U.S. patent application number 11/446429 was filed with the patent office on 2007-01-25 for pharmacological chaperones for treating obesity.
Invention is credited to Michel Bouvier, Jian-Qiang Fan, Gary Lee, Ken Valenzano.
Application Number | 20070021433 11/446429 |
Document ID | / |
Family ID | 37499005 |
Filed Date | 2007-01-25 |
United States Patent
Application |
20070021433 |
Kind Code |
A1 |
Fan; Jian-Qiang ; et
al. |
January 25, 2007 |
Pharmacological chaperones for treating obesity
Abstract
The invention relates to methods of enhancing normal
melanocortin-4 receptor (MC4R) activity, and to enhancing activity
of an MC4R having a mutation which affects protein folding and/or
processing of the MC4R. The invention provides a method of treating
an individual having a condition in which increased activity of an
MC4R at the cell surface would be beneficial, for example in
obesity, by administering an effective amount of a pharmacological
chaperone for the MC4R. The invention provides MC4R pharmacological
chaperones which enhance the activity of MC4R. The invention
further provides a method of screening to identify pharmacological
chaperones which enhance folding of an MC4R in the endoplasmic
reticulum (ER), in order to enhance the activity of the MC4R at the
cell surface.
Inventors: |
Fan; Jian-Qiang; (Demarest,
NJ) ; Valenzano; Ken; (Cranbury, NJ) ; Lee;
Gary; (Cranbury, NJ) ; Bouvier; Michel;
(Montreal, CA) |
Correspondence
Address: |
BAKER & BOTTS
30 ROCKEFELLER PLAZA
44TH FLOOR
NEW YORK
NY
10112-4498
US
|
Family ID: |
37499005 |
Appl. No.: |
11/446429 |
Filed: |
June 2, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60687648 |
Jun 3, 2005 |
|
|
|
60799968 |
May 12, 2006 |
|
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|
Current U.S.
Class: |
514/252.11 ;
514/256 |
Current CPC
Class: |
C07K 14/723 20130101;
A61K 31/505 20130101; A61P 43/00 20180101; A61K 31/495 20130101;
A61P 3/04 20180101; A61K 31/497 20130101; A61K 9/0053 20130101 |
Class at
Publication: |
514/252.11 ;
514/256 |
International
Class: |
A61K 31/497 20070101
A61K031/497; A61K 31/505 20070101 A61K031/505 |
Claims
1. A method for enhancing activity of an MC4R polypeptide, which
method comprises contacting an MC4R-expressing cell with a
pharmacological chaperone that binds to the MC4R polypeptide in an
amount effective to increase MC4R activity.
2. The method of claim 1, wherein the MC4R polypeptide is a
wild-type MC4R polypeptide.
3. The method of claim 2, wherein the MC4R polypeptide has a
sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID
NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8.
4. The method of claim 1, wherein the MC4R polypeptide is a mutant
MC4R polypeptide.
5. The method of claim 4, wherein the mutant MC4R polypeptide
comprises a mutation associated with misfolding of the MC4R
polypeptide.
6. The method of claim 4, wherein the mutant MC4R polypeptide
comprises a mutation selected from the group consisting of P78L,
R165Q, R165W, I125K, C271Y, T11A, A175T, I316L, I316S, I317T, N97D,
G98R, N62S, C271R, S58C, N62S, N97D, Y157S, I102S, L106P, L250Q,
Y287X, and P299H.
7. The method of claim 4, wherein the mutant MC4R polypeptide is
R165Q, R165W, S58C, N62S, or P299H.
8. The method of claim 1, wherein the pharmacological chaperone is
an MC4R antagonist.
9. The method of claim 8, wherein the MC4R antagonist has a
structure as set forth in FIG. 5 or FIG. 6.
10. The method of claim 1, wherein the pharmacological chaperone
binds to the MC4R polypeptide as it is being folded into a
functional conformation.
11. The method of claim 1, wherein the activity enhanced by the
pharmacological chaperone is adenylyl cyclase activation.
12. A method for enhancing cell surface expression of an MC4R
polypeptide, which method comprises contacting a MC4R-expressing
cell with a pharmacological chaperone that binds to the MC4R
polypeptide in an amount effective to increase MC4R cell surface
expression.
13. The method of claim 12, wherein the pharmacological chaperone
dissociates from the MC4R polypeptide when it is folded into a
functional conformation.
14. The method of claim 12, wherein the MC4R polypeptide is a
wild-type MC4R polypeptide.
15. The method of claim 12, wherein the MC4R polypeptide has a
sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID
NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8.
16. The method of claim 12, wherein the MC4R polypeptide is a
mutant MC4R polypeptide.
17. The method of claim 16, wherein the mutant MC4R polypeptide
comprises a mutation associated with misfolding of the MC4R
polypeptide.
18. The method of claim 16, wherein the mutant MC4R polypeptide
comprises a mutation selected from the group consisting of P78L,
R165Q, R165W, I125K, C271Y, T11A, A175T, I316L, I316S, I317T, N97D,
G98R, N62S, C271R, S58C, N62S, N97D, Y157S, I102S, L106P, L250Q,
Y287X, and P299H.
19. The method of claim 16, wherein the mutant MC4R polypeptide is.
R165Q, RI 65W, S58C, N62S, or P299H.
20. The method of claim 12, wherein the pharmacological chaperone
is an MC4R antagonist.
21. The method of claim 20, wherein the MC4R antagonist has a
structure as set forth in FIG. 5 or FIG. 6.
22. A method of treating an individual in need of increased
stability of an MC4R polypeptide, which method comprises
administering to the individual a pharmacological chaperone that
binds to the MC4R polypeptide in an amount effective to increase
stability.
23. The method of claim 22, wherein the pharmacological chaperone
is an MC4R antagonist having a structure as set forth in FIG. 5 or
FIG. 6.
24. The method of claim 22, wherein the pharmacological chaperone
increases trafficking of the MC4R polypeptide to the cell
membrane.
25. The method of claim 22, wherein the individual is obese or is
at risk for becoming obese.
26. The method of claim 22, wherein the individual comprises a
mutation in an MC4R gene which has been associated with
obesity.
27. The method of claim 22, wherein the pharmacological chaperone
is administered in-a pharmaceutically-acceptable carrier.
28. The method of claim 22, wherein the MC4R polypeptide is a
wild-type MC4R polypeptide.
29. The method of claim 22, wherein the MC4R polypeptide is a
mutant MC4R polypeptide.
30. A method for identifying a pharmacological chaperone for an
MC4R, which method comprises: (a) contacting a test compound to a
reaction mixture that comprises a cell or cell extract expressing
an MC4R polypeptide, wherein the reaction mixture conditions permit
binding of the test compound to the MC4R polypeptide; (b) detecting
stability, activity, or cell surface localization of the MC4R
polypeptide in the reaction mixture in the presence of the test
compound; and (c) comparing stability, activity, or cell surface
localization of the MC4R polypeptide in the presence of the test
compound to stability, activity, or cell surface localization of
the MC4R polypeptide in the absence of the test compound, wherein
detection of enhanced stability, activity, or cell surface
localization in the presence of the test compound relative to the
absence of the test compound indicates that the test compound is a
pharmacological chaperone for the MC4R.
31. The method of claim 30, wherein the MC4R polypeptide is a
wild-type MC4R polypeptide.
32. The method of claim 31, wherein the MC4R polypeptide comprises
the amino acid sequence set forth in SEQ ID NO: 2.
33. The method of claim 30, wherein the MC4R polypeptide is a
mutant MC4R polypeptide.
34. The method of claim 33, wherein the MC4R polypeptide comprises
a mutation associated with misfolding of the MC4R polypeptide.
35. The method of claim 34, wherein the MC4R misfolding mutation is
selected from the group consisting of P78L, R165Q, R165W, I125K,
C271Y, T11A, A175T, I316L, I316S, I317T, N97D, G98R, N62S, C271R,
S58C, N62S, N97D, Y157S, I102S, L106P, L250Q, Y287X, P299H, and
S58C, relative to the wild-type MC4R polypeptide sequence as set
forth in SEQ ID NO: 2.
36. The method of claim 30, wherein the reaction mixture is
cell-based.
37. The method of claim 30, wherein the reaction mixture is
cell-free.
38. The method of claim 30, further comprising detecting activity
of an MC4R polypeptide located at the cell surface.
39. The method of claim 38, wherein the activity detected is cAMP
activation.
40. The method of claim 1, wherein the pharmacological chaperone
has a structure as depicted in FIG. 13.
