonists are
particularly useful for treating a primary tumor, e.g., a tumor in
a tissue that expresses a peptide hormone receptor, e.g., a tumor
in the pancreas, the pituitary, or the adrenal gland.
[0020] In other embodiments of the invention, an inverse agonist of
the LH receptor can be a useful compound for treating or preventing
precocious puberty; an inverse agonist of the FSH receptor can be a
useful compound for treating or preventing infertility; an inverse
agonist of the TSH receptor can be a useful compound for treating
or preventing thyroid adenomas. An inverse or positive agonist of
the G-LP1 receptor can be a useful compound for treating or
preventing obesity or diabetes, e.g., type-I or type-II
diabetes.
[0021] Non-peptide agonists are further useful for treating or
preventing a disorder involving the gastrointestinal tract, or a
disorder involving, e.g., sleep, anxiety, panic, appetite
regulation, stress, or pain, or a disorder discussed below.
[0022] A candidate compound useful in a method of treatment or
prevention of a physiological disorder of the invention is a ligand
that binds with specificity to a peptide hormone receptor, e.g., a
peptide, peptoid, or non-peptide ligand, preferably a non-peptide
ligand. A candidate compound is shown to be a positive or inverse
agonist by a screening assay disclosed herein. Exemplified
candidate compounds include the peptoid compounds ((see, e.g.,
Horwell et al., Eur. J. Med. Chem., 30 Suppl.:537S-550S, 1995;
Horwell et al., J. Med. Chem., 34:404-14, 1991); the dipeptoid
analogues of CCK (see, e.g., Horwell et al. J. Med. Chem.,
34:404-14, 1991); cyclic nucleotides and modified amino acids (see,
e.g., Dethloff et al., Drug Metab., 24:267-93, 1992), the
benzodiazepine derivatives, e.g.., the compounds described in Bock
et al., J. Med. Chem., 33:450-55, 1990, or a derivative thereof.
Additional benzodiazepine derivatives having a peptide hormone
receptor agonist activity are described in the following patents
and patent applications, each of which is hereby incorporated by
reference: EPA 167919, EPA 284256, EPA 434360, EPA 434364, EPA
434369, EPA 514125, EPA 51426, EPA 514133, EPA 508796, EPA 508797,
EPA 508798, EPA 508799, EPA 523845, EPA 523846, EPA 559170, EPA
549039, EPA 667,334, WO 9211246, WO 93032078, WO 9308175, WO
9307131, WO 9317011, WO 9319053, WO 9308175, WO 9413648, WO
9403437, WO 9611689, and U.S. Pat. No. 5,521,175. Also encompassed
are those compounds described in Henke et al., J. Med. Chem.,
39:2655-58, 1996; or in Willson et al., J. Med. Chem., 39:3030-34,
1996 (both hereby incorporated by reference) which are shown to be
inverse agonists.
[0023] As a further example, a benzodiazepine compound useful in a
method of treatment or prevention of a physiological disorder
resulting from a peptide hormone receptor is a benzodiazepine
compound of formula (I): 1
[0024] wherein:
[0025] R.sup.1 represents H, C.sub.1-6 alkyl optionally substituted
by one or more halo, C.sub.3-7 cycloalkyl, cyclopropylmethyl,
(CH.sub.2).sub.rimidazolyl, (CH.sub.2).sub.rtriazolyl,
CH.sub.2)).sub.rtetrazolyl (where r is 1, 2 or 3),
CH.sub.2CO.sub.2R.sup.11 (where R.sup.11 is C.sub.1-4alkyl) or
CH.sub.2CONR.sup.6R.sup.7 (where R.sup.6 and R.sup.7 each
independently represents H or C.sub.1-4alkyl, or R.sup.6 and
R.sup.7 together form a chain CH.sub.2p where p is 4 or 5;
[0026] R.sup.2 represents NHR.sup.12 or (CH.sub.2).sub.qR.sup.13
(where .sub.s is 0, 1, 2, or 3);
[0027] R.sup.3 represents C.sub.1-6alkyl, halo or NR.sup.6R.sup.7,
where R.sup.6 and R.sup.7 are as previously defined;
[0028] R.sup.4 and R.sup.5 each independently represents H,
C.sub.1-12alkyl optionally substituted by NR.sup.9R.sup.9, (R.sup.9
and R.sup.9, are as previously defined) or an azacyclic or
azabicyclic group, C.sub.4-9cycloalkyl optionally substituted by
one or more C.sub.1-4alkyl groups,
C.sub.4-9cycloalkylC.sub.1-4alkyl optionally substituted in the
cycloalkyl ring by one or more C.sub.1-4alkyl groups, optionally
substituted aryl, optionally substituted arylC.sub.1-6alkyl or
azacyclic or azabicyclic groups, or R.sup.4 and R.sup.5 together
form the residue of an optionally substituted azacyclic or
azabicyclic ring system;
[0029] x is 0, 1, 2, or 3;
[0030] R.sup.12 represent a phenyl or pyridyl group optionally
substituted by one or more substituents selected form
C.sub.1-6alkyl, halo, hydroxy, C.sub.1-4alkoxy,
(CH.sub.2)q-tetrazolyl optionally substituted in the tetrazole ring
by C.sub.1-4alkyl, (CH.sub.2)q-imidazolyl, (CH.sub.2)q-triazolyl
(qhere q is 0, 1, 2, or 3), 5-hydroxy-4-pyrone, NR.sup.6R.sup.7,
NR.sup.9COR.sup.11, NR.sup.9CONR.sup.9'R.sup.11 (where R.sup.9 and
R.sup.9, are each independently H or C.sub.1-4alkyl and R.sup.11 is
as previously defined), SO(C.sub.1-6alkyl),
SO.sub.2(C.sub.1-6alkyl), trifluoromethyl, CONHSO.sub.2R.sup.8,
SO.sub.2NHCOR.sup.8 (where R.sup.8 is C.sub.1-6alkyl, optionally
substituted aryl, 2,2-difluorocyclopropane or trifluoromethyl),
SO.sub.2NHR.sup.10 (where R.sup.10 is a nitrogen containing
heterocycle), B(OH).sub.2, (CH.sub.2)qCO.sub.2H, where q is as
previously defined; or
[0031] R.sup.12 represents a group: 2
[0032] where X.sup.1 represents CH or N; W represents CH.sub.2 or
NR.sup.9, where R.sup.9 is as previously defined, and W.sup.1
represents CH.sub.2, or W and W.sup.1 each represent O; or
[0033] R.sup.12 represents phenyl substituted by a group: 3
[0034] wherein X.sup.2 is O, S or NR.sup.9, where R.sup.9 is as
previously defined; z is a bond, O or S; m is 1, 2 or 3; n is 1, 2,
or 3; and y is 0, 1, 2, or 3;
[0035] R.sup.13 represents a group: 4
[0036] where R.sup.14 represents H or C.sub.1-6alkyl, R.sup.15
represents H, C.sub.1-6alkyl, halo or NR.sup.6R.sup.7, where
R.sup.6 and R.sup.7 are as previously defined; and the dotted line
represents an optional covalent bond; and pharmaceutically
acceptable salts or prodrugs thereof, with the provision that, when
NR.sup.4R.sup.5 represents an unsubstituted azacyclic ring system,
R.sup.2 does not represent NHR.sup.12 where R.sup.12 is optionally
substituted phenyl or: 5
[0037] Compounds of formula (I) are intended to embrace all
possible racemers and isomers, including optical isomers, and
mixtures thereof. Each expression, where occurring more than once
in any structure, intended to be independent of its definition
elsewhere in the same structure. The present invention includes
within its scope prodrugs of the compounds of formula (I) above. In
general, such prodrugs will be functional derivatives of the
compounds of formula (I) which are readily convertible in vivo into
the required compound of formula (I). Conventional procedures for
the selection and preparation of suitable prodrug derivatives are
described, for example, in "Design of Prodrugs" ed. H. Bungaard,
Elsevier, 1985.
[0038] As used herein, unless otherwise indicated, alkyl means
straight or branched chain saturated hydrocarbon;; halo includes
fluoro, chloro, bromo, and iodo; as used herein, unless otherwise
indicated, alkyl means straight or branched chain saturated
hydrocarbon; azacyclic means non-aromatic nitrogen-containing
monocyclic, and azabicyclic means non-aromatic nitrogen-containing
bicyclic; aryl means optionally substituted carbocyclic or
heterocyclic aromatic groups, especially phenyl; heteroaryl means
aromatic rings preferably having r or 6 ring atoms and containing
at least one atom selected from O, S, and N. Compounds of formula
(I) are prepared according to the methods of WO 94/03437, hereby
incorporated by reference.
[0039] By using the screening assay of the invention, those skilled
in the art can easily identify which candidate ligand is optimal
for therapeutic or preventive use. For example, preferred agonists
and their respective derivatives include, but are not limited to,
L-740,093
[3(R,S)-Amino-1,3-dihydro-5-((1S,4S)-5-methyl-2,5-diazabicyclo[2,2,1]hept-
an-2-vl)-2H-1-propyl-1,4-benzodiazepin-2-one]; L-740,093 R
[(-)-N-[5-(3-azabicyclo[3.2.2]nonan-3-yl)-2,3-dihydro-1-methyl-2-oxo-1H-1-
,4-benzodiazepin-3-yl]-N'-[3-methylphenyl]urea]; L-740.093 S
[(+)-N-[5-(3-azabicyclo[3.2.2]nonan-3-yl)-2,3-dihydro-1-methyl-2-oxo-1H-1-
,4-benzodiazepin-3-yl]-N'-[3-methylphenyl]urea]; L-365,260
[3R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3-yl)-
-N'-(3-methylphenylurea)]; and L-364,718.
[0040] Other terms used in the various embodiments of the invention
will be understood from the following definitions. For example, by
a "peptide hormone" is meant a polypeptide that interacts with a
target cell by contacting an extracellular receptor, i.e., a
"peptide hormone receptor." A "peptide" is used loosely herein to
refer to a molecule comprised, at least in part, of amino acid
residues that are connected to each other by peptide bonds. A
"mutant receptor" is understood to be a form of the receptor in
which one or more amino acid residues in the corresponding receptor
which predominates in nature, e.g., in a naturally-occurring
wild-type receptor, have been either deleted or replaced with a
different type of amino acid residue. By a "constitutively active
receptor" is meant a receptor with a higher basal activity level
than the corresponding wild-type receptor, where "activity" refers
to the spontaneous ability of a receptor to signal in the absence
of further activation by a positive agonist. The basal activity of
a constitutively active receptor can also be decreased by an
inverse agonist. A "naturally-occurring" receptor refers to a form
or sequence of the receptor as it exists in an animal, or to a form
of the receptor that is synonymous with the sequence known to those
skilled in the art as the "wild-type" sequence. Those skilled in
the art will understand a "wild-type" receptor to refer to the
conventionally accepted "wild-type" amino acid consensus sequence
of the receptor, or to a "naturally-occurring" receptor with normal
physiological patterns of ligand binding and signaling. A "second
messenger signaling activity" refers to production of an
intracellular stimulus (including, but not limited to, cAMP, cGMP,
ppGpp, inositol phosphate, or calcium ion) in response to
activation of the receptor, or to activation of a protein in
response to receptor activation, including but not limited to a
kinase, a phosphatase, or to activation or inhibition of a membrane
channel.