41. The method of claim 1, wherein the pharmacological chaperone
has a structure as depicted in FIG. 14.
42. The method of claim 1, wherein the pharmacological chaperone
has a structure as depicted in FIG. 15.
43. The method of claim 1, wherein the pharmacological chaperone
has a structure as depicted in FIG. 16.
Description
[0001] This application claims priority from U.S. Provisional
Patent Application Nos. 60/687,648 filed Jun. 3, 2005, and
60/799,968 filed May 12, 2006, each of which is incorporated by
reference herein in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to methods of enhancing normal
melanocortin-4 receptor (MC4R) activity, and to enhancing activity
of an MC4R having a mutation or mutations which affects protein
folding and/or processing of the MC4R. The invention provides a
method of treating an individual having a condition in which
increased activity of an MC4R at the cell surface would be
beneficial, such as in obesity, by administering an effective
amount of a pharmacological chaperone for the MC4R. The invention
provides MC4R pharmacological chaperones which enhance activity of
MC4R. The invention further provides a method of screening to
identify pharmacological chaperones which enhance folding of an
MC4R in the endoplasmic reticulum (ER), in order to enhance
activity of the MC4R at the cell surface.
BACKGROUND OF THE INVENTION
Obesity
[0003] Obesity represents the most prevalent of body weight
disorders, and it is the most important nutritional disorder in the
Western world, with estimates of its prevalence ranging from 30% to
50% of the middle-aged population. The number of overweight and
obese Americans has continued to increase since 1960, a trend that
is not slowing down. Today, 64.5 percent of adult Americans (about
127 million) are categorized as being overweight or obese. Each
year, obesity causes at least 300,000 deaths in the U.S., and
healthcare costs of American adults with obesity amount to
approximately $100 billion (American Obesity Association).
[0004] Obesity increases an individual's risk of developing
conditions such as high blood pressure, diabetes (type 2),
hyperlipidemia, heart disease, hypertension, stroke, gallbladder
disease, and cancer of the breast, prostate, and colon (see, e.g.,
Nishina, P. M. et al., 1994, Metab. 43: 554-558; Grundy, S. M.
& Barnett, J. P., 1990, Dis. Mon. 36: 641-731). In the U.S.,
the incidence of being overweight or obese occurs at higher rates
in racial/ethnic minority populations such as African American and
Hispanic Americans, compared with Caucasian Americans. Women and
persons of low socioeconomic status within minority populations
appear to particularly be affected by excess weight and obesity.
This trend is not limited to adults. Approximately 30.3 percent of
children (ages 6 to 11) are overweight and 15.3 percent are obese.
For adolescents (ages 12 to 19), 30.4 percent are overweight and
15.5 percent are obese. Diabetes, hypertension and other
obesity-related chronic diseases that are prevalent among adults
have now become more common in children and young adults. Poor
dietary habits and inactivity are reported to contribute to the
increase of obesity in youth.
[0005] Additionally, risk factors for developing childhood obesity
include having overweight parents, or parents unconcerned about
their child's weight, increased energy intake due to larger serving
sizes, increased sedentary lifestyle and decreased
transport-related activity (walking to school or to the bus stop),
having a temperament with high levels of anger/frustration (which
may cause parents to give their child extra food and calories to
decrease tantrums); having Down's Syndrome, mother's pregnancy Body
Mass Index (BMI), and first born status (increased prevalence of
obesity).
[0006] One tool used for diagnosing obesity in adults is
calculating an individual's BMI, which is a measure of body weight
for height (Garrow and Webster, International Journal of Obesity
1985; 9:147-153). A BMI of 25 to 29.9 indicates that an individual
is overweight, while a BMI of 30 or above is indicative of obesity.
For children, BMI is gender and age specific (Pietrobelli et al.,
Journal of pediatrics 1998; 132:204-210).
[0007] Risk factors for developing obesity in adulthood include
poor diet (high-calorie, low nutrients); lack of physical activity;
working varied shifts; quitting smoking, having certain medical
conditions such as rare hereditary diseases, and hormonal
imbalances (such as hypothyroid, Cushing's disease and polycystic
ovarian syndrome); certain medications (steroids and some
antidepressants); being a racial or ethnic minority (especially a
female minority); low socioeconomic status; age (increased risk
from 20-55), pregnancy; and retirement (due to altered
schedule).
Melanocortin 4 Receptor and Obesity
[0008] The melanocortin 4 receptor (MC4R) has been implicated in
the regulation of body weight (Graham et al, Nat. Genetics 1997;
17: 273-4). MC4R is expressed in the brain, including the
hypothalamus, which influences food intake. Numerous mutations
affecting MC4R activity have been found and many are associated
with obesity including early-onset (childhood) obesity (Nijenhuis
et al., J. Biol. Chem. 2003, 278:22939-45; Branson et al., New Eng.
J. Med. 2003, 348:1096-1103; Gu et al., Diabetes 1999, 48:635-39;
Farooqi et al., New Eng. J. Med. 2003, 348:1085-95; Tao et al.,
Endocrinology 2003, 144:4544-51).
Current Treatments
[0009] Current anti-obesity drugs have limited efficacy and
numerous side effects (Crowley, V. E., Yeo, G. S. & O'Rahilly,
S., Nat. Rev. Drug Discov. 2002; 1, 276-86). With obesity reaching
epidemic proportions worldwide, there is a pressing need for the
development of adequate therapeutics in this area. In recent years,
hormones and neuropeptides involved in the regulation of appetite,
body energy expenditure, and fat mass accumulation have emerged as
potential anti-obesity drugs (McMinn, J. E., Baskin, D. G. &
Schwartz, M. W., Obes Rev 2000; 1:37-46; Drazen, D. L. & Woods,
S. C., Curr Opin Clin Nutr Metab Care 2003; 6:621-629). At present,
however, these peptides require parenteral administration. The
prospect of daily injections to control obesity for extended
periods of time (since obesity is a chronic condition) is not very
encouraging and limits the use of these drugs.
Molecular Chaperones Stabilize Proper Protein Folding
[0010] Proteins are synthesized in the cytoplasm, and the newly
synthesized proteins are secreted into the lumen of the endoplasmic
reticulum (ER) in a largely unfolded state. In general, protein
folding is governed by the principle of self assembly. Newly
synthesized polypeptides fold into their native conformation based
on their amino acid sequences (Anfinsen et al., Adv. Protein Chem.
1975; 29:205-300). In vivo, protein folding is complicated, because
the combination of ambient temperature and high protein
concentration stimulates the process of aggregation, in which amino
acids normally buried in the hydrophobic core interact with their
neighbors non-specifically. To avoid this problem, protein folding
is usually facilitated by a special group of proteins called
chaperones, which prevent nascent polypeptide chains from
aggregating by binding to unfolded protein such that the protein
refolds in the native conformation (Hartl, Nature 1996;
381:571-580).
[0011] Endogenous molecular chaperones are present in virtually all
types of cells and in most cellular compartments. Some are involved
in the transport of proteins and permit cells to survive under
stresses such as heat shock and glucose starvation (Gething et al.,
Nature 1992; 355:33-45; Caplan, Trends Cell. Biol. 1999; 9:262-268;
Lin et al., Mol. Biol. Cell. 1993; 4:109-1119; Bergeron et al.,
Trends Biochem. Sci. 1994; 19:124-128). Among the endogenous
chaperones, BiP (immunoglobulin heavy-chain binding protein, Grp78)
is the best characterized chaperone of the ER (Haas, Curr. Top.
Microbiol. Immunol. 1991; 167:71-82). Like other chaperones, BiP
interacts with many secretory and membrane proteins within the ER
throughout their maturation. When nascent protein folding proceeds
smoothly, this interaction is normally weak and short-lived. Once
the native protein conformation is achieved, the molecular
chaperone no longer interacts with the protein. BiP binding to a
protein that fails to fold, assemble, or be properly glycosylated
becomes stable, and usually leads to degradation of the protein
through the ER-associated degradation pathway. This process serves
as a "quality control" system in the ER, ensuring that only those
properly folded and assembled proteins are transported out of the
ER for further maturation, and improperly folded proteins are
retained for subsequent degradation (Hurtley et al., Annu. Rev.
Cell. Biol. 1989; 5:277-307). Due to the combined actions of the
inefficiency of the thermodynamic protein folding process and the
ER quality control system, only a fraction of nascent (non-mutated)
proteins become folded into a functional conformation and
successfully exit the ER.