[0041] "Sequence identity," as used herein, refers to the subunit
sequence similarity between two nucleic acid or polypeptide
molecules. When a given position in both of the two molecules is
occupied by the same nucleotide or amino acid residue, e.g., if a
given position (as determined by conventionally known methods of
sequence alignment) in each of two polypeptides is occupied by
serine, then they are identical at that position. The identity
between two sequences is a direct function of the number of
matching or identical positions, e.g., if 90% of the positions in
two polypeptide sequences are identical, e.g., 9 of 10, are
matched, the two sequences share 90% sequence identity. Methods of
sequence analysis and alignment for the purpose of comparing the
sequence identity of two comparison sequences are well known by
those skilled in the art. "Biological activity," as used herein,
refers to the ability of a peptide hormone receptor to bind to a
ligand, e.g., an agonist or an antagonist, and to induce
signaling.
[0042] Other features and advantages of the invention will be
apparent from the following detailed description and from the
claims.
DETAILED DESCRIPTION
[0043] We first briefly describe the drawings.
DRAWINGS
[0044] FIG. 1 is a schematic diagram showing the relationship
between a full or partial agonist, an inverse agonist, and an
antagonist.
[0045] FIG. 2 is an illustration showing a multiple alignment of
cloned CCK receptor deduced amino acid sequences: mastomys CCK-B
(SEQ ID NO: 1), rat CCK-B (SEQ ID NO: 2), human CCK-B (SEQ ID NO:
3), canine CCK-B (SEQ ID NO: 4), human CCK-A (SEQ ID NO: 5), rat
CCK-A (SEQ ID NO: 6), and xenopus CCK-XL (SEQ ID NO: 7). `A` marks
the position in the hCCK-A receptor where an E to Q substitution
results in an increase in PD 135,158 intrinsic activity without
increasing basal receptor activity. `B` marks the position in the
hCCK-B receptor where an L to either S or E substitution results in
an increase in basal activity. The corresponding L to S in the
hCCK-A receptor does not result in an increase in basal activity.
`C` marks the position in the hCCK-B receptor where a V to E
substitution results in an increase in basal activity. The
corresponding I to E substitution in the human CCK-A receptor does
not result in an increase in basal activity. The numbering shown is
generic; each receptor is different based on deletions or
insertions.
[0046] FIG. 3 is a bar graph showing that the intrinsic activity of
peptide, peptide-derived and non-peptide ligands at the wild-type
CCK-B/gastrin receptor (top panel) is amplified in a constitutively
active receptor mutant (bottom panel).
[0047] FIG. 4 is an illustration of inositol phosphate production
by the non-peptide agonist L-740,093. Top panel: L-740,093 S
stimulated inositol phosphate production in COS-7 cells expressing
a constitutively active human CCK-B/gastrin receptor. Bottom panel:
YM022 antagonizes the partial agonist activity induced by 10 nM
L-740,093-S.
[0048] FIG. 5 is an illustration of the inhibition of inositol
phosphate production of the non-peptide inverse agonist L-740,093R.
Top panel: L-740,093 R inhibits basal inositol phosphate production
in COS-7 cells expressing a constitutively active human
CCK-B/gastrin receptor. Bottom panel: The inverse agonist activity
induced by 10 nM L-740,093 R is partially abolished by YM022 in a
concentration-dependent fashion.
[0049] FIG. 6 is a comparison of intrinsic activities of
CCK-B/gastrin receptor ligands utilizing the wild-type and the
constitutively active receptors. Values for all compounds follow a
logarithmic-linear correlation (r2=0.93).
[0050] FIG. 7 is a competition binding curve showing the extent of
.sup.125I-CCK-8 receptor binding. Binding of .sup.125I-CCK-8 to
COS-7 cells, transiently transfected with hCCK-B-pcDNAI is shown in
the presence of increasing concentrations of CCK-8, gastrin I, and
CCK-4 (part A) and L-364,718 and L-365,260 (part B).
[0051] FIG. 8 is a graph showing second messenger signaling (i.e.,
mobilization of intracellular calcium) in COS-7 cells that express
the recombinant human brain CCK-B receptor with (part A, left
panel) and without (part A, right panel) the addition of the
calcium chelator, EGTA. This is paralleled by an increased
production of inositol phosphate (part B).
[0052] FIG. 9 is a schematic representation of the seven
transmembrane (TM) domain structure of the human CCK-B/gastrin
receptor. The C-terminal domain of the third intracellular loop is
highlighted in black.
[0053] FIG. 10 is a bar graph of basal inositol phosphate
accumulation in COS-7 cells transfected with wild-type
CCK-B/gastrin receptor (WT), or with one of two constitutively
active mutants (Mut.1, Mut.2).
[0054] FIG. 11 is a bar graph showing a functional comparison of
the CCK receptors human CCK-A (hCCK-A), human CCK-B (hCCK-B), dog
CCK-B (dCCK-B), mouse CCK-B (mCCK-B), and the mastomys CCK
receptor.
[0055] FIG. 12 is a bar graph showing a comparison of the
efficiency by which the agonists CCK-8, L-365,260, and PD136,450
stimulate inositol phosphate (IP) production when contacting a
wild-type CCK-A receptor or a constitutively active CCK-A receptor
(MHA21/35).
[0056] FIG. 13 is a graph showing the level of inositol phosphate
(IP) response at varying concentrations of the peptoid PD 136,450,
a partial positive agonist. .box-solid.: mutant CCK-A receptor
MHA21/35; .tangle-soliddn.: wild-type human CCK-A receptor.
[0057] Recent drug development efforts have yielded small molecules
which competitively block G-protein coupled peptide hormone
receptors by acting as antagonists. In contrast, very few
non-peptide ligands have been identified which activate this family
of receptors. Here, applicants demonstrate that chemical
modifications of non-peptide ligands, known to act as antagonists
for the CCK-B/gastrin receptor, can result in converting the
ligands from antagonists to either positive agonists or to inverse
agonists.
[0058] Changes in the intrinsic activity of the ligand resulting
from such modifications were detectable because applicants designed
a screening assay which employed a constitutively active mutant of
the human CCK-B/gastrin receptor (.sup.325L.fwdarw.E, MH162).
Several peptide, peptoid and benzodiazepine-based nonpeptide
ligands were tested in this assay, and evaluated for the ability to
activate the recombinant wild-type or constitutively active mutant
receptor, respectively. Full positive agonists had similar
signaling efficacy in both receptors when compared to the intrinsic
activity of the peptide agonist CCK-8s. The signalling efficacy of
ligands with lower intrinsic activity was logarithmically amplified
when a constitutively active mutant receptor was used in the assay.
The prototype benzodiazepine-derived non-peptide antagonist
L-365,260 barely increased basal activity of the human wild-type
CCK-B/gastrin receptor, but was identified as a partial agonist
using the .sup.325L.fwdarw.E mutant MH162. Minor chemical
modification of L-365,260 resulted in compounds which were pure
antagonists (YM022), partial agonists (L-740,093 S) or inverse
agonists (L-740,093 R). Thus, the process of discovering novel
non-peptide agonists, e.g., those with positive inverse or
intrinsic activity, should be expedited by using enhanced
receptors, e.g., constitutively active mutant receptors, in the
screening assay.
[0059] I. Working Examples:
[0060] A. CCK-B/gastrin receptor: The following example
demonstrates the use of an enhanced peptide hormone receptor, the
constitutive CCK-B/gastrin receptor MH162 (.sup.325L.fwdarw.E), to
screen for an agonist.
[0061] Using a constitutively active mutant of the human
CCK-B/gastrin receptor it was discovered that several
benzodiazepine-based putative non-peptide `antagonists` had
detectable intrinsic activity as agonists when binding to this
receptor.
[0062] The constitutively active CCK-B/gastrin receptor mutant
MH162 (.sup.325L.fwdarw.E) was transiently overexpressed in COS-7
cells. The fact that it was constitutively active was evident from
ligand-independent production of inositol phosphate; the wild-type
receptor, in contrast, exhibits only ligand-dependent inositol
phosphate production. Both mutant and wild-type receptors induced
similar inositol phosphate production when maximally stimulated
with the peptide agonists CCK-8s or gastrin I (FIG. 3). In
contrast, the different intrinsic activities of three
benzodiazepine-derived compounds, L-740,093 R, YM022 and L-365,260
were only detected by using the constitutive CCK-B receptor mutant
MH162 in the assay. Each of these compounds was previously
considered a prototype non-peptide antagonist of the wild-type
CCK-B/gastrin receptor (Castro Pineiro et al., WO 94/03437; Lotti
et al., Eur. J. Pharmacol., 162:273-280, 1989; Nashida et al., J.
Pharmacol. Exp. Ther., 270:1256-61, 1994; Nashida et al., J.
Pharmacol. Exp. Ther., 269:725-31, 1994). The intrinsic activity
(percent maximal stimulation of inositol phosphate formation) of
all compounds was tested at concentrations that were at least
100-fold higher than the corresponding receptor affinities.
[0063] The benzodiazepine L-365,260 had 62% efficacy when compared
to the full agonist CCK-8s, and was on that basis identified as a
partial agonist in the .sup.325L.fwdarw.E constitutively active
mutant receptor MH162 (FIG. 3, right section). In fact, close
re-examination of the function of L-365,260 with the wild-type
CCK-B/gastrin receptor also revealed a barely detectable, yet
significant, increase in inositol phosphate production that had not
been seen with other non-peptide compounds.
[0064] From the above results it was concluded that minor changes
in the chemical groups attached to the benzodiazepine backbone can
result in marked alterations in intrinsic activity of small
non-peptide compounds. The stereochemistry of
benzodiazepine-derived CCK receptor ligands is another feature
which can alter binding affinity as well as receptor selectivity
(Showell et al. J. Med. Chem. 37:719-721, 1994).
[0065] The following additional observations confirmed that
differences in ligand stereochemistry determine the functional
properties of the CCK-B/gastrin receptor specific compounds. For
example, it was noted that L-740,093 S was almost a full agonist in
the .sup.325L.fwdarw.E CCK-B/gastrin receptor mutant MH162 (FIG.
3). When tested with the human wild-type CCK-B/gastrin receptor,
L-740,093 S functions as a partial agonist (25% efficacy compared
with CCK-8s). As such, L-740,093 S is the first non-peptide agonist
known to be specific to the CCK-B/gastrin receptor. The mirror
image of L-740,093 S, L-740,093 R, has properties opposite to those
of the S enantiomer. L-740,093 R reduces the basal activity of the
constitutively active receptor almost to wild-type levels, and is
therefore acting as an inverse agonist.
[0066] One advantage of the instant screening assay is its ability
to distinguish between an agonist, e.g., an inverse agonist, and an
antagonist. When testing an agonist, the level of signaling
activity observed with an enhanced form of a peptide hormone
receptor (e.g., a constitutive receptor) differs from the activity
observed with a human wild-type receptor. In contrast, an
antagonist shows similar signalling activities with both enhanced
and human wild-type receptor. A pure antagonist is further expected
to attenuate the effects of both positive and inverse agonists.
[0067] Of the compounds tested, YM022 came closest to being a
`perfect` antagonist, having almost no intrinsic activity on either
the wild-type or the constitutively active CCK-B/gastrin receptor.
In both the wild-type and the constitutively active receptors,
YM022 blocked CCK-8s induced inositol phosphate production with
almost identical affinity, reflected by similar pA2 values (9.78
and 9.37, respectively). Consistent with the functional
classification of L-740,093 S as a non-peptide agonist, the
inositol phosphate production induced by this compound could be
blocked by YM022 (pA2=9.54; FIG. 4). YM022 was also able to
attenuate the inverse agonist activity of L-740,093 R on the
constitutively active CCK-B/gastrin receptor (FIG. 5). In a
concentration-dependent manner, YM022 partially restored basal
activity to the constitutively active receptor which had been
inhibited by 20 nM L-740,093 R. The fact that basal activity was
not restored completely is explained by the fact that YM022 itself
is a weak inverse agonist in this mutant rather than a pure
receptor antagonist.