Pharmacological Chaperones Derived From Specific Enzyme Inhibitors
Rescue Mutant Enzymes and Enhance Wild-Type Enzymes
[0012] It has previously been shown that small molecule inhibitors
of enzymes associated with lysosomal storage disorders (LSDs) can
both rescue folding and activity of the mutant enzyme, and enhance
folding and activity of the wild-type enzyme (see U.S. Pat. Nos.
6,274,597; 6,583,158; 6,589,964; 6,599,919; and 6,916,829, all
incorporated herein by reference). In particular, it was discovered
that administration of small molecule derivatives of glucose and
galactose, which were specific competitive inhibitors of mutant
enzymes associated with LSDs, effectively increased in vitro and in
vivo stability of the mutant enzymes and enhanced the mutant enzyme
activity. The original theory behind this strategy is as follows:
since the mutant enzyme protein folds improperly in the ER (Ishii
et al., Biochem. Biophys. Res. Comm. 1996; 220: 812-815), the
enzyme protein is retarded in the normal transport pathway
(ER.fwdarw.Golgi apparatus.fwdarw.endosome.fwdarw.lysosome) and
rapidly degraded. Therefore, a compound which stabilizes the
correct folding of a mutant protein will serve as an active
site-specific chaperone for the mutant protein to promote its
smooth escape from the ER quality control system. Enzyme inhibitors
occupy the catalytic center, resulting in stabilization of enzyme
conformation in cells in culture and in animals. These specific
chaperones were designated "active site-specific chaperones
(ASSCs)" since they bound in the active site of the enzyme.
[0013] In addition to rescuing the mutant enzymes, the ASSCs
enhance ER secretion and activity of recombinant wild-type enzymes.
An ASSC facilitates folding of overexpressed wild-type enzyme,
which is otherwise retarded in the ER quality control system
because overexpression and over production of the enzyme exceeds
the capacity of the ER and leads to protein aggregation and
degradation. Thus, a compound that induces a stable molecular
conformation of an enzyme during folding serves as a "chaperone" to
stabilize the enzyme in a proper conformation for exit from the ER.
As noted above, for enzymes, one such compound unexpectedly turned
out to be a competitive inhibitor of the enzyme.
Enhancement of Other Proteins with Chaperones
[0014] In addition to the LSDs, a large and diverse number of
diseases are now recognized as "conformational diseases" that are
caused by adoption of non-native protein conformations, which may
lead to retardation of the protein in the ER and ultimate
degradation of the proteins (Kuznetsov et al., N. Engl. J. Med.
1998; 339:1688-1695; Thomas et al., Trends Biochem. Sci. 1995;
20:456-459; Bychkova et al., FEBS Lett. 1995; 359:6-8; Brooks, FEBS
Lett. 1997; 409:115-120).
[0015] For example, small synthetic compounds were found to
stabilize the DNA binding domain of mutant forms of the tumor
suppressor protein p53, thereby allowing the protein to maintain an
active conformation (Foster et al., Science 1999; 286:2507-10).
Synthesis of receptors has been shown to be rescued by small
molecule receptor antagonists and ligands (Morello et al., J. Clin.
Invest. 2000; 105: 887-95; Petaja-Repo et al., EMBO J. 2002;
21:1628-37). Even pharmacological rescue of membrane channel
proteins and other plasma membrane transporters has been
demonstrated using channel-blocking drugs or substrates (Rajamani
et al., Circulation 2002; 105:2830-5; Zhou et al., J. Biol. Chem.
1999; 274:31123-26; Loo et al., J. Biol. Chem. 1997; 272: 709-12;
Pedemonte et al., J. Clin. Inves. 2005; 115: 2564-71).
[0016] There remains in the art a particular need to address
deficiencies in MC4R protein function which are both related and
unrelated to MC4R mutation.
SUMMARY OF THE INVENTION
[0017] As described herein, the present invention provides a method
for enhancing the activity of the melanocortin-4 receptor (MC4R),
e.g., for the treatment of obesity, in subjects who have a folding
mutation in the gene encoding MC4R, or in subjects for whom an
increase in wild-type MC4R activity would be beneficial.
[0018] In one embodiment, the present invention provides a method
for enhancing intracellular folding of an MC4R polypeptide into a
functional conformation by contacting an MC4R-expressing cell with
an effective amount of a pharmacological chaperone. Enhancing
intracellular folding of MC4R, resulting in enhanced expression on
the cell surface of, e.g., neurons of the hypothalamus, reduces the
urge to eat, and, therefore, is useful in the treatment of
overeating disorders, such as binge-eating.
[0019] In one embodiment, the MC4R polypeptide is a wild-type MC4R
polypeptide, which, for example, has a sequence as depicted in SEQ
ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.
[0020] In another embodiment, the MC4R polypeptide is a mutant MC4R
polypeptide. In this embodiment, the mutant polypeptide contains
one or more mutations that result in reduced or improper
intracellular folding of the MC4R polypeptide. Exemplary mutations
are as follows: P78L, R165Q, R165W, I125K, C271Y, T11A, A175T,
I316L, I316S, I317T, N97D, G98R, N62S, C271R, S58C, N62S, N97D,
Y157S, I102S, L106P, L250Q, Y287X, P299H, S58C, CTCT at codon 211,
and TGAT insertion at codon 244.
[0021] In one embodiment, the pharmacological chaperone is an MC4R
antagonist. In another embodiment, the pharmacological chaperone is
an MC4R agonist. In other embodiments, the pharmacological
chaperone is an MC4R partial agonist and/or inverse agonist.
[0022] The present invention also provides a method for enhancing
cell surface expression of an MC4R polypeptide. This method
comprises contacting an MC4R-expressing cell with an effective
amount of a pharmacological chaperone. This embodiment of the
invention pertains to both wild-type MC4R polypeptides and mutant
MC4R polypeptides, and the pharmacological chaperones set forth
above, for methods of enhancing intracellular folding of MC4R
polypeptides.
[0023] The present invention also provides a screening method for
identifying a chaperone for an MC4R polypeptide by contacting a
test compound to a reaction mixture that comprises a cell
expressing an MC4R polypeptide; detecting stability, activity,
and/or cell surface localization of the MC4R polypeptide in the
reaction mixture in the presence of the test compound; and
comparing stability, activity, and/or cell surface localization of
the MC4R polypeptide in the presence of the test compound to the
stability, activity, and/or cell surface localization of the MC4R
polypeptide in the absence of said test compound, where detection
of increased stability, activity, and/or cell surface localization
in the presence of the test compound relative to the absence of the
test compound indicates that the test compound is a chaperone for
the MC4R polypeptide.
[0024] In one embodiment of this screening method, the MC4R
polypeptide comprises an amino acid sequence selected from the
group consisting of SEQ ID NOs: 2, 4, 6 and 8.
[0025] In another embodiment of this screening method, the MC4R
polypeptide comprises a mutation associated with misfolding of the
MC4R polypeptide. In specific embodiments, the misfolding mutation
is one or more of the following alterations: P78L, R165Q or R165W,
I125K, C271Y, T11A, A175T, I316L, I316S, I317T, N97D, G98R, N62S,
C271R, S58C, N62S, N97D, Y157S, I102S, L106P, L250Q, Y287X, P299H,
S58C, CTCT at codon 211, or TGAT insertion at codon 244.
[0026] In one embodiment, the reaction mixture is cell-based. In
another embodiment, the reaction mixture is cell-free.
[0027] In one embodiment, the screening method further includes
detecting activity of an MC4R polypeptide, e.g., on the cell
surface. In another embodiment, the activity is measured through
cAMP activation.
[0028] The present invention will be further understood by
reference to the Detailed Description and the Examples.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1. An agonist of the rat and human melanocortin-4
receptors, as reported by Sebhat, 2002, J Med Chem, 45, 4589-4593
(compound 1).
[0030] FIG. 2. An agonist of human MC4R, as reported by Richardson,
2004, J Med Chem 47, 744-755 (compound 2).
[0031] FIG. 3. Synthetic scheme for compound 1.
[0032] FIG. 4. Synthetic scheme for compound of 2.
[0033] FIG. 5. An antagonist of MC4R, as reported by Arasasingham,
2003, J Med Chem 46, 9-11 (compound 3).
[0034] FIG. 6. An antagonist of MC4R (compound 4); the biological
activity of this compound is reported in WO 02/062766 using a
scintillation proximity assay.
[0035] FIG. 7. Synthetic scheme for compound 3.
[0036] FIG. 8. Synthetic scheme for compound 4.
[0037] FIG. 9. A bisaminothiazole compound described in Pedemonte
et al., J. Clin. Inves. 2005; 115: 2564-71 (compound 5).
[0038] FIG. 1OA-D. Compounds 6-25 described infra.
[0039] FIG. 11. MC4R signaling assay in MC4R mutants treated with
ligand agonist and with and without antagonist chaperones.
[0040] FIG. 12. Mean .alpha.-galactosidase A activity in white
blood cells from normal, healthy volunteers who received 50 mg
1-deoxygalactonojirimycin (DGJ) b.i.d. (triangles), 150 mg DGJ
b.i.d. (squares), or placebo (open circles).
[0041] FIG. 13. Structure of compound class based upon compounds 1,
2, 6, 7, and 12-17.
[0042] FIG. 14. Structure of compound class based upon compounds 3,
9, 10, 11, and 21.
[0043] FIG. 15. Structure of compound class based upon compounds 4,
8, 24, and 25.
[0044] FIG. 16. Structure of compound class based upon compounds
18-20.
DETAILED DESCRIPTION
[0045] The present invention relates to the discovery that small
molecules can be identified to rescue protein folding and
processing of mutant and wild-type MC4R polypeptides and enhance
protein stability on the cell surface of neurons, which in turn,
decreases hunger and overeating. The pharmacological chaperones
bind specifically to the MC4R protein and induce or stabilize a
functional conformation of the mutant or wild-type MC4R. The
invention therefore permits specific rescue of mutant MC4R, as well
as enhanced expression of wild-type MC4R at the cell surface.
Accordingly, pharmacological chaperones for MC4R can be used for
the treatment of disorders where rescue of, or increased stability
or activity of, MC4R is desired, e.g., the condition of being
overweight or obese.
[0046] The invention is based, in part, on the discovery that
administration of a pharmacological chaperone to a human resulted
in a meaningful increase in the level of activity of a wild-type
protein. This discovery, combined with an understanding of a
pharmacological chaperone's ability to promote proper protein
folding in the ER, leading to correct protein trafficking and
significantly increased protein activity, advantageously provides
the ability to achieve sufficient protein activity to reverse or
ameliorate a disease, disorder, or condition, particularly in a
human subject. This phenomenon is highly specific to the protein
specifically bound by the particular pharmacological chaperone, in
contrast to methods using compounds that operate generally to
increase expression of all proteins, called "chemical
chaperones."
[0047] Certain experimental results underlie the present invention:
pharmacological chaperones increased endogenous wild-type protein
activity in humans to about 120% of normal, 130% of normal, and
145% of normal at a lower dose, and to 150% and 185% of normal at a
higher dose after administration of a pharmacological chaperone
(see Example 7 and FIG. 12).This level of increase in vivo was not
predictable from results with cells in tissue culture which remain
exposed to the pharmacological chaperone. For example, U.S. Pat.
No. 6,274,597 describes a 30% increase of a-galactosidase A
(.alpha.-Gal A) activity in normal lymphoblasts cultured in vitro
with deoxygalactonojirimycin (DGJ), a pharmacological chaperone.
Given the expectation that physiological clearance processes would
be expected to reduce the effects of pharmacological chaperones on
normal proteins in vivo, it was not expected that a pharmacological
chaperone would yield a significant increase in wild-type protein
activity. Example 10 of U.S. Pat. No. 6,274,597 describes an
increase in activity of a mutant enzyme in transgenic mice treated
for one week with a pharmacological chaperone. However, these
experiments involved mutant forms of the rescued protein, not
wild-type, and were conducted in mice, so the results were not
predictive or suggestive of the results observed for wild-type
protein in humans.
[0048] There was no basis to expect that a pharmacological
chaperone could increase the level of activity of a wild-type
protein in vivo by at least 20-25%, i.e., by at least 1.2-fold or
120% of normal, or by 30% (1.3-fold, 130% of normal), 40%
(1.4-fold, 140% of normal), and particularly not by at least about
50% (1.5-fold, 150% of normal). Yet, as exemplified herein,
administration of DGJ to subjects resulted in a dose-dependent
increase in .alpha.-Gal A. This extraordinary effect results from
titrating the pharmacological chaperone, which is already
demonstrated in accordance with existing technology to rescue a
mutant form of the protein, to achieve the disclosed increase in
activity or wild-type protein. Accordingly, the invention provides
for titrating a dose of a pharmacological chaperone that has been
found to rescue activity of a mutant protein to increase the level
of activity of a wild-type protein by a defined amount.
Definitions
[0049] The terms used in this specification generally have their
ordinary meanings in the art, within the context of this invention
and in the specific context where each term is used. Certain terms
are discussed below, or elsewhere in the specification, to provide
additional guidance to the practitioner in describing the
compositions and methods of the invention and how to make and use
them.
[0050] As used herein, the term "pharmacological chaperone," or
sometimes "specific pharmacological chaperone" ("SPC"), refers to a
molecule that specifically binds to MC4R and has one or more of the
following effects: (i) enhancing the formation of a stable
molecular conformation of the protein; (ii) enhances proper
trafficking of the protein from the ER to another cellular
location, preferably a native cellular location, i.e., preventing
ER-associated degradation of the protein; (iii) preventing
aggregation of conformationally unstable, i.e., misfolded proteins;
(iv) restoring or enhancing at least partial wild-type function,
stability, and/or activity of the protein; and/or (v) improving the
phenotype or function of the cell harboring MC4R. Thus, a
pharmacological chaperone for MC4R is a molecule that binds to
MC4R, resulting in proper folding, trafficking, non-aggregation,
and activity of MC4R. As used herein, this term does not refer to
endogenous chaperones, such as BiP, or to non-specific agents which
have demonstrated non-specific chaperone activity against various
proteins, such as glycerol, DMSO or deuterated water, i.e.,
chemical chaperones (see Welch et al., Cell Stress and Chaperones
1996; 1(2):109-115; Welch et al., Journal of Bioenergetics and
Biomembranes 1997; 29(5):491-502; U.S. Pat. No. 5,900,360; U.S.
Pat. No. 6,270,954; and U.S. Pat. No. 6,541,195). It includes
specific binding molecules, e.g. specific pharmacological
chaperones (discussed above), inhibitors or antagonists, and
agonists.
[0051] As used herein, the term "specifically binds" refers to the
interaction of a pharmacological chaperone with MC4R, specifically,
an interaction with amino acid residues of MC4R that directly
participate in contacting the pharmacological chaperone. A
pharmacological chaperone specifically binds to a target protein,
here MC4R, to exert a chaperone effect on MC4R, and not on a
generic group of related or unrelated proteins. The amino acid
residues of MC4R that interact with any given MC4R pharmacological
chaperone may or may not be within the MC4R ligand-binding domain,
i.e., the domain that binds the natural ligand MSH, or any other
MC4R "active site," e.g., the G-protein binding domain. Specific
binding can be evaluated through routine binding assays or through
structural studies, e.g., co-crystallization, NMR, and the like.
Examples of amino acids in the MSH ligand-binding domain of MC4R
include but are not limited to Phe284 and Tyr268 (using, e.g., SEQ
ID NO: 2 as a reference sequence).
[0052] In one non-limiting embodiment, the pharmacological
chaperone is an inhibitor or antagonist of MC4R. In another
non-limiting embodiment, the pharmacological chaperone is an
agonist of MC4R. In yet another embodiment, the pharmacological
chaperone is a mixed agonist/antagonist. As used herein, the term
"antagonist" refers to any molecule that binds to a protein and
either partially or completely blocks, inhibits, reduces, or
neutralizes an activity of MC4R. The term "agonist" refers to any
molecule that binds to a protein and at least partially increases,
enhances, restores, or mimics an activity of MC4R. As discussed
below, such molecules are known for MC4R.
[0053] As used herein, the terms "enhance MC4R conformational
stability" or "increase MC4R conformational stability" refer to
increasing the amount or proportion of MC4R that adopts a
finctional conformation in a cell contacted with a pharmacological
chaperone specific for MC4R, relative to MC4R in a cell (preferably
of the same cell-type or the same cell, e.g., at an earlier time)
not contacted with the pharmacological chaperone specific for MC4R.
In one embodiment, the cells do not express a conformation mutant
MC4R. In another embodiment, the cells do express a mutant MC4R
polynucleotide encoding a polypeptide e.g., a conformational mutant
MC4R.