[0068] The pA2 value measures the functional affinity of a
competitive antagonist. In contrast to IC.sub.50 values (50%
inhibitory concentration), pA.sub.2 values are independent of which
agonist concentrations are used to measure antagonist affinities.
Ideally, pA.sub.2 values should also be independent of what
specific agonist compounds are tested to assess antagonist
affinities. The pA2 value is defined as the negative logarithm of
the specific antagonist concentration which shifts the agonist
concentration-response curve by a factor of two to the right. In
other words, in the presence of a given antagonist concentration,
one would need twice as much agonist as would be required in the
absence of antagonist to induce the same effect. pA.sub.2 values of
competitive antagonists are typically assessed by Schift plots, but
can also be measured by simplified `null` methods (Lazareno et al.,
Trends in Pharmacol. Sci., 14:237-239, 1993).
[0069] In addition to non-peptide ligands, the constitutively
active mutant CCK-B receptor MH162 (.sup.325L.fwdarw.E) amplified
the intrinsic activity of peptide-derived partial agonists, called
peptoids (Horwell et al., Eur. J. Med. Chem., 30 Suppl.:537S-550S,
1995; Horwell et al., J. Med. Chem., 34:404-14, 1991). The peptoids
used in the following experiments were obtained by sequentially
modifying CCK-4, which is a tetrapeptide comprising the four
carboxyterminal amino acids of CCK. Two prototype peptoid
compounds, PD 135,158 and PD 136,450, were converted from partial
agonists when tested in the presence of wild-type CCK-B/gastin
receptor to almost full agonists in the presence of constitutively
active CCK-B/gastrin receptor. Thus, peptide-derived as well as
non-peptide compounds can exhibit an increased efficacy when tested
with the constitutively active versus the wild-type CCK-B/gastrin
receptor. Despite these marked alterations in efficacy, the ratio
of wild-type versus mutant receptor affinities, as determined by
.sup.125I-CCK-8 competition binding experiments, fell within a
two-fold range (Table 1A). There was no apparent correlation
between the intrinsic activity of CCK-B/gastrin receptor ligands
and potency shifts between the wild-type and the constitutively
active receptors (Table 1B).
[0070] The intrinsic activity of L-740,093 S was comparable to that
observed for the `peptoid` ligand PD 135,158, a compound that has
been recently demonstrated to be a partial agonist in vivo (Ding et
al., Gastroenterology, 109:1181-87, 1995).
[0071] Precedent with the constitutively active CCK-B/gastrin
receptor illustrates a new strategy using mutant receptors as a
`magnifying glass` to screen for non-peptide lead compounds with
some degree of intrinsic activity. The constitutively active
.sup.325L.fwdarw.E mutant MH162 reliably enhanced detection of the
intrinsic activity a compound possesses for stimulating the
wild-type receptor (FIG. 6). This was true over the spectrum of
peptide, `peptoid,` and non-peptide ligands tested.
1TABLE 1 A) .sup.125I CCK-8 binding affinities of tested ligands
Ratio Wild-type receptor MH162 Mutant (Wild-type/ Compound Ki (nM)
Ki (nM) Mutant) Gastrin I 1.35 .+-. 0.28 0.80 .+-. 0.16 1.69 CCK-8s
0.12 .+-. 0.01 0.07 .+-. 0.01 1.71 PD 135, 158 2.25 .+-. 0.61 1.01
.+-. 0.19 2.23 PD 136, 450 0.99 .+-. 0.1 0.59 .+-. 0.12 1.68 L-740,
093 R 0.19 .+-. 0.02 0.18 .+-. 0.04 1.06 YM022 0.07 .+-. 0.01 0.08
.+-. 0.01 0.88 L-364, 718 150 .+-. 42 170 .+-. 34 0.88 L-365, 158
7.16 .+-. 0.87 7.83 .+-. 1.51 0.91 L-740, 093 S 19.5 .+-. 1.5 16
.+-. 1.4 1.22 B) Signaling potencies of tested ligands Ratio
Wild-type receptor MH162 Mutant (Wild-type/ Compound IC50 (nM) 95%
C.I. IC50 (nM) 95% C.I. Mutant) Gastrin I 0.24 (0.12-0.48) 0.20
(0.06-0.69) 1.20 CCK-8s 0.14 (0.11-0.19) 0.15 (0.08-0.30) 0.93 PD
135, 158 1.05 (0.29-3.83) 1.01 (0.21-4.80) 1.04 PD 136, 450 0.36
(0.04-3.35) 0.58 (0.23-1.46) 0.62
[0072] B. CCK-A receptor: In another example of using an enhanced
receptor to identify an agonist, a CCK-A receptor mutant, MHA21/35
(.sup.138E.fwdarw.Q and .sup.303ANLM.fwdarw.HVSA modifications of
SEQ ID NO: 5), was used to test the peptoid compound PD 136,450.
FIG. 12 illustrates the ability of the MHA 21/35 CCK-A mutant
receptor to enhance the intrinsic agonist activity of the peptide
CCK-8, the non-peptide L-365, 260, and the peptoid PD 136,450
relative to human wild-type CCK-A receptor. FIG. 13 illustrates the
amplification of partial agonist efficacy by the constitutive CCK-A
receptor MHA21/35 as a function of the concentration of peptoid
agonist PD 136,450.
[0073] II. Receptor Binding and Activity Assays:
[0074] A. Receptor Binding Assays: The binding of a ligand to a CCK
receptor, e.g., the CCK-A or the CCK-B/gastrin receptor, can be
measured according to the following example. In this example, the
binding affinity of a ligand to the human CCK-B/gastrin receptor is
measured.
[0075] COS-7 cells (1.5.times.10.sup.6) were plated in 10-cm
culture dishes (Nunc) and grown in Dulbecco's modified Eagle's
medium containing 10% fetal calf serum in a 5% CO.sub.2, 95% air
incubator at 37.degree. C. After an overnight incubation, cells
were transfected (Pacholczyk et al., Nature 350:350-354, 1991) with
5-7 .mu.g of a pcDNA I expression vector containing hCCKB
(HCCKB-pcDNA I). Twenty-four hours after transfection cells were
split into 24-well dishes (2.times.10.sup.4 cells/well) (Costar).
After an additional 24 hours, competition binding experiments were
performed in Hank's buffer supplemented with 25 mM
phenylmethylsulfonyl fluoride (PMSF). Twenty pM of .sup.125I CCK-8
(DuPont-New England Nuclear) was used as radioligand. Equilibrium
binding occurred after incubation for 80 min. at 37.degree. C. Cell
monolayers were then washed three times, hydrolyzed in 1 N NaOH,
and the amount of radioactivity to the receptor was quantified.
Unlabeled agonists (e.g., CCK-8s, unsulphated CCK-8 (CCK-8us),
gastrin I, CCK-4 (Peninsula)) and antagonists (L364,718 and
L365,260 (Merck)) were tested over the concentration range of 0.1
pM to 10 .mu.M. All binding experiments were repeated three to five
times.
[0076] The competition data were analyzed using computer software
which is specifically designed for the purpose of radioligand
binding assays (Inplot 4.0, GraphPad, San Diego, Calif.). Analyses
of competition and saturation binding data can also be performed
using computerized non-linear curve fitting (McPherson, G. A., J
Pharmacol Methods, 14:213-28, 1985).
[0077] The affinities of all agonists and antagonists were
confirmed by repeating the above assay using Chinese hamster ovary
(CHO) cells stably transfected with human CCK-B/gastrin receptor
cDNA. This CHO cell line was established by transfecting a
hCCKB-pcDNAI Neo expression vector (Invitrogen) into CHO cells
using a standard lipofection protocol (Bethesda Research
Laboratories) followed by G418 selection.
[0078] Where binding parameters are determined in isolated plasma
membranes, binding can be performed, e.g., for 60 min. at
22.degree. C. (Kopin et al., Proc. Natl. Acad. Sci. USA,
89:3605-09, 1992). Separation of bound and free radioligand can be
achieved by receptor-binding filtermat filtration (Klueppelberg, U.
G., et al., 1989, Biochemistry 28:3463-8).
[0079] In order to compare the binding specificity of CCK-B/gastrin
mutant receptors of the invention with the binding specificity
typical of wild-type CCK-B/gastrin receptors see Matsumoto et al.
(Am J Physiol., 252:G143-G147, 1987) and Lee et al. (J. Biol.
Chem., 268(11):8164-69, 1993).
[0080] Comparison of binding affinity to that of a wild-type human
CCK-B receptor: A base line value for binding of a radiolabelled
ligand to a human wild-type receptor, e.g., the human CCK-B/gastrin
receptor was determined (see Lee et al., J. Biol. Chem.,
268(11):8164-69, 1993). Agonist affinities of the human brain
CCK-B/gastrin receptor expressed in COS-7 cells were characterized
(FIG. 7). The structurally related agonists CCK-8s, gastrin I, and
CCK-4 all competed in a concentration-dependent manner for binding
of .sup.125I-CCK-8 to COS-7 cells expressing the recombinant
receptor. The calculated IC.sub.50 values for CCK-8s, gastrin I,
and CCK-4 are 0.14, 0.94, and 32 nM respectively (FIG. 7, part A).
Similar .sup.125I-CCK-8 competition curves were assessed with
L-364,718 and L-365,260 (FIG. 7, part B), and revealed IC.sub.50
values of 145 and 3.8 nM, respectively. Untransfected cells showed
no displaceable binding.
[0081] B. Receptor Signaling Activity Assays:
[0082] Binding of an agonist to a CCK receptor elicits an increase
in the intracellular calcium concentration and in
phosphatidylinositol hydrolysis.
[0083] Measurement of [Ca.sup.2+]: Forty-eight hours after
transfection with hCCKB-pcDNAI, COS-7 cells were loaded with the
Ca.sup.2+ fluorophore fura-2 in modified Krebs-Ringer bicarbonate
buffer. Changes in the fluorescence emission ratios (340:380 nm)
after stimulation of cells with 10.sup.-7M CCK-8s or 10.sup.-6M
gastrin I were measured as previously described (Rajan et al.,
Diabetes, 38:874-80, 1989). Extracellular calcium can be chelated
with EGTA (2.5 mM) to confirm that a gastrin-induced increase in
[Ca.sup.2+] originates primarily from intracellular [Ca.sup.2+]
pools.
[0084] Measurement of Inositol phosphate Metabolites: COS-7 cells
transfected with hCCKB-pcDNAI were cultured in inositol-free
Dulbecco's modified Eagle's medium (DMEM, GIBCO) which was
supplemented with 10 .mu.Ci/ml [.sup.3H] myo-inositol (ARC) for 24
hours prior to analysis. After 1 hour of equilibration in modified
Krebs-Ringer bicarbonate, the cells were stimulated with 10.sup.-7M
CCK-8s for 10 seconds and harvested in methanol-HCl. The aqueous
phase was extracted with chloroform, lyophilized, and analyzed for
inositol 1,4,5-triphosphate (Ins-1,3,4,5-P.sub.3) and inositol
1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P.sub.4) by strong
anion-exchange high performance liquid chromatography (Auger et
al., Cell, 57:167-75, 1989).
[0085] Comparison of signaling activity to that of a wild-type
human CCK-B receptor: A baseline level of human wild-type CCK-B
receptor second messenger signaling activity was measured in
response to CCK-8s stimulation of COS-7 cells expressing the
receptor (FIG. 8; see Lee et al., J. Biol. Chem., 268(11):8164-69,
1993). CCK-8s (10.sup.-7M) triggered a marked increase in free
cytosolic calcium, [Ca.sup.2+].sub.i (FIG. 8, part A, left panel).