[0054] As used herein, the terms "enhance MC4R trafficking" or
"increase MC4R trafficking" refer to increasing the efficiency of
transport of MC4R to the plasma membrane in a cell contacted with a
pharmacological chaperone specific for MC4R, relative to the
efficiency of transport of MC4R in a cell (preferably of the same
cell-type or the same cell, e.g., at an earlier time) not contacted
with the pharmacological chaperone specific for MC4R.
[0055] As used herein, the terms "enhance MC4R activity" or
"increase MC4R activity" refer to increasing the activity of MC4R,
as described herein, in a cell contacted with a pharmacological
chaperone specific for MC4R, relative to the activity of MC4R in a
cell (preferably of the same cell-type or the same cell, e.g., at
an earlier time) not contacted with the pharmacological chaperone
specific for MC4R.
[0056] As used herein, the terms "enhance MC4R level" or "increase
MC4R level" refer to increasing the level of MC4R in a cell
contacted with a pharmacological chaperone specific for MC4R,
relative to the level of MC4R in a cell (preferably of the same
cell-type or the same cell, e.g., at an earlier time) not contacted
with the pharmacological chaperone specific for MC4R.
[0057] The term "stabilize a proper conformation" refers to the
ability of a MC4R pharmacological chaperone to induce or stabilize
a conformation of a mutated MC4R protein that is fuictionally
identical to the conformation of the wild-type MC4R protein. The
term "functionally identical" means that while there may be minor
variations in the conformation (almost all proteins exhibit some
conformational flexibility in their physiological state),
conformational flexibility does not result in (1) protein
aggregation, (2) elimination through the endoplasmic
reticulum-associated degradation pathway, (3) impairment of protein
function, e.g., the ability to bind ligand and/or activate adenylyl
cyclase activity, and/or (4) improper transport within the cell,
e.g., localization to the plasma membrane, to a greater or lesser
degree than that of the wild-type protein.
[0058] The term "stable molecular conformation" refers to a
conformation of a protein, i.e., MC4R, induced by a pharmacological
chaperone, that provides at least partial wild-type function in the
cell. For example, a stable molecular conformation of a mutant MC4R
would be one where MC4R escapes from the ER and is trafficked to
the cell membrane as for a wild-type MC4R, instead of misfolding
and being degraded. In addition, a stable molecular conformation of
a mutated MC4R may also possess full or partial MC4R activity,
e.g., adenylyl cyclase activating activity for enhanced cAMP
generation via its cognate physiologic G protein. However, it is
not necessary that the stable molecular conformation have all of
the functional attributes of the wild-type protein.
[0059] The term "MC4R activity" refers to the normal physiological
function of a wild-type MC4R in a cell. For example, upon binding
by an agonist, MC4R signals via interaction with a G-protein,
G.alpha..sub.s, and activation of adenylate cyclase (see e.g.,
VanLeeuwen et al., J Biol. Chem. 2003; 18: 15935-40). This results
in the intracellular accumulation of cAMP and activation of protein
kinase A (PKA). Such functionality can be tested by any method
known in the art. For example, binding assays of the .alpha.-,
.beta.-, or .gamma.-MSH ligand, or
.sup.125I-[Nle.sup.4,D-Phe.sup.7].alpha.-MSH agonist to MC4R, or
using adenylyl cyclase activation assays, or luciferase reporter
gene assays, can be used to determine increases in intracellular
cAMP. Cyclic AMP accumulation assays are well known in the art (see
e.g., VanLeeuwen et al., J Biol. Chem. 2003; 18: 15935-40).
[0060] "MC4R" refers to a polypeptide encoded by a nucleotide
sequence having the sequence as depicted in any one of: SEQ ID NO:
1 (human; GenBank Accession No. BC069172); 3 (human; GenBank
Accession No. NM.sub.--005912); 5 (rat; GenBank Accession No.
NM.sub.--013099); or 7 (murine; GenBank Accession No.
NM.sub.--016977).
[0061] An "MC4R polypeptide" also refers to an amino acid sequence
as depicted in SEQ ID NOs: 2 (human; GenBank Accession No.
AAI01803); 4 (human; GenBank Accession No. NM.sub.--005912); 6
(rat; GenBank Accession No. NM.sub.--013099); or 8 (murine; GenBank
Accession No. AF201662), and any other amino acid sequence that
encodes an MC4R polypeptide having the same function and ligand
binding affinity as any one of SEQ ID NOs: 2, 4, 6 or 8.
[0062] The term "wild-type MC4R" refers to the nucleotide (SEQ ID
NOs: 1, 3, 5 and 7) sequences encoding MC4R, and polypeptide (SEQ
ID NOs: 2, 4, 6, and 8) sequences encoded by the aforementioned
nucleotide sequences (human MC4R-GenBank Accession AAI01803; human
MC4R-GenBank Accession No. NM.sub.--005912; rat MC4R-GenBank
Accession No. NM.sub.--013099; and mouse MC4R-GenBank Accession
AF201662), and any other nucleotide sequence that encodes MC4R
polypeptide (having the same functional properties and binding
affinities as the aforementioned polypeptide sequences), such as
allelic variants in normal individuals, that have the ability to
achieve a functional conformation in the ER, achieve proper
localization within the cell, and exhibit wild-type activity (e.g.,
MC4R stimulation of cAMP accumulation).
[0063] As used herein the term "mutant MC4R" refers to a MC4R
polypeptide translated from a gene containing a genetic mutation
that results in an altered MC4R amino acid sequence. In one
embodiment, the mutation results in a MC4R protein that does not
achieve a native conformation under the conditions normally present
in the ER, when compared with wild-type MC4R, or exhibits decreased
stability or activity as compared with wild-type MC4R. This type of
mutation is referred to herein as a "conformational mutation," and
the protein bearing such a mutation is referred as a
"conformational mutant." The failure to achieve this conformation
results in MC4R protein being degraded or aggregated, rather than
being transported through a normal pathway in the protein transport
system to its native location in the cell or into the extracellular
environment. In some embodiments, a mutation may occur in a
non-coding part of the gene encoding MC4R that results in less
efficient expression of the protein, e.g., a mutation that affects
transcription efficiency, splicing efficiency, mRNA stability, and
the like. By enhancing the level of expression of wild-type as well
as conformational mutant variants of MC4R, administration of a MC4R
pharmacological chaperone can ameliorate a deficit resulting from
such inefficient protein expression.
[0064] Exemplary mutations (using the polypeptide of SEQ ID NO: 2
as a reference) include P78L, R165Q, and R165W. Other MC4R mutants
include I125K, C271Y, T11A, A175T, I316L, I316S, I317T, N97D, G98R,
N62S, C271R, S58C, N62S, N97D, Y157S, I102S, L106P, L250Q, Y287X,
P299H, S58C, CTCT at codon 211, and TGAT insertion at codon 244. In
addition, other MC4R mutations (again using SEQ ID NO: 2 as a
reference) include those described in Table 1, infra.
[0065] Certain tests may evaluate attributes of a protein that may
or may not correspond to its actual in vivo activity, but
nevertheless are appropriate surrogates of protein functionality,
and wild-type behavior in such tests demonstrates evidence to
support the protein folding rescue or enhancement techniques of the
invention. One such activity in accordance with the invention is
appropriate transport of a functional MC4R from the endoplasmic
reticulum to the cell membrane.
[0066] The terms "endogenous expression" and "endogenously
expressed" refers to the normal physiological expression of MC4R in
cells in an individual not having or suspected of having a disease
or disorder associated with MC4R deficiency, overexpression of a
dominant negative mutant, or other defect, e.g., obesity, such as a
mutation in MC4R nucleic acid or polypeptide sequence that alters,
e.g., inhibits its expression, activity, or stability. This term
also refers to the expression of MC4R in cells or cell types in
which it is normally expressed in healthy individuals, and does not
include expression of MC4R in cells or cell types, e.g., tumor
cells, in which MC4R is not expressed in healthy individuals.
[0067] As used herein, the term "efficiency of transport" refers to
the ability of a mutant protein to be transported out of the
endoplasmic reticulum to its native location within the cell, cell
membrane, or into the extracellular environment.
[0068] The terms "therapeutically effective dose" and "effective
amount" refer to an amount sufficient to enhance protein processing
in the ER (permitting a functional conformation), without
inhibiting protein already expressed at the appropriate cellular
location (in the case of an antagonist), or without inducing
ligand-mediated receptor internalization of protein from the
appropriate cellular location (in the case of an agonist), and
enhance activity of the target protein, thus resulting in a
therapeutic response in a subject. A therapeutic response may be
any response that a user (e.g., a clinician) will recognize as an
effective response to the therapy, including the foregoing symptoms
and surrogate clinical markers. Thus, a therapeutic response will
generally be an amelioration or inhibition of one or more symptoms
of a disease or disorder, e.g., obesity or binge eating.
[0069] The phrase "pharmaceutically acceptable" refers to molecular
entities and compositions that are physiologically tolerable and do
not typically produce untoward reactions when administered to a
human. Preferably, as used herein, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the compound is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils. Water or aqueous saline solutions and
aqueous dextrose and glycerol solutions are preferably employed as
carriers, particularly for injectable solutions. Suitable
pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin, 18th Edition, or other
editions.
[0070] The terms "about" and "approximately" shall generally mean
an acceptable degree of error for the quantity measured given the
nature or precision of the measurements. Typical, exemplary degrees
of error are within 20 percent (%), preferably within 10%, and more
preferably within 5% of a given value or range of values.
Alternatively, and particularly in biological systems, the terms
"about" and "approximately" may mean values that are within an
order of magnitude, preferably within 5-fold and more preferably
within 2-fold of a given value. Numerical quantities given herein
are approximate unless stated otherwise, meaning that the term
"about" or "approximately" can be inferred when not expressly
stated.
[0071] As used herein, the term "isolated" means that the
referenced material is removed from the environment in which it is
normally found. Thus, an isolated biological material can be free
of cellular components, i.e., components of the cells in which the
material is found or produced. In the case of nucleic acid
molecules, an isolated nucleic acid includes a PCR product, an mRNA
band on a gel, a cDNA, or a restriction fragment. In another
embodiment, an isolated nucleic acid is preferably excised from the
chromosome in which it may be found, and more preferably is no
longer joined to non-regulatory, non-coding regions, or to other
genes, located upstream or downstream of the gene contained by the
isolated nucleic acid molecule when found in the chromosome. In yet
another embodiment, the isolated nucleic acid lacks one or more
introns. Isolated nucleic acids include sequences inserted into
plasmids, cosmids, artificial chromosomes, and the like. Thus, in a
specific embodiment, a recombinant nucleic acid is an isolated
nucleic acid. An isolated protein may be associated with other
proteins or nucleic acids, or both, with which it associates in the
cell, or with cellular membranes if it is a membrane-associated
protein. An isolated organelle, cell, or tissue is removed from the
anatomical site in which it is found in an organism. An isolated
material may be, but need not be, purified.
[0072] The term "purified" as used herein refers to material, such
as a MC4R nucleic acid or polypeptide, that has been isolated under
conditions that reduce or eliminate unrelated materials, i.e.,
contaminants. For example, a purified protein is preferably
substantially free of other proteins or nucleic acids with which it
is associated in a cell. As used herein, the term "substantially
free" is used operationally, in the context of analytical testing
of the material. Preferably, purified material substantially free
of contaminants is at least 50% pure; more preferably, at least 90%
pure, and more preferably still at least 99% pure. Purity can be
evaluated by conventional means, e.g., chromatography, gel
electrophoresis, immunoassay, composition analysis, biological
assay, and other methods known in the art.
[0073] The term "Me" means methyl, "Et" means ethyl, and "Ac" means
acetyl.
[0074] The term "halo", unless otherwise indicated, means fluoro,
chloro, bromo or iodo. Preferred halo groups are fluoro, chloro and
bromo.
[0075] The term "alkyl", unless otherwise indicated, includes
saturated monovalent hydrocarbon radicals having straight,
branched, or cyclic moieties (including fused and bridged bicyclic
and spirocyclic moieties), or a combination of the foregoing
moieties. For an alkyl group to have cyclic moieties, the group
must have at least three carbon atoms.
[0076] The term "cycloalkyl", unless otherwise indicated, includes
cyclic alkyl moieties wherein alkyl is as defined above. The use of
the term "cycloalkyl" shall not be construed as limiting the term
"alkyl" to non-cyclic moieties.
[0077] The term "alkenyl", unless otherwise indicated, includes
alkyl moieties having at least one carbon-carbon double bond
wherein alkyl is as defined above and including E and Z isomers of
said alkenyl moiety.
[0078] The term "alkynyl", unless otherwise indicated, includes
alkyl moieties having at least one carbon-carbon triple bond
wherein alkyl is as defined above.
[0079] The term "alkoxy", unless otherwise indicated, includes
O-alkyl groups wherein alkyl is as defined above.
[0080] The term "aryl", unless otherwise indicated, includes an
organic radical derived from an aromatic hydrocarbon by removal of
one hydrogen, such as phenyl or naphthyl.
[0081] The term "4 to 10 membered heterocyclic", unless otherwise
indicated, includes aromatic and non-aromatic heterocyclic groups
containing one to four heteroatoms each selected from O, S and N,
wherein each heterocyclic group has from 4 to 10 atoms in its ring
system, and with the proviso that the ring of said group does not
contain two adjacent O or S atoms. Non-aromatic heterocyclic groups
include groups having only 4 atoms in their ring system, but
aromatic heterocyclic groups must have at least 5 atoms in their
ring system. The heterocyclic groups include benzo-fused ring
systems. An example of a 4 membered heterocyclic group is
azetidinyl (derived from azetidine). An example of a 5 membered
heterocyclic group is thiazolyl and an example of a 10 membered
heterocyclic group is quinolinyl. Examples of non-aromatic
heterocyclic groups are pyrrolidinyl, tetrahydrofuranyl,
dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl,
dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino,
thiomorpholino, thioxanyl, piperazinyl, homopiperazinyl,
azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl,
thiepanyl, oxazepinyl, diazepinyl, thiazepinyl,
1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl,
2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl,
dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl,
dihydrofuranyl, pyrazolidinylimidazolinyl, imidazolidinyl,
3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl,
azabicyclo[2.2.2]hexanyl, 3H-indolyl and quinolizinyl. Examples of
aromatic heterocyclic groups are pyridinyl, imidazolyl,
pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl,
thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl,
quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl,
cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl,
triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl,
thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl,
benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl,
naphthyridinyl, and furopyridinyl. Spiro moieties are also included
within the scope of this definition including
1-oxa-6-aza-spiro[2.5]oct-6-yl. The foregoing groups, as derived
from the groups listed above, may be C-attached or N-attached where
such is possible. For instance, a group derived from pyrrole may be
pyrrol-1-yl (N-attached) or pyrrol-3-yl (C-attached). Further, a
group derived from imidazole may be imidazol-1-yl (N-attached) or
imidazol-3-yl (C-attached).
[0082] The phrase "pharmaceutically acceptable salt(s)", unless
otherwise indicated, includes salts of acidic or basic groups which
may be present in a compound used in the methods of the invention.
Compounds that are basic in nature are capable of forming a wide
variety of salts with various inorganic and organic acids. The
acids that may be used to prepare pharmaceutically acceptable acid
addition salts of such basic compounds are those that form
non-toxic acid addition salts, i.e., salts containing
pharmacologically acceptable anions, such as the acetate,
benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate,
borate, bromide, calcium edetate, camsylate, carbonate, chloride,
clavulanate, citrate, dihydrochloride, edetate, dislyate, estolate,
esylate, ethylsuccinate, fumarate, gluceptate, gluconate,
glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,
hydrobromide, hydrochloride, iodide, isothionate, lactate,
lactobionate, laurate, malate, maleate, mandelate, mesylate,
methylsulfate, mucate, napsylate, nitrate, oleate, oxalate, pamoate
(embonate), palmitate, pantothenate, phospate/diphosphate,
polygalacturonate, salicylate, stearate, subacetate, succinate,
tannate, tartrate, teoclate, tosylate, triethiodode, and valerate
salts. Since a single compound may include more than one acidic or
basic moiety, such a compound may include mono, di or tri-salts in
a single compound.
Melanocortin 4 Receptor
[0083] Melanocortin (MC) receptors are members of the
seven-transmembrane-domain G protein-coupled receptor superfamily
that activate generation of the second messenger cyclic AMP (cAMP).
There are five MC receptors isolated to date: MC1R, MC2R, MC3R,
MC4R and MC5R. MC2R is the receptor for adrenocorticotropic hormone
(ACTH). Human MC4R is 332 amino acids in length.