There was no change in free cytosolic calcium in cells transfected
with the empty expression vector, pcDNAI. After chelation of
extracellular calcium (1.5 mM Ca.sup.2+ in the buffer) by 2.5 mM
EGTA, addition of CCK-8s (10.sup.-7 M) still transiently increased
[Ca.sup.2+].sub.i (FIG. 8, part A right panel), suggesting that the
initial peak of the CCK-induced increase in [Ca.sup.2+].sub.i
originated primarily from intracellular Ca.sup.2+ pools. The arrows
indicate the addition of CCK-8s (0.1 .mu.M) or EGTA (2.5 mM). The
pattern of [Ca.sup.2+].sub.i response suggests that the binding of
CCK-8s to the recombinant receptor triggers intracellular signaling
through activation of phospholipase C. This was confirmed by
measurement of inositol phosphate metabolites in hCCKB-pcDNA
I-transfected COS-7 cells 10 seconds after CCK-8s stimulation (FIG.
8, part B). This time point was chosen because it immediately
precedes the CCK-8-induced [Ca.sup.2+].sub.i peak. CCK-8s
(10.sup.-7M) increased the level of Ins-1,4,5-P.sub.3 by 453% over
control, unstimulated hCCKB-pcDNA I-transfected COS-7 cells (n=3,
p<0.001). The level of Ins-1,3,4,5-P.sub.4, an immediate
metabolite of Ins-1,4,5,-P.sub.3, also increased by 186% over
control (n=3, p<0.01).
[0086] A simplified method for measuring total inositol phosphate
content: While the above method specifically assesses
Ins(1,4,5)P.sub.3 content, a simplified screening method can be
used to test for the total concentration of inositol phosphate; the
simplified method does not distinguish between specific isoforms.
(This method was used to measure inositol phosphate generation for
the experiments shown in FIGS. 3, 4, 5, 6, and 10.)
[0087] COS-7 cells transfected with receptor cDNA-pcDNAI were
cultured in inositol-free, serum-free Dulbecco's modified Eagle's
medium (DMEM, GIBCO), supplemented with 3 .mu.Ci/ml
.sup.3H-myo-inositol (NEN, 45-80 Ci/mmol), for 18 hours prior to
analysis. The cells were then washed twice with DMEM/10 mM
LiCl.sub.2 and twice with phosphate-buffered saline/10 mM
LiCl.sub.2. After stimulation with putative agonists in
phosphate-buffered saline 10/mM LiCl.sub.2 for 30 minutes at
37.degree. C., cells were scraped in ice-cold methanol. Lipids were
extracted with chloroform (Pfeiffer et al., FEBS Lett.,
204:352-356, 1986). The upper phase was analyzed for inositol
phosphates by strong anion exchange chromatography, using Dowex
1-X8 columns (BIORAD) and differential elution with water/60 mM
ammonium fornate/2 M ammonium fornate. Eluted radioactivity was
measured by liquid scintillation counting, and inositol phosphate
content was expressed as a percentage of total
.sup.3H-radioactivity applied to the columns.
[0088] Further information on the second messenger pathways linked
to the native parietal cell gastrin receptor can be obtained in the
following references: Muallem, S. et al., 1984, Biochim Biophys
Acta 805:181-5; Chew, C. S. et al., 1986, Biochim Biophys Acta
888:116-25; Roche,S. et al., 1991, FEBS Letts., 282:147-51.
[0089] In addition to inositol phosphate production, second
messenger signaling activity can be measured according to, e.g.,
cAMP, cGMP, ppGpp, or calcium ion production, or using as
indicators, e.g., intracellular pH, pH-sensitive dyes, or
expression of a reporter gene, e.g., a luciferase gene, or
measuring channel activity or cell depolarization or
hyperpolarization by electrophysiological techniques.
[0090] III. Suitable Peptide Hormone Receptors with the Ability to
Amplify the Intrinsic Activity of a Non-peptide Agonist:
[0091] The screening assay of the invention can be performed using
peptide hormone receptors that have a higher activity than the
corresponding human wild-type receptor. An enhanced basal activity
amplifies the intrinsic activity of ligands, and is useful for
detecting either activation of the receptor by a partial agonist,
or inhibition by an inverse agonist. Receptors that do not have an
enhanced basal activity relative to the corresponding wild-type
receptor, but still amplify the intrinsic activity of a partial
agonist, are also useful.
[0092] Examples of peptide hormone receptors that are useful for
screening non-peptide agonists include various forms of the
receptors that interact with the following peptide hormones (along
with references for their respective wild-type amino acid
sequences): amylin, angiotensin, bombesin, bradykinin, C5a
anaphylatoxin, calcitonin, calcitonin-gene related peptide (CGRP),
chemokines, cholecystokinin (CCK), endothelin, follicle stimulating
hormone (FSH), formyl-methionyl peptides, galanin, gastrin, gastrin
releasing peptide, glucagon, glucagon-like peptide 1, glycoprotein
hormones, gonadotrophin-releasing hormone, leptin, luteinizing
hormone (LH), melanocortins, neuropeptide Y, neurotensin, opioid,
oxytocin, parathyroid hormone, secretin, somatostatin, tachykinins,
thrombin, thyrotrophin, thyrotrophin releasing hormone, vasoactive
intestinal polypeptide (VIP), and vasopressin. An enhanced
receptor. can further embrace a single transmembrane domain peptide
hormone receptor, e.g., an insulin receptor. The wild-type amino
acid sequences of the above peptide hormone receptors is available
in, and/or referenced in, Watson and Arkinstall, The G-Protein
Linked Receptor, Academic Press, NY., 1994.
[0093] Forms of a peptide hormone receptor that are capable of
amplifying the intrinsic activity of an agonist include, but are
not limited to, the following forms of receptors:
[0094] 1. Mutant peptide hormone receptors that are capable of
amplifying the intrinsic activity of partial. agonists.
[0095] An example is given of a mutant human CCK-A receptor that
enhances the intrinsic activity of the partial `peptoid` agonist PD
135,158, yet causes no apparent increase in agonist-independent
basal receptor activity, is the mutant CCK-A receptor MHA35. MHA35
was made by replacing amino acids 138-ERY-140 of the human
wild-type CCK-A receptor with QRY in the vector pcDNAI. (See FIG. 2
for an illustration of the wild-type CCK-A receptor amino acid
sequence.)
[0096] 2. CCK-A receptors in which one or more of residues
.sup.138E, .sup.305L, and .sup.312I are replaced with any other
amino acid residue, e.g., a serine, aspartic acid, glutamine, or
glutamic acid residue. For example, a constitutively active CCK-A
receptor mutant was constructed by replacing five amino acids
(.sup.138E.fwdarw.Q and .sup.303ANLM.fwdarw.HVSA modifications of
SEQ ID NO: 5); the resulting modified receptor is called MHA
21/35.
[0097] 3. CCK-B/gastrin receptors in which one or more of residues
.sup.151E, .sup.325L, and .sup.332V are replaced with any other
amino acid residue, e.g., a serine, aspartic acid, glutamine, or
glutamic acid residue.
[0098] 4. Naturally-occurring Mutant Receptors, including but not
limited to naturally-occurring constitutively active mutant
receptors, that are associated with a disease or other adverse
phenotype, e.g., a phenotype that results from a constitutively
active naturally-occurring mutant receptor. Examples include, but
are not limited to, the following peptide hormone receptors:
[0099] a) Point mutations in the luteinizing hormone (LH) receptor
gene are responsible for some incidences of precocious puberty.
Mutant receptors of the invention can be constructed by altering
the following amino acid residues of the LH receptor: the alanine
residue at position 568 to another amino acid, e.g., to a valine
(Latronico et al., J. Clin. Endo. & Meta., 80(8):2490-94,
1995); the asparagine residue at position 578 to another amino
acid, e.g., to a glycine or a tyrosine (Kosugi et al., Human Mol.
Genet., 4(2):183-88, 1995; Laue et al., Proc. Natl. Acad. Sci. USA,
92(6):1906-10, 1995); the Met residue at position 571 to another
amino acid, e.g., to an Ile, or the Thr residue at 577 to another
amino acid residue, e.g., to an Ile (Kosugi et al., supra; Laue et
al. supra); the Ile residue at position 542 to another amino acid,
the Asp residue at position 564 to another amino acid, the Cys
residue at position 581 to another amino acid, or the Asp residue
at position 578 to another amino acid (Laue et al., supra); amino
acid residues within transmembrane helices 5 or 6, e.g., in the
intracellular domain-proximal portion of transmembrane helix 6, or
in intracellular loop 3 (Laue et al. supra).
[0100] Also embraced are mutations at the corresponding residues of
the follicle stimulating hormone (FSH) receptor and the thyroid
stimulating hormone (TSH) receptor (Latronico et al., supra).
[0101] b) A naturally-occurring constitutively active parathyroid
(PTH) receptor can result from a His to Arg substitution at
conserved position 223 (Schipani et al., Science, 268:98-100,
1995). A constitutively active mutant G-LP1 receptor can be
constructed by substituting alternative amino acids at the
corresponding residues in related receptors, e.g., substituting
another amino acid for the homologue His in the glucagon-like
peptide 1 (G-LP1) receptor. A similar change in any of the
receptors related to PTH or G-LP1 by amino acid homology including,
but not limited to, secretin, vasoactive intestinal polypeptide,
glucagon, G-LP1, and calcitonin.
[0102] Non-peptide positive or inverse agonists identified in a
screening assay employing any of the above-listed naturally
occurring mutant receptors can be therapeutically useful against a
corresponding adverse pnenotype.
[0103] 5. Strategies to identify synthetic mutant receptors.
[0104] Deletional analysis defines intracellular receptor domains
important in second messenger signaling: Recombinant CCK-A and
CCK-B/gastrin receptors are both coupled to phospholipase-C
activation. Applicants hypothesized that the third intracellular
loop of the CCK-B/gastrin receptor would include residues that are
important in second messenger signaling. To test this hypothesis, a
series of deletion mutants was made, each lacking between six and
55 amino acids located in the third intracellular loop. Each mutant
receptor was expressed in COS-7 cells and was tested for
[.sup.125I]CCK-8s binding and .sup.3H inositol phosphate formation.
Deletion of a twelve amino acid segment in the carboxy-terminal end
of the third intracellular loop resulted in normal affinity and
capacity for CCK-8s binding, but caused a 90% reduction of maximal
CCK-8s induced inositol phosphate formation; all other receptors in
this series signaled normally. The region containing the twelve
amino acids that proved to functionally important was then screened
for constitutively active point mutations, as described below.
[0105] Strategy 1: Domain swapping with cAMP generating receptors
results in constitutive receptor activity: A method for rapidly
identifying constitutively active mutant receptors relies on
exchanging functional domains between two receptors, the domains
being, e.g., approximately 5-10 amino acids in length. One of the
two receptors is a form of the receptor which is the main template
of the desired mutant receptor, e.g., a wild-type receptor; the
second receptor is a different peptide hormone receptor from the
first. Candidate receptors are coupled to different signal
transduction pathways, e.g., a signal via a same or different
second messenger pathways, yet are closely related in their amino
acid sequence. These criteria are based on the idea that stretches
of amino acids which function normally in their native context can
confer agonist-independent signaling when transplanted into a
closely related receptor which is linked to a different
second-messenger signaling pathway.
[0106] The domain swapping strategy was used to identify
constitutively active mutants of the CCK-B/gastrin receptor. A
series of short segments in the third intracellular loop were
sequentially replaced with homologous amino acid sequence from the
vasopressin 2 receptor, which is the receptor most nearly identical
in sequence to hCCK-B. Vasopressin 2 is also a good candidate for
swapping domains with the CCK-B/gastrin receptor because it is,
different from the latter, linked to the adenylate cyclase
signaling pathway.
2 309 transmembrane domain VI 359 LT APGPGSGSRP TQAKLLAKKR
VVRMLLVIVV LFFLCWLPVY SANTWR AFD AHVSA [MH40] SA [MH128] S [MH156]
E [MH162]
[0107] When tested, a five amino acid substitution (QAKLL (SEQ ID
NO: 13) to AHVSA (SEQ ID NO: 14)) into the homologous position of
the CCK-B/gastrin receptor resulted in constitutive activity of the
CCK-B/gastrin receptor. The QAKLL (SEQ ID NO: 13) to AHVSA (SEQ ID
NO: 14) substitution caused an increased level of basal inositol
phosphate formation to 290% of the wild-type CCK-B/gastrin receptor
(FIG. 9, Mutant 2). In addition, mutations causing constitutive
activity include replacement of LL to SA, L to S, and L to E.
[0108] Strategy 2: Glutamic acid scanning mutagenesis identifies
constitutively active receptors: In addition to, or as a substitute
for, deletion analysis or domain swapping, mutant receptors can be
made using a process applicants have named `amino acid scanning
mutagenesis.` Amino acid scanning mutagenesis involves sequentially
replacing each amino acid found in either an intracellular loop or
in the half of the transmembrane domain flanking the intracellular
portion of the receptor. An experimental option is to change the
charge of the amino acid, e.g., to exchange a negative for a
positive amino acid, a positive for a negative amino acid, or a
positive or negative amino acid for a neutral amino acid. Another
option would be to exchange each amino acid, e.g., each neutral
amino acid, with another neutral amino acid.
[0109] In the case of the CCK-B/gastrin receptor, deletion analysis
was initially used to define a functionally important twelve amino
acid segment within the third intracellular loop which was
important for second messenger signaling. Subsequently, each of the
neutral amino acids within the 12 of this segment was replaced
sequentially with another amino acid, preferably with glutamic
acid. The scanning analysis technique revealed that one of the
glutamic acid substitutions caused a 228% increase in the
basal-level of inositol phosphate accumulation, relative to the
wild-type value, in transiently transfected COS-7 cells (FIG. 9,
Mutant 1).
[0110] In this example, applicants focused on the region limited to
the carboxy end of the third intracellular (IC) loop and the
portion of the sixth transmembrane domain which flanks the third IC
loop. Glutamic acid residues (E) were introduced in place of
neutral amino acid residues.
3 309 359 25 LT APGPGSGSRP TQAKLLAKKR VVRMLLVIVV LFFLCWLPVY SANTWR
AFD E E E EE
[0111] Constitutively active receptors include an amino acid
replacement at .sup.325L.fwdarw.E (MH162; SEQ ID NO: 19), and at
.sup.332V.fwdarw.E (MH129; SEQ ID NO: 22). Each mutant was
constructed in a pcDNAI vector as described above.
[0112] FIG. 9 is a schematic representation of the seven
transmembrane (TM) domain structure of the human CCK-B/gastrin
receptor. The C-terminal domain of the third intracellular loop,
which is crucial for intracellular signaling, is highlighted in
black. Within this segment, two mutations were found to confer
constitutive activity on the receptor. One of the mutations was
constructed by glutamic acid substitution scanning (Mutant 1;
MH129); a second mutation was constructed by domain swapping
(Mutant 2; MH162). A bar graph showing the basal inositol phosphate
accumulation in COS-7 cells, which had been transfected with the
wild-type CCK-B/gastrin receptor or with two different
constitutively active mutants, is shown in FIG. 10.
[0113] Strategy 3: A third method for making a mutant receptor is
to align the receptor of interest with a known constitutively
active mutant receptor, including, but not limited to, peptide
hormone, biogenic amine, rhodopsin, or other G-protein coupled
receptors. An example of such an alignment is shown in FIG. 2.
Generally, mutations which result in constitutive activity in the
known mutant can be introduced into the corresponding position of
the receptor of interest. Examples of known constitutively active
mutant receptors include, but are not limited to, the follicle
stimulating hormone (FSH) receptor, the thyroid stimulating hormone
(TSH) receptor, and the luteinizing hormone receptor, e.g., a 568
Ala to Val mutation in the LH receptor (Latronico et al., J. Clin.
Endo. & Meta., 80(8):2490-94, 1995).
[0114] This method, based on alignment, was employed to construct a
CCK-A mutant receptor. A multiple alignment map was made which
included the human and rat CCK-A sequences, the mastomys, rat,
human, and canine CCK-B/gastrin receptor, and a Xenopus CCK-A/CCK-B
intermediate receptor (CCK-XL; FIG. 2). Based on this map,
conserved amino acids 138-ERY-140 of the CCK-A receptor were
replaced with amino acids QRY, based on a known constitutively
active rhodopsin mutant with enhanced transducing activation (Arnis
et al., J. Biol. Chem., 269:23879-81, 1994). The altered amino acid
residues are positioned in transmembrane domain III and flank the
second intracellular loop. Although the basal level of signaling
was not increased, the intrinsic activity of the non-peptide ligand
PD 135,158 was significantly increased.
[0115] Strategy 4: Additional mutant receptors can be made by
sequentially deleting intracellular portions of the receptor, and
looking for an increase in basal activity, or for overactivity of a
partial agonist, relative to the wild-type receptor.
[0116] Wild-type Receptors with Enhanced Basal Activity:
[0117] Peptide hormone receptors useful in the method of the
invention can include non-human receptors which have the ability to
amplify the intrinsic activity of non-peptide agonist as compared
to the corresponding human wild-type receptor, or which have a
higher basal level of activity than does the human wild-type
receptor.
[0118] In FIG. 11, basal levels of inositol phosphate production
were measured for human CCK-A (hCCK-A), human CCK-B (hCCK-B), dog
CCK-B (dCCK-B), mouse CCK-B (mCCK-B), and the mastomys CCK receptor
(FIG. 11, part A), and expressed relative to the basal level of
hCCK-B.
[0119] Single experiments were also performed for the rat
CCK-B/gastrin receptor and for the related Xenopus CCK receptor
(Table 2). The human .sup.325L to E mutant served as a positive
control (n=14).
4TABLE 2 CCK-8s stimulated receptor basal (% of human basal) (%
human basal) rat CCK-A 77 684 Xenopus CCK 74 442 MH162 231 .+-. 7
771 .+-. 36
[0120] The wild-type human CCK-A and CCK-B/gastrin receptors
induced only insignificant changes of basal inositol phosphate
production in COS-7 cells (as compared to control cells transfected
with the empty plasmid vector, pcDNAI). Similarly, the wild-type
rat CCK-A and canine CCK-B/gastrin receptors, as well as the
closely related Xenopus CCK receptor all appeared more or less
functionally silent in the basal state. In contrast, the wild type
mouse CCK-B/gastrin receptor and its homologue from mastomys
natalensis significantly increased basal inositol phosphate
production in COS-7 cells over pcDNAI controls. When compared with
the slight basal activity of the wild type human CCK-B/gastrin
receptor, it was estimated that the basal activities of the wild
type mouse and mastomys homologues were 7- and 11-fold higher,
respectively. For comparison, the .sup.325L-E mutant of the human
CCK-B/gastrin receptor appeared to be at least 16-fold more active
than the human wild type receptor in its basal state. It should be
noted that the described species differences in basal activities
were clearly not related to different degrees of receptor
expression, since the maximal response to stimulation with CCK-8s
was comparable for all tested receptors (positive control).
[0121] IV. Therapeutic Use.
[0122] The ability to pharmacologically modulate wild-type or
constitutively active receptor activity opens the door for a new
class of clinically useful drugs. Enhanced receptors enable the
discovery of drugs with the ability to act as agonists, thereby
having advantages for treatment or prevention of a broad spectrum
of diseases. Constitutively active mutants of the thyrotropin,
luteinizing hormone, and parathyroid hormone receptors are already
known to occur in nature (see above) and might provide a starting
point for non-peptide agonist/inverse agonist screening. For
example, drugs which silence constitutively active thyroid
stimulating hormone receptors, which are implicated in the etiology
of thyroid adenomas, could be used to inhibit tumor growth.
Similarly, in patients with constitutively active luteinizing
hormone receptors, inverse agonists could delay the onset of
precocious puberty.
[0123] Non-peptide agonists, including but not limited to the
compounds of formula (I), are useful as agonists for treating and
preventing central nervous system disorders wherein CCK and/or
gastrin receptors are involved. Examples of such disease states
include gastrointestinal diseases, including gastrointestinal
ulcers such as peptic and duodenal ulcers, irritable bowel
syndrome, gastroesophagenal reflux disease or excess pancreatic or
gastrin secretion, acute pancreatitis, or motility disorders;
central nervous system disorders, including central nervous system
disorders caused by CCK interaction with dopamine, serotonin and
other monoamine neurotransmitters, such as neuroleptic disorders,
tardive dyskinesia, Parkinson's disease, psychosis, or Gilles de la
Tourette syndrome; depression; schizophrenia; disorders of appetite
regulatory systems; Zollinger-Ellison syndrome, antral and cell
hyperplasia, or pain.
[0124] Non-peptide agonists are further useful for:
[0125] 1) the treatment or prevention of neurological disorders
involving anxiety or panic, wherein a CCK and/or gastrin receptor
is involved;
[0126] 2) directly inducing analgesia, opiate or non-opiate
mediated, as well as anesthesia or loss of the sensation of
pain;
[0127] 3) preventing or treating the withdrawal response produced
by chronic treatment or abuse of drugs or alcohol. Such drugs
include, but are not limited to, benzodiazepines, cocaine, alcohol,
and nicotine;
[0128] 4) the treatment of stress and its relationship with drug
abuse;
[0129] 5) the treating oncologic disorders wherein a CCK receptor
may be involved. Examples of such oncologic disorders include small
cell adenocarcinomas and primary tumors of the central nervous
system glial and neuronal cells. Examples include, but are not
limited to, tumors of the lower esophagus, stomach, intestine,
colon, and lung, including small cell lung carcinoma;
[0130] 6) the treating or preventing of neurodegenerative disorders
arising as a consequence of a pathological condition, e.g., stroke,
hypoglycemia, cerebral palsy, transient cerebral ischemic attack,
cerebral ischaemia during cardiac pulmonary surgery or cardiac
arrest, perinatal asphyxia, epilepsy, Huntington's chorea,
Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's
disease, Olivo-ponto-cerebellar atrophy, anoxia such as from
drowning, spinal cord and head injury, and poisoning by
neurotoxins, including environmental neurotoxins.
[0131] The compounds of formula (I) may further be used to induce
miosis for therapeutic purposes after certain types of examination
and intraocular surgery. An example of intraocular surgery would
include cataract surgery with implantation of an artificial lens.
The CCK inverse agonist compounds can be used to prevent miosis
occurring in association with, e.g., iritis, uveitis and trauma.
Conversely, agonist derivatives can be used to induce miosis, e.g.,
for the treatment of glaucoma. The present invention therefore
provides a compound of formula (I) or a salt or prodrug thereof for
use in the preparation of a medicament.
[0132] The present invention also provides a compound of formula
(I) for use in therapy.
[0133] In a further or alternative embodiment the present invention
provides a method for the treatment or prevention of a
physiological disorder involving CCK and/or gastrin receptor, which
method comprises administration to a patient in need thereof of a
CCK and/or gastrin receptor agonist or inverse agonist amount of a
compound of formula (I).
[0134] When a compound is used as an agonist or inverse agonist of
a peptide hormone receptor in a human subject, the daily dosage
will normally be determined by the prescribing physician. The
dosage generally will vary with the age, weight, and response of
the individual patient, as well with as the severity of the
patient's symptoms. However, in most instances, an effective daily
dosage will be in the range of from about 0.001 mg/kg to about 2
.mu.g/kg of body weight; preferably, of from about 0.01 mg/kg to
about 200 mg/kg, e.g., from about 0.1 mg/kg to about 100 mg/kg of
body weight, administered in single or divided doses. One skilled
in the art knows that in some cases it will be necessary to use
dosages outside these limits.