[0084] The melanocortin 4 receptor (MC4R) has been implicated in
the regulation of body weight (Graham et al, Nat. Genetics 1997;
17: 273-4). MC4R is expressed in the brain, including the
hypothalamus, which influences food intake. Signaling via MC4R
stimulates anorexigenic neural pathways. MC4R null mice develop
late onset obesity with hyperglycemia and hyperinsulinemia. Mice
lacking one MC4R allele (heterozygotes) have intermediate body
weight between wild-type and homozygous null mice. In humans, MC4R
deficiency is the most common monogenic form of obesity (Farooqi et
al., New Engl. J. Med. 2003; 348: 1085-95). Transgenic mice
overexpressing an endogenous MC4R antagonist, agouti-related
protein (AgRP), exhibited increased weight gain, food consumption,
and body length compared with non-transgenic littermates (Ollman et
al., Science 1997; 278: 135-37).
[0085] Numerous mutations, found mostly in obese individuals, have
been identified in the human MC4R gene, including frameshift,
nonsense and missense mutations (Nijenhuis et al., J. Biol. Chem.
2003; 278: 22939-45). At least two groups of researchers have
confirmed that MC4R is mutated in about 5% of obese individuals.
Carriers of MC4R mutations demonstrated hyperphagia and
hyperinsulinemia, had above-average bone mineral density, and more
rapid linear growth than control subjects matched for BMI. Farooqi
et al. also have found that signaling properties of the mutant MC4R
receptors correlated with the severity of obesity.
[0086] Several authors have now reviewed the recent advances in our
understanding of the genetics of MC4R in early onset obesity (see
e.g., Farooqi IS, O'Rahilly S, Int J Obes (Lond), 2005 Oct.,
29(10), 1149-52; Govaerts C, Srinivasan S, Shapiro A, Zhang S,
Picard F, Clement K, Lubrano-Berthelier C, Vaisse C, Peptides, 2005
Oct., 26(10), 1909-19; Tao YX, Mol Cell Endocrinol, 2005 Jul. 15,
239(1-2), 1-14; Farooqi IS, O'Rahilly S, Annu Rev Med, 2005, 56,
443-58). For example, in one patient with severe early-onset
obesity, an autosomal-dominant mode of inheritance of an MC4R
mutation has been found to be due to a dominant-negative effect
caused by receptor dimerization (Biebermann H, Krude H, Elsner A,
Chubanov V, Gudermann T, Gruters A, Diabetes, 2003 December,
52(12), 2984-8).
[0087] Loss of function is expected for MC4R with some mutations,
since most of the mutations identified to date are non-conservative
amino acid substitutions. This has been demonstrated for several
MC4Rs found in obese individuals. In addition, a number of
mutations have been associated with reduced expression of MC4R at
the cell surface (Gu et al., Diabetes 1999, 48: 635-39; Nijenhuis
et al., supra). For example, in a screen of eleven MC4R missense
mutations that were only found in obese individuals, and which were
located outside of the N-terminal region of MC4R (which is not
involved in ligand binding), ten exhibited lower specific binding
at the cell surface to the labeled .alpha.-melanocyte stimulating
hormone (.alpha.-MSH) ligand
.sup.125I-[Nle.sup.4,D-Phe7].alpha.-MSH, compared with wild-type
MC4R. Nijenhuis et al., supra, at 22941. The decreased specific
binding was determined to reflect a lower cell surface expression,
since the affinity for ligand among the mutants was largely similar
to the wild-type receptor, as depicted in Table 1 below (IC.sub.50
values in nM +/-S.E.): TABLE-US-00001 TABLE 1 IC.sub.50 IC.sub.50
WT 9.1 .+-. 0.64 WT 9.1 .+-. 0.64 WT 55 .+-. 7.4
.sup.125I-[Nle.sup.4, D- WT 55 .+-. 7.4 .sup.125I-[Nle.sup.4, D-
Mutant .alpha.-MSH Phe.sup.7].alpha.-MSH Mutant .alpha.-MSH
Phe.sup.7].alpha.-MSH T112M 28 .+-. 2.7 5.4 .+-. 0.64 I317T 38 .+-.
3.0 7.8 .+-. 0.44 V253I 43 .+-. 2.0 8.1 .+-. 0.25 I301T 24 .+-. 4.8
5.8 .+-. 0.78 (A700G) S30F/ 67 .+-. 8.8 6.7 .+-. 0.30 R165W 40 .+-.
13 8.7 .+-. 1.0 G252S (C886T) L250Q 5.8 .+-. 0.35 3.4 .+-. 0.69
R165Q 40 .+-. 11 8.9 .+-. 1.2 (C886A) I170V 59 .+-. 5.7 8.9 .+-.
1.7 P78L -- -- Nijenhuis et al., J. Biol. Chem. 2003; 278:
22939-22945, at 22942.
[0088] Even two mutants with higher binding affinity (L250Q and
T112M) demonstrated lower cell surface expression according to
saturation binding experiments. In addition, all mutants
demonstrated decreased maximal response (receptor activation as
measured using an adenylyl cyclase assay) upon .alpha.-MSH binding.
In particular, Nijenhuis et al. concluded from results of
immunocytochemical data that the P78L, R165Q and R165W mutants are
expressed, but are retained intracellularly.
[0089] An additional study identified the following MC4R mutations:
I125K; C271Y; T11A (A434G); A175T; I316L; N97D; N62S; and C271R
(Farooqi et al., New Eng. J. Med. 2003; 348; 1085-95). Of these
mutations, all exhibited reduced activity, or no activity, in vitro
evaluated using a luciferase reporter gene assay responsive to
cAMP. However, this group found that three variants V1 03I; I251L;
and T112M have no effect on MC4R signaling. Mutations associated
with childhood, i.e., early onset obesity were S58C, N62S, Y157S,
C271Y, P78L, G98R that resulted in either decreased (S58C, N62S,
Y157S, C271Y) or no (P78L, G98R) ligand binding, also demonstrated
proportional impairments in
[Nle.sup.4,D-Phe.sup.7].alpha.-MSH-stimulated cAMP production (Tao
et al., Endocrinology 2003; 144(10):4544-5 1).
[0090] A final study identified the following mutants in MC4R;
I125L (A1144C); F51L (T544C); M200V (A991G); T5T (C408T) (Branson
et al., New Eng. J. Med. 2003; 348: 1096-1103).
[0091] In addition to obesity, MC4R has been implicated in binge
eating. According to the Diagnostic and Statistical Manual of
Mental Disorders-Text Revision (DSM-IV-TR.TM., Fourth Ed.), binge
eating involves recurrent episodes of eating an abnormally large
amount of food and experiencing feelings of lack of control over
the behavior. In one study of 469 white obese subjects, it was
found that while only a small percentage of obese subjects were
diagnosed with binge-eating, all of the obese subjects with MC4R
mutations were diagnosed with binge-eating (Branson et al.,
supra).
MC4R Structure and Ligand Binding
[0092] Endogenous melanocortin agonists contain the sequence
His-Phe-Arg-Trp, which is important for melanocortin receptor
molecular recognition and stimulation. The molecular determinants
of MC4R ligand binding were determined in one study by employing a
large array of ligands (Nickolls et al., Pharmacol Exp Ther 2003;
304(3):1217-27). Molecular modeling of the receptor was used to
identify Phe284, in transmembrane (TM) domain 7 (TM7), as a
potential site of ligand interaction. Mutation of Phe284 to alanine
reduced binding affinity and potency of peptides containing L-Phe
by up to 71-fold but did not affect binding of linear peptides
containing D-Phe. This data was consistent with a hydrophobic
interaction between the Phe7 of .alpha.-MSH and Phe284. Second, the
effect of a naturally occurring mutation in TM3 (I137T), which, as
described above is linked to obesity, was examined. This mutation
decreased affinity and potency of cyclic, rigid peptides but not
more flexible peptides, consistent with an indirect effect of the
mutation on the tertiary structure of the receptor. The residues
that support ligand selectivity for the MC4R over the MC3R were
also determined. Mutation of Ile125 (TM3) of the MC4R to the
equivalent residue of the MC3R (phenylalanine) selectively
decreased affinity and potency of MC4R-selective ligands. This
effect was mirrored by the reciprocal MC3R mutation F157I. The
magnitude of this effect indicates that this locus is not of major
importance. However, it was proposed that an
isoleucine/phenylalanine mutation may affect the orientation of
Asp122, which has been identified as a major determinant of ligand
binding affinity.
[0093] Others have determined that Tyr268 was required for the
selective interaction with the endogenous MC4R antagonist Agouti
protein, as well as for the selectivity of another MC4R agonist
(Oosterom et al., J. Biol. Chem. 2001; 276(2):931-6). Agouti
protein is normally expressed in the skin and is a natural
antagonist of the MC4R (Kiefer et al., Biochemistry 1997; 36:
2084-2090).