[0135] Further guidance from in vitro tests are instructive; e.g.,
agonists of the invention are effective in the subnanomolar range
(e.g., L-740,093 R) and in the 20 nM range (e.g., L-740,093 S). An
agonist of the invention can be administered orally in single or
divided doses, or systemically, or by other means known to one
skilled in the art.
[0136] Other Embodiments
[0137] The invention can further embrace additional methods of
amplifying the activity of, so as to detect, an agonist or inverse
agonist which affects the activity of a peptide hormone receptor.
For example, an amplification scheme can involve modifying an
intracellular factor which is involved neutralizing the expression
of a peptide hormone receptor regulator, e.g., either a G-protein
or a molecule which acts as a downstream messenger. An
amplification scheme can alternatively involve optimizing the
kinetic conditions of a signal transduction assay which is used for
detecting receptor signaling. For example, the incubation time can
be prolonged so as to detect agonist activity with a particular
assay, e.g., a .beta.-galactosidase assay, or shortened so as to
avoid desensitization.
[0138] Further information on peptide hormone receptor amino acid
sequences, receptor-specific agonists and antagonists, receptor
conformation, pharmacology, receptor-encoding genes, animal models
for subsequent follow-up studies, and database accession numbers
can be obtained from: Watson and Arkinstall, The G-Protein Linked
Receptor, Academic Press, NY., 1994; see also, Kolakowski, L. F.,
"The G Protein-Coupled Receptor Database," World-Wide-Web Site,
GCRDB-WWW.
[0139] From the above description, one skilled in the art can
easily ascertain the essential characteristics of the present
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions.
[0140] Other embodiments are within the following claims.
Sequence CWU 1
1
23 1 449 PRT Mastomys natalensis 1 Met Glu Leu Leu Lys Leu Asn Ser
Ser Val Gln Gly Pro Gly Pro Gly 1 5 10 15 Ser Gly Ser Ser Leu Cys
His Pro Gly Val Ser Leu Leu Asn Ser Ser 20 25 30 Ala Gly Asn Leu
Ser Cys Glu Pro Pro Arg Ile Arg Gly Thr Gly Thr 35 40 45 Arg Glu
Leu Glu Leu Ala Ile Arg Ile Thr Leu Tyr Ala Val Ile Phe 50 55 60
Leu Met Ser Ile Gly Gly Asn Met Leu Ile Ile Val Val Leu Gly Leu 65
70 75 80 Ser Arg Arg Leu Arg Thr Val Thr Asn Ala Phe Leu Leu Ser
Leu Ala 85 90 95 Val Ser Asp Leu Leu Leu Ala Val Ala Cys Met Pro
Phe Thr Leu Leu 100 105 110 Pro Asn Leu Met Gly Thr Phe Ile Phe Gly
Thr Val Ile Cys Lys Ala 115 120 125 Val Ser Tyr Leu Met Gly Val Ser
Val Ser Val Ser Thr Leu Asn Leu 130 135 140 Val Ala Ile Ala Leu Glu
Arg Tyr Ser Ala Ile Cys Arg Pro Leu Gln 145 150 155 160 Ala Arg Val
Trp Gln Thr Arg Ser His Ala Ala Arg Val Ile Leu Ala 165 170 175 Thr
Trp Leu Leu Ser Gly Leu Leu Met Val Pro Tyr Pro Val Tyr Thr 180 185
190 Val Val Gln Pro Val Gly Pro Arg Val Leu Gln Cys Met His Arg Trp
195 200 205 Pro Ser Ala Arg Val Arg Gln Thr Trp Ser Val Leu Leu Leu
Met Leu 210 215 220 Leu Phe Phe Ile Pro Gly Val Val Met Ala Val Ala
Tyr Gly Leu Ile 225 230 235 240 Ser Arg Glu Leu Tyr Leu Gly Leu Arg
Phe Asp Gly Asp Asn Asp Ser 245 250 255 Asp Thr Gln Ser Arg Val Arg
Asn Gln Gly Gly Leu Pro Gly Gly Thr 260 265 270 Ala Pro Gly Pro Val
His Gln Asn Gly Gly Cys Arg His Val Thr Val 275 280 285 Ala Gly Glu
Asp Asn Asp Gly Cys Tyr Val Gln Leu Pro Arg Ser Arg 290 295 300 Leu
Glu Met Thr Thr Leu Thr Thr Pro Thr Pro Gly Pro Gly Leu Ala 305 310
315 320 Ser Ala Asn Gln Ala Lys Leu Leu Ala Lys Lys Arg Val Val Arg
Met 325 330 335 Leu Leu Val Ile Val Leu Leu Phe Phe Leu Cys Trp Leu
Pro Ile Tyr 340 345 350 Ser Ala Asn Thr Trp Cys Ala Phe Asp Gly Pro
Gly Ala Met Arg Ala 355 360 365 Leu Ser Gly Ala Pro Ile Ser Phe Ile
His Leu Leu Ser Tyr Ala Ser 370 375 380 Ala Cys Val Asn Pro Leu Val
Tyr Cys Phe Met His Arg Arg Phe Arg 385 390 395 400 Gln Ala Cys Leu
Asp Thr Cys Ala Arg Cys Cys Pro Arg Pro Pro Arg 405 410 415 Ala Arg
Pro Arg Pro Leu Pro Asp Glu Asp Pro Pro Thr Pro Ser Ile 420 425 430
Ala Ser Leu Ser Arg Leu Ser Tyr Thr Thr Ile Ser Thr Leu Gly Pro 435
440 445 Gly 2 451 PRT Rattus norvegicus 2 Met Glu Leu Leu Lys Leu
Asn Arg Ser Val Gln Gly Pro Gly Pro Gly 1 5 10 15 Ser Gly Ser Ser
Leu Cys Arg Pro Gly Val Ser Leu Leu Asn Ser Ser 20 25 30 Ser Ala
Gly Asn Leu Ser Cys Asp Pro Pro Arg Ile Arg Gly Thr Gly 35 40 45
Thr Arg Glu Leu Glu Met Ala Ile Arg Ile Thr Leu Tyr Ala Val Ile 50
55 60 Phe Leu Met Ser Val Gly Gly Asn Val Leu Ile Ile Val Val Leu
Gly 65 70 75 80 Leu Ser Arg Arg Leu Arg Thr Val Thr Asn Ala Phe Leu
Leu Ser Leu 85 90 95 Ala Val Ser Asp Leu Leu Leu Ala Val Ala Cys
Met Pro Phe Thr Leu 100 105 110 Leu Pro Asn Leu Met Gly Thr Phe Ile
Phe Gly Thr Val Ile Cys Lys 115 120 125 Ala Ile Ser Tyr Leu Met Gly
Val Ser Val Ser Val Ser Thr Leu Asn 130 135 140 Leu Val Ala Ile Ala
Leu Glu Arg Tyr Ser Ala Ile Cys Arg Pro Leu 145 150 155 160 Gln Ala
Arg Val Trp Gln Thr Arg Ser His Ala Ala Arg Val Ile Leu 165 170 175
Ala Thr Trp Leu Leu Ser Gly Leu Leu Met Val Pro Tyr Pro Val Tyr 180
185 190 Thr Met Val Gln Pro Val Gly Pro Arg Val Leu Gln Cys Met His
Arg 195 200 205 Trp Pro Ser Ala Arg Val Gln Gln Thr Trp Ser Val Leu
Leu Leu Leu 210 215 220 Leu Leu Phe Phe Ile Pro Gly Val Val Ile Ala
Val Ala Tyr Gly Leu 225 230 235 240 Ile Ser Arg Glu Leu Tyr Leu Gly
Leu His Phe Asp Gly Glu Asn Asp 245 250 255 Ser Glu Thr Gln Ser Arg
Ala Arg Asn Gln Gly Gly Leu Pro Gly Gly 260 265 270 Ala Ala Pro Gly
Pro Val His Gln Asn Gly Gly Cys Arg Pro Val Thr 275 280 285 Ser Val
Ala Gly Glu Asp Ser Asp Gly Cys Cys Val Gln Leu Pro Arg 290 295 300
Ser Arg Leu Glu Met Thr Thr Leu Thr Thr Pro Thr Gly Pro Val Pro 305
310 315 320 Gly Pro Arg Pro Asn Gln Ala Lys Leu Leu Ala Lys Lys Arg
Val Val 325 330 335 Arg Met Leu Leu Val Ile Val Leu Leu Phe Phe Leu
Cys Trp Leu Pro 340 345 350 Val Tyr Ser Val Asn Thr Trp Arg Ala Phe
Asp Gly Pro Gly Ala Gln 355 360 365 Arg Ala Leu Ser Gly Ala Pro Ile
Ser Phe Ile His Leu Leu Ser Tyr 370 375 380 Val Ser Ala Cys Val Asn
Pro Leu Val Tyr Cys Phe Met His Arg Arg 385 390 395 400 Phe Arg Gln
Ala Cys Leu Asp Thr Cys Ala Arg Cys Cys Pro Arg Pro 405 410 415 Pro
Arg Ala Arg Pro Gln Pro Leu Pro Asp Glu Asp Pro Pro Thr Pro 420 425
430 Ser Ile Ala Ser Leu Ser Arg Leu Ser Tyr Thr Thr Ile Ser Thr Leu
435 440 445 Gly Pro Gly 450 3 448 PRT Homo sapiens 3 Met Glu Leu
Leu Lys Leu Asn Arg Ser Val Gln Gly Thr Gly Pro Gly 1 5 10 15 Pro
Gly Ala Ser Leu Cys Arg Pro Gly Ala Pro Leu Leu Asn Ser Ser 20 25
30 Ser Val Gly Asn Leu Ser Cys Glu Pro Pro Arg Ile Arg Gly Ala Gly
35 40 45 Thr Arg Glu Leu Glu Leu Ala Ile Arg Ile Thr Leu Tyr Ala
Val Ile 50 55 60 Phe Leu Met Ser Val Gly Gly Asn Met Leu Ile Ile
Val Val Leu Gly 65 70 75 80 Leu Ser Arg Arg Leu Arg Thr Val Thr Asn
Ala Phe Leu Leu Ser Leu 85 90 95 Ala Val Ser Asp Leu Leu Leu Ala
Val Ala Cys Met Pro Phe Thr Leu 100 105 110 Leu Pro Asn Leu Met Gly
Thr Phe Ile Phe Gly Thr Val Ile Cys Lys 115 120 125 Ala Val Ser Tyr
Leu Met Gly Val Ser Val Ser Val Ser Thr Leu Ser 130 135 140 Leu Val
Ala Ile Ala Leu Glu Arg Tyr Ser Ala Ile Cys Arg Pro Leu 145 150 155
160 Gln Ala Arg Val Trp Gln Thr Arg Ser His Ala Ala Arg Val Ile Val
165 170 175 Ala Thr Trp Leu Leu Ser Gly Leu Leu Met Val Pro Tyr Pro
Val Tyr 180 185 190 Thr Val Val Gln Pro Val Gly Pro Arg Val Leu Gln
Cys Val His Arg 195 200 205 Trp Pro Ser Ala Arg Val Arg Gln Thr Trp
Ser Val Leu Leu Leu Leu 210 215 220 Leu Leu Phe Phe Ile Pro Gly Val
Val Met Ala Val Ala Tyr Gly Leu 225 230 235 240 Ile Ser Arg Glu Leu
Tyr Leu Gly Leu Arg Phe Asp Gly Asp Ser Asp 245 250 255 Ser Asp Ser
Gln Ser Arg Val Arg Asn Gln Gly Gly Leu Pro Gly Ala 260 265 270 Val
His Gln Asn Gly Arg Cys Arg Pro Glu Thr Gly Ala Val Gly Glu 275 280
285 Asp Ser Asp Gly Cys Tyr Val Gln Leu Pro Arg Ser Arg Pro Ala Leu
290 295 300 Glu Leu Thr Ala Leu Thr Ala Pro Gly Pro Gly Gly Ser Gly
Ser Arg 305 310 315 320 Pro Thr Gln Ala Lys Leu Leu Ala Lys Lys Arg
Val Val Arg Met Leu 325 330 335 Leu Val Ile Val Val Leu Phe Phe Leu
Cys Trp Leu Pro Val Tyr Ser 340 345 350 Ala Asn Thr Trp Arg Ala Phe
Asp Gly Pro Gly Ala His Arg Ala Leu 355 360 365 Ser Gly Ala Pro Ile
Ser Phe Ile His Leu Leu Ser Tyr Ala Ser Ala 370 375 380 Cys Val Asn
Pro Leu Val Tyr Cys Phe Met His Arg Arg Phe Arg Gln 385 390 395 400
Ala Cys Leu Glu Thr Cys Ala Arg Cys Cys Pro Arg Pro Pro Arg Ala 405
410 415 Arg Pro Arg Ala Leu Pro Asp Glu Asp Pro Pro Thr Pro Ser Ile
Ala 420 425 430 Ser Leu Ser Arg Leu Ser Tyr Thr Thr Ile Ser Thr Leu
Gly Pro Gly 435 440 445 4 453 PRT Canis familiaris 4 Met Glu Leu
Leu Lys Leu Asn Arg Ser Ala Gln Gly Ser Gly Ala Gly 1 5 10 15 Pro
Gly Ala Ser Leu Cys Arg Ala Gly Gly Ala Leu Leu Asn Ser Ser 20 25
30 Gly Ala Gly Asn Leu Ser Cys Glu Pro Pro Arg Leu Arg Gly Ala Gly
35 40 45 Thr Arg Glu Leu Glu Leu Ala Ile Arg Val Thr Leu Tyr Ala
Val Ile 50 55 60 Phe Leu Met Ser Val Gly Gly Asn Val Leu Ile Ile
Val Val Leu Gly 65 70 75 80 Leu Ser Arg Arg Leu Arg Thr Val Thr Asn
Ala Phe Leu Leu Ser Leu 85 90 95 Ala Val Ser Asp Leu Leu Leu Ala
Val Ala Cys Met Pro Phe Thr Leu 100 105 110 Leu Pro Asn Leu Met Gly
Thr Phe Ile Phe Gly Thr Val Val Cys Lys 115 120 125 Ala Val Ser Tyr
Leu Met Gly Val Ser Val Ser Val Ser Thr Leu Ser 130 135 140 Leu Val
Ala Ile Ala Leu Glu Arg Tyr Ser Ala Ile Cys Arg Pro Leu 145 150 155
160 Gln Ala Arg Val Trp Gln Thr Arg Ser His Ala Ala Arg Val Ile Ile
165 170 175 Ala Thr Trp Met Leu Ser Gly Leu Leu Met Val Pro Tyr Pro
Val Tyr 180 185 190 Thr Ala Val Gln Pro Ala Gly Gly Ala Arg Ala Leu
Gln Cys Val His 195 200 205 Arg Trp Pro Ser Ala Arg Val Arg Gln Thr
Trp Ser Val Leu Leu Leu 210 215 220 Leu Leu Leu Phe Phe Val Pro Gly
Val Val Met Ala Val Ala Tyr Gly 225 230 235 240 Leu Ile Ser Arg Glu
Leu Tyr Leu Gly Leu Arg Phe Asp Glu Asp Ser 245 250 255 Asp Ser Glu
Ser Arg Val Arg Ser Gln Gly Gly Leu Arg Gly Gly Ala 260 265 270 Gly
Pro Gly Pro Ala Pro Pro Asn Gly Ser Cys Arg Pro Glu Gly Gly 275 280
285 Leu Ala Gly Glu Asp Gly Asp Gly Cys Tyr Val Gln Leu Pro Arg Ser
290 295 300 Arg Gln Thr Leu Glu Leu Ser Ala Leu Thr Ala Pro Thr Pro
Gly Pro 305 310 315 320 Gly Gly Gly Pro Arg Pro Tyr Gln Ala Lys Leu
Leu Ala Lys Lys Arg 325 330 335 Val Val Arg Met Leu Leu Val Ile Val
Val Leu Phe Phe Leu Cys Trp 340 345 350 Leu Pro Leu Tyr Ser Ala Asn
Thr Trp Arg Ala Phe Asp Ser Ser Gly 355 360 365 Ala His Arg Ala Leu
Ser Gly Ala Pro Ile Ser Phe Ile His Leu Leu 370 375 380 Ser Tyr Ala
Ser Ala Cys Val Asn Pro Leu Val Tyr Cys Phe Met His 385 390 395 400
Arg Arg Phe Arg Gln Ala Cys Leu Glu Thr Cys Ala Arg Cys Cys Pro 405
410 415 Arg Pro Pro Arg Ala Arg Pro Arg Pro Leu Pro Asp Glu Asp Pro
Pro 420 425 430 Thr Pro Ser Ile Ala Ser Leu Ser Arg Leu Ser Tyr Thr
Thr Ile Ser 435 440 445 Thr Leu Gly Pro Gly 450 5 428 PRT Homo
sapiens 5 Met Asp Val Val Asp Ser Leu Leu Val Asn Gly Ser Asn Ile
Thr Pro 1 5 10 15 Pro Cys Glu Leu Gly Leu Glu Asn Glu Thr Leu Phe
Cys Leu Asp Gln 20 25 30 Pro Arg Pro Ser Lys Glu Trp Gln Pro Ala
Val Gln Ile Leu Leu Tyr 35 40 45 Ser Leu Ile Phe Leu Leu Ser Val
Leu Gly Asn Thr Leu Val Ile Thr 50 55 60 Val Leu Ile Arg Asn Lys
Arg Met Arg Thr Val Thr Asn Ile Phe Leu 65 70 75 80 Leu Ser Leu Ala
Val Ser Asp Leu Met Leu Cys Leu Phe Cys Met Pro 85 90 95 Phe Asn
Leu Ile Pro Asn Leu Leu Lys Asp Phe Ile Phe Gly Ser Ala 100 105 110
Val Cys Lys Thr Thr Thr Tyr Phe Met Gly Thr Ser Val Ser Val Ser 115
120 125 Thr Phe Asn Leu Val Ala Ile Ser Leu Glu Arg Tyr Gly Ala Ile
Cys 130 135 140 Lys Pro Leu Gln Ser Arg Val Trp Gln Thr Lys Ser His
Ala Leu Lys 145 150 155 160 Val Ile Ala Ala Thr Trp Cys Leu Ser Phe
Thr Ile Met Thr Pro Tyr 165 170 175 Pro Ile Tyr Ser Asn Leu Val Pro
Phe Thr Lys Asn Asn Asn Gln Thr 180 185 190 Ala Asn Met Cys Arg Phe
Leu Leu Pro Asn Asp Val Met Gln Gln Ser 195 200 205 Trp His Thr Phe
Leu Leu Leu Ile Leu Phe Leu Ile Pro Gly Ile Val 210 215 220 Met Met
Val Ala Tyr Gly Leu Ile Ser Leu Glu Leu Tyr Gln Gly Ile 225 230 235
240 Lys Phe Glu Ala Ser Gln Lys Lys Ser Ala Lys Glu Arg Lys Pro Ser
245 250 255 Thr Thr Ser Ser Gly Lys Tyr Glu Asp Ser Asp Gly Cys Tyr
Leu Gln 260 265 270 Lys Thr Arg Pro Pro Arg Lys Leu Glu Leu Arg Gln
Leu Ser Thr Gly 275 280 285 Ser Ser Ser Arg Ala Asn Arg Ile Arg Ser
Asn Ser Ser Ala Ala Asn 290 295 300 Leu Met Ala Lys Lys Arg Val Ile
Arg Met Leu Ile Val Ile Val Val 305 310 315 320 Leu Phe Phe Leu Cys
Trp Met Pro Ile Phe Ser Ala Asn Ala Trp Arg 325 330 335 Ala Tyr Asp
Thr Ala Ser Ala Glu Arg Arg Leu Ser Gly Thr Pro Ile 340 345 350 Ser
Phe Ile Leu Leu Leu Ser Tyr Thr Ser Ser Cys Val Asn Pro Ile 355 360
365 Ile Tyr Cys Phe Met Asn Lys Arg Phe Arg Leu Gly Phe Met Ala Thr
370 375 380 Phe Pro Cys Cys Pro Asn Pro Gly Pro Pro Gly Ala Arg Gly
Glu Val 385 390 395 400 Gly Glu Glu Glu Glu Gly Gly Thr Thr Gly Ala
Ser Leu Ser Arg Phe 405 410 415 Ser Tyr Ser His Met Ser Ala Ser Val
Pro Pro Gln 420 425 6 443 PRT Rattus norvegicus 6 Met Ser His Ser
Pro Ala Arg Gln His Leu Val Glu Ser Ser Arg Met 1 5 10 15 Asp Val
Val Asp Ser Leu Leu Met Asn Gly Ser Asn Ile Thr Pro Pro 20 25 30
Cys Glu Leu Gly Leu Glu Asn Glu Thr Leu Phe Cys Leu Asp Gln Pro 35
40 45 Gln Pro Ser Lys Glu Trp Gln Ser Ala Leu Gln Ile Leu Leu Tyr
Ser 50 55 60 Ile Ile Phe Leu Leu Ser Val Leu Gly Asn Thr Leu Val
Ile Thr Val 65 70 75 80 Leu Ile Arg Asn Lys Arg Met Arg Thr Val Thr
Asn Ile Phe Leu Leu 85 90 95 Ser Leu Ala Val Ser Asp Leu Met Leu
Cys Phe Cys Met Pro Phe Asn 100 105 110 Leu Ile Pro Asn Leu Leu Lys
Asp Phe Ile Phe Gly Ser Ala Val Cys 115 120 125 Lys Thr Thr Thr Tyr
Phe Met Gly Thr Ser Val Ser Val Ser Thr Phe 130 135 140 Asn Leu Val
Ala Ile Ser Leu Glu Arg Tyr Gly Ala Ile Cys Arg Pro 145 150 155 160
Leu Gln Ser Arg Val Trp Gln Thr Lys Ser His Ala Leu Lys Val Ile 165
170 175 Ala Ala Thr Trp Cys Leu Ser Phe Thr Ile Met Thr Pro Tyr Pro
Ile 180 185 190 Tyr Ser Asn Leu Val Pro Phe Thr Lys Asn Asn Asn Gln
Thr
Ala Asn 195 200 205 Met Cys Arg Phe Leu Leu Pro Ser Asp Ala Met Gln
Gln Ser Trp Gln 210 215 220 Thr Phe Leu Leu Leu Ile Leu Phe Leu Leu
Pro Gly Ile Val Met Val 225 230 235 240 Val Ala Tyr Gly Leu Ile Ser