MC4R Agonists and Antagonists
[0094] According to the invention, MC4R agonists and antagonists
include the compounds depicted in FIGS. 1-8 and 10 herein and
further described in Examples 3 and 4 below.
[0095] Natural agonists (ligands) of MC4R include .alpha.-MSH,
ACTH, .beta.-MSH, and .gamma.-MSH (in order from highest to lowest
affinity). Other MC4R ligands, including agonists and antagonists,
which have been described to date are predominantly peptides (U.S.
Pat. No. 6,060,589) and cyclic peptide analogs (U.S. Pat. No.
6,613,874 to Mazur et al.). A series of MC4R peptide agonists have
also been designed (Sun et al., Bioorg Med Chem 2004;
12(10):2671-7). In addition, Nijenhuis et al. (Peptides 2003;
24(2):271-80) described the development and evaluation of
melanocortin antagonist compounds that were selective for the MC4R.
One compound, designated Ac-Nle-Gly-Lys-D-Phe-Arg-Trp-Gly-NH(2)
(SEQ ID NO:9), was found to be the most selective MC4R compound,
with a 90- and 110-fold selectivity for the MC4R as compared to the
MC3R and MC5R, respectively. Subsequent modification yielded
compound Ac-Nle-Gly-Lys-D-Nal(2)-Arg-Trp-Gly-NH(2) (SEQ ID NO:10),
a selective MC4R antagonist with 34-fold MC4R/MC3R and 109-fold
MC4RIMC5R selectivity. Both compounds were active in vivo, and
crossed the blood-brain barrier. Further, U.S. Pat. Nos. 6,054,556
and 5,731,408 describe families of agonists and antagonists for
MC4R that are lactam heptapeptides having a cyclic structure.
[0096] Other high-affinity MC4R antagonists are described in Grieco
et al. (J Med Chem 2002; 24:5287-94). These cyclic antagonists were
designed based on the known high affinity antagonist SHU9119
(Ac-Nle4-[Asp5-His6-DNal(2')7-Arg8-Trp9-Lys10]-NH(2)) (SEQ ID NO:
11). The SHU9119 analogues were modified in position 6 (His) with
non-conventional amino acids. One compound containing a Che
substitution at position 6 is a high affinity MC4R antagonist
(IC.sub.50=0.48 nM) with 100-fold selectivity over MC3R. Another
compound with a Cpe substitution at position 6 also was a high
affinity MC4R antagonist (IC.sub.50=0.51 nM) with a 200-fold
selectivity over MC3R. Molecular modeling was used to examine the
conformational properties of the cyclic peptides modified in
position 6 with conformationally restricted amino acids. See also,
Grieco et al., Peptides 2006; 27(2):472-81.
[0097] Several non-peptide MC4R ligands have been disclosed in U.S.
published patent applications 2003/0158209 to Dyck et al. and
2004/082590 to Briner et al. Also, U.S. Pat. No. 6,638,927 to
Renhowe et al. describes small, low-molecular weight
guanidobenzamides as specific MC4R agonists. Richardson et al. have
described novel arylpiperizines that are agonists of MC4R (J Med
Chem 2004; 47(3):744-55). U.S. Pat. Nos. 6,979,691 to Yu et al. and
6,699,873 to Maguire also describe non-peptide compounds which bind
selectively to MC4R.
[0098] WO 99/55679 to Basu et al. discloses isoquinoline
derivatives, small molecule non-peptide compounds, which show low
(micromolar) affinities for the MC1R and MC4R, reduction of dermal
inflammation induced by arachidonic acids, and reductions of body
weight and food intake.
[0099] WO 99/64002 to Nargund et al. also discloses spiropiperidine
derivatives as melanocortin receptor agonists, useful for the
treatment of diseases and disorders such as obesity, diabetes, and
sexual dysfunction.
[0100] Other non-peptide MC4R antagonists have been described.
Thus, U.S. published patent applications 2003/0176425 and
2003/0162819 to Eisinger disclose novel 1,2,4-thiadiazole and
1,2,4-thiadiazolium derivatives, respectively, as MC4R antagonists
or agonists. These applications also disclose use of these
compounds to treat obesity.
[0101] Several antagonists of melanocortin receptors have been
demonstrated to be competitive antagonists, i.e., competing for
binding with a ligand. For example, the melanocortin antagonist
agouti signaling protein (ASIP) was shown to have characteristics
consistent with competitive antagonism observed at the hMC1R, and
more complex behavior observed at the other receptors (Yang et al.,
Mol. Endocrinology 1997; 11(3): 274-280). Similarly, ACTH, the
natural ligand for MC2R, cannot be out-competed for binding by
.alpha.-, .beta.-, or .gamma.-MSH (Abdel-Malek et al., Cell. Mol.
Life Sci. 2001; 48: 434-41.
[0102] Other MC4R binding compounds are described in the following:
Bednarek and Fong, Exp Opn Ther Patents 2004; 14: 327-36;
Ujjainwalla et al., Bioorg. Med. Chem. Lett. 2005; 15(18):4023-8;
WO 03/07949 (Merck); WO 03/61660 (Eli Lilly); WO 03/09847 (Amgen);
WO 03/09850 (Amgen); WO 03/31410 (Neurocrine Biosciences); WO
03/94918 (Neurocrine Biosciences); WO 03/68738 (Neurocrine
Biosciences); WO 03/92690 (Procter and Gamble); WO 03/93234
(Procter and Gamble); WO 03/72056 (Chiron); WO 03/66597 (Chiron);
WO 03/66587 (Chiron); WO 03/66587 (Chiron); WO 02/67869 (Merck); WO
02/68387 (Merck); WO 02/00259 (Taisho); WO 02/92566 (Taisho); Tran
et al, Bioorg Med Chem Lett. 2006 [epub ahead of print]; Pontillo
et al., Bioorg Med Chem Lett. 2005; 15(23):5237-40; Pontillo et
al., Bioorg Med Chem Lett. 2005; 15(10):2541-6; Pontillo et al.,
Bioorg Med Chem Lett. 2004; 14(22):5605-9; Cheung et al., Bioorg
Med Chem Lett. 2005; 15(24):5504-8; Yan et al., Bioorg Med Chem
Lett. 2004; 15(20): 4611-4; Hsiung et al., Endocrinology. 2005
December; 146(12):5257-66; and Todorovic et al., Peptides. 2005
October; 26(10):2026-36.
[0103] Specific MC4R non-peptide agonists or antagonists
contemplated for use in the presently claimed methods are described
in Sebhat et al., J Med Chem 2002; 45: 4589 (compounds 1 and 6);
Richardson et al., J Med Chem. 2004; 47: 744 (compound 2);
Arasasingham et al., J Med Chem. 2003; 46: 9 (compound 3); WO
02/062766 to Millennium Pharmaceuticals (compound 4); Pedemonte et
al., J. Clin. Inves. 2005; 115: 2564-71 (compound 5); Tran et al.,
Bioorg Med Chem Lett. 2005; 15: 3434-38 (compound 7); Xi et al.,
Bioorg Med Chem Lett. 2004; 14: 377-81 (compound 8); Vos et al., J
Med Chem. 2004; 47: 1602-04 (compound 9); Pan et al., Bioorg Med
Chem Lett. 2003; 11: 185 (compound 10); Marsilje et al., Bioorg Med
Chem Lett. 2004. 14: 3721 (compound 11); Ujjainwalla et al., Bioorg
Med Chem Lett. 2003; 133: 4431 (compound 12); Nickolls et al., J
Pharmacol Exp Therap. 2005; 313: 1281-1288 (compounds 13-17);
Schioth et al., Biophys Biochem Res Comm. 2003; 399-405 (compound
18); Benoit et al., J Neurosci. 2000; 20: 3442-48 (compounds 19 and
20); Vos et al., Bioorg Med Chem Lett. 2006; 15: 2302 (compound
21); Tucci et al., Bioorg Med Chem Lett 2005; 15: 4389 (compound
22); Pontillo et al., Bioorg Med Chem Lett. 2005; 15: 4615-18
(compound 23); Chaki et al., J Pharmacol Exp Ther. 2003; 304: 818
(compound 24); Chaki et al., Pharmacol Biochem Behav. 2005; 82: 621
(compound 25).
[0104] Compounds 1, 2, 5, 6, 8, 10, 12, 13-17, and 19 described
above are MC4R agonists, while compounds 3, 4, 7, 9, 11, 18, and 20
are antagonists.
[0105] Specific MC4R peptide antagonists