Leu Glu Leu Tyr Gln Gly Ile Lys Phe 245 250 255 Asp Ala Ser Gln Lys
Lys Ser Ala Lys Glu Lys Lys Pro Ser Thr Gly 260 265 270 Ser Ser Thr
Arg Tyr Glu Asp Ser Asp Gly Cys Tyr Leu Gln Lys Ser 275 280 285 Arg
Pro Pro Arg Lys Leu Glu Leu Gln Gln Leu Ser Ser Gly Ser Gly 290 295
300 Gly Ser Arg Leu Asn Arg Ile Arg Ser Ser Ser Ser Ala Ala Asn Leu
305 310 315 320 Ile Ala Lys Lys Arg Val Ile Arg Met Leu Ile Val Ile
Val Val Leu 325 330 335 Phe Phe Leu Cys Trp Met Pro Ile Phe Ser Ala
Asn Ala Trp Arg Ala 340 345 350 Tyr Asp Thr Val Ser Ala Glu Lys His
Leu Ser Gly Thr Pro Ile Ser 355 360 365 Phe Ile Leu Leu Leu Ser Tyr
Thr Ser Ser Cys Val Asn Pro Ile Ile 370 375 380 Tyr Cys Phe Met Asn
Lys Arg Phe Arg Leu Gly Phe Met Ala Thr Phe 385 390 395 400 Pro Cys
Cys Pro Asn Pro Gly Pro Pro Gly Val Arg Gly Glu Val Gly 405 410 415
Glu Glu Glu Asp Gly Arg Thr Ile Arg Ala Leu Leu Ser Arg Tyr Ser 420
425 430 Tyr Ser His Met Ser Thr Ser Ala Pro Pro Pro 435 440 7 453
PRT Xenopus laevis 7 Met Glu Ser Leu Arg Ser Leu Ser Asn Ile Ser
Ala Leu His Glu Leu 1 5 10 15 Leu Cys Arg Tyr Ser Asn Leu Ser Gly
Thr Leu Thr Trp Asn Leu Ser 20 25 30 Ser Thr Asn Gly Thr His Asn
Leu Thr Thr Ala Asn Trp Pro Pro Trp 35 40 45 Asn Leu Asn Cys Thr
Pro Ile Leu Asp Arg Lys Lys Pro Ser Pro Ser 50 55 60 Asp Leu Asn
Leu Trp Val Arg Ile Val Met Tyr Ser Val Ile Phe Leu 65 70 75 80 Leu
Ser Val Phe Gly Asn Thr Leu Ile Ile Ile Val Leu Val Met Asn 85 90
95 Lys Arg Leu Arg Thr Ile Thr Asn Ser Phe Leu Leu Ser Leu Ala Leu
100 105 110 Ser Asp Leu Met Val Ala Val Leu Cys Met Pro Phe Thr Leu
Ile Pro 115 120 125 Asn Leu Met Glu Asn Phe Ile Phe Gly Glu Val Ile
Cys Arg Ala Ala 130 135 140 Ala Tyr Phe Met Gly Leu Ser Val Ser Val
Ser Thr Phe Asn Leu Val 145 150 155 160 Ala Ile Ser Ile Glu Arg Tyr
Ser Ala Ile Cys Asn Pro Leu Xaa Ser 165 170 175 Arg Val Trp Gln Thr
Arg Ser His Ala Tyr Arg Val Ile Ala Ala Thr 180 185 190 Trp Val Leu
Ser Ser Ile Ile Met Ile Pro Tyr Leu Val Tyr Asn Lys 195 200 205 Thr
Val Thr Phe Pro Met Lys Asp Arg Arg Val Gly His Gln Cys Arg 210 215
220 Leu Val Trp Pro Ser Lys Gln Val Gln Gln Ala Trp Tyr Val Leu Leu
225 230 235 240 Leu Thr Ile Leu Phe Phe Ile Pro Gly Val Val Met Ile
Val Ala Tyr 245 250 255 Gly Leu Ile Ser Arg Glu Leu Tyr Arg Gly Ile
Gln Phe Glu Met Asp 260 265 270 Leu Asn Lys Glu Ala Lys Ala His Lys
Asn Gly Val Ser Thr Pro Thr 275 280 285 Thr Ile Pro Ser Gly Asp Glu
Gly Asp Gly Cys Tyr Ile Gln Val Thr 290 295 300 Lys Arg Arg Asn Thr
Met Glu Met Ser Thr Leu Thr Pro Ser Val Cys 305 310 315 320 Thr Lys
Met Asp Arg Ala Arg Ile Asn Asn Ser Glu Ala Lys Leu Met 325 330 335
Ala Lys Lys Arg Val Ile Arg Met Leu Ile Val Ile Val Ala Met Phe 340
345 350 Phe Ile Cys Trp Met Pro Ile Phe Val Ala Asn Thr Trp Lys Ala
Phe 355 360 365 Asp Glu Leu Ser Ala Phe Asn Thr Leu Thr Gly Ala Pro
Ile Ser Phe 370 375 380 Ile His Leu Leu Ser Tyr Thr Ser Ala Cys Val
Asn Pro Leu Ile Tyr 385 390 395 400 Cys Phe Met Asn Lys Arg Phe Arg
Lys Ala Phe Leu Gly Thr Phe Ser 405 410 415 Ser Cys Ile Lys Pro Cys
Arg Asn Phe Arg Asp Thr Asp Glu Asp Ile 420 425 430 Ala Ala Thr Gly
Ala Ser Leu Ser Lys Phe Ser Tyr Thr Thr Val Ser 435 440 445 Ser Leu
Gly Pro Ala 450 8 51 PRT Homo sapiens 8 Leu Thr Ala Pro Gly Pro Gly
Ser Gly Ser Arg Pro Thr Gln Ala Lys 1 5 10 15 Leu Leu Ala Lys Lys
Arg Val Val Arg Met Leu Leu Val Ile Val Val 20 25 30 Leu Phe Phe
Leu Cys Trp Leu Pro Val Tyr Ser Ala Asn Thr Trp Arg 35 40 45 Ala
Phe Asp 50 9 51 PRT Homo sapiens 9 Leu Thr Ala Pro Gly Pro Gly Ser
Gly Ser Arg Pro Thr Ala His Val 1 5 10 15 Ser Ala Ala Lys Lys Arg
Val Val Arg Met Leu Leu Val Ile Val Val 20 25 30 Leu Phe Phe Leu
Cys Trp Leu Pro Val Tyr Ser Ala Asn Thr Trp Arg 35 40 45 Ala Phe
Asp 50 10 51 PRT Homo sapiens 10 Leu Thr Ala Pro Gly Pro Gly Ser
Gly Ser Arg Pro Thr Gln Ala Lys 1 5 10 15 Ser Ala Ala Lys Lys Arg
Val Val Arg Met Leu Leu Val Ile Val Val 20 25 30 Leu Phe Phe Leu
Cys Trp Leu Pro Val Tyr Ser Ala Asn Thr Trp Arg 35 40 45 Ala Phe
Asp 50 11 51 PRT Homo sapiens 11 Leu Thr Ala Pro Gly Pro Gly Ser
Gly Ser Arg Pro Thr Gln Ala Lys 1 5 10 15 Ser Leu Ala Lys Lys Arg
Val Val Arg Met Leu Leu Val Ile Val Val 20 25 30 Leu Phe Phe Leu
Cys Trp Leu Pro Val Tyr Ser Ala Asn Thr Trp Arg 35 40 45 Ala Phe
Asp 50 12 51 PRT Homo sapiens 12 Leu Thr Ala Pro Gly Pro Gly Ser
Gly Ser Arg Pro Thr Gln Ala Lys 1 5 10 15 Glu Leu Ala Lys Lys Arg
Val Val Arg Met Leu Leu Val Ile Val Val 20 25 30 Leu Phe Phe Leu
Cys Trp Leu Pro Val Tyr Ser Ala Asn Thr Trp Arg 35 40 45 Ala Phe
Asp 50 13 5 PRT Homo sapiens 13 Gln Ala Lys Leu Leu 1 5 14 5 PRT
Homo sapiens 14 Ala His Val Ser Ala 1 5 15 51 PRT Homo sapiens 15
Leu Thr Ala Pro Gly Pro Gly Ser Gly Ser Arg Pro Thr Gln Ala Lys 1 5
10 15 Leu Leu Ala Lys Lys Arg Val Val Arg Met Leu Leu Val Ile Val
Val 20 25 30 Leu Phe Phe Leu Cys Trp Leu Pro Val Tyr Ser Ala Asn
Thr Trp Arg 35 40 45 Ala Phe Asp 50 16 51 PRT Homo sapiens 16 Leu
Thr Ala Pro Gly Pro Gly Ser Gly Ser Arg Pro Thr Gln Glu Lys 1 5 10
15 Glu Leu Glu Lys Lys Arg Glu Glu Arg Met Leu Leu Val Ile Val Val
20 25 30 Leu Phe Phe Leu Cys Trp Leu Pro Val Tyr Ser Ala Asn Thr
Trp Arg 35 40 45 Ala Phe Asp 50 17 51 PRT Homo sapiens 17 Leu Thr
Ala Pro Gly Pro Gly Ser Gly Ser Arg Pro Thr Gln Glu Lys 1 5 10 15
Leu Leu Ala Lys Lys Arg Val Val Arg Met Leu Leu Val Ile Val Val 20
25 30 Leu Phe Phe Leu Cys Trp Leu Pro Val Tyr Ser Ala Asn Thr Trp
Arg 35 40 45 Ala Phe Asp 50 18 51 PRT Homo sapiens 18 Leu Thr Ala
Pro Gly Pro Gly Ser Gly Ser Arg Pro Thr Gln Ala Glu 1 5 10 15 Leu
Leu Ala Lys Lys Arg Val Val Arg Met Leu Leu Val Ile Val Val 20 25
30 Leu Phe Phe Leu Cys Trp Leu Pro Val Tyr Ser Ala Asn Thr Trp Arg
35 40 45 Ala Phe Asp 50 19 51 PRT Homo sapiens 19 Leu Thr Ala Pro
Gly Pro Gly Ser Gly Ser Arg Pro Thr Gln Ala Lys 1 5 10 15 Glu Leu
Ala Lys Lys Arg Val Val Arg Met Leu Leu Val Ile Val Val 20 25 30
Leu Phe Phe Leu Cys Trp Leu Pro Val Tyr Ser Ala Asn Thr Trp Arg 35
40 45 Ala Phe Asp 50 20 51 PRT Homo sapiens 20 Leu Thr Ala Pro Gly
Pro Gly Ser Gly Ser Arg Pro Thr Gln Ala Lys 1 5 10 15 Leu Leu Glu
Lys Lys Arg Val Val Arg Met Leu Leu Val Ile Val Val 20 25 30 Leu
Phe Phe Leu Cys Trp Leu Pro Val Tyr Ser Ala Asn Thr Trp Arg 35 40
45 Ala Phe Asp 50 21 51 PRT Homo sapiens 21 Leu Thr Ala Pro Gly Pro
Gly Ser Gly Ser Arg Pro Thr Gln Ala Lys 1 5 10 15 Leu Leu Ala Lys
Lys Arg Glu Val Arg Met Leu Leu Val Ile Val Val 20 25 30 Leu Phe
Phe Leu Cys Trp Leu Pro Val Tyr Ser Ala Asn Thr Trp Arg 35 40 45
Ala Phe Asp 50 22 51 PRT Homo sapiens 22 Leu Thr Ala Pro Gly Pro
Gly Ser Gly Ser Arg Pro Thr Gln Ala Lys 1 5 10 15 Leu Leu Ala Lys
Lys Arg Val Glu Arg Met Leu Leu Val Ile Val Val 20 25 30 Leu Phe
Phe Leu Cys Trp Leu Pro Val Tyr Ser Ala Asn Thr Trp Arg 35 40 45
Ala Phe Asp 50 23 51 PRT Homo sapiens 23 Leu Thr Ala Pro Gly Pro
Gly Ser Gly Ser Arg Pro Thr Gln Ala Lys 1 5 10 15 Leu Leu Ala Lys
Lys Arg Glu Glu Arg Met Leu Leu Val Ile Val Val 20 25 30 Leu Phe
Phe Leu Cys Trp Leu Pro Val Tyr Ser Ala Asn Thr Trp Arg 35 40 45
Ala Phe Asp 50
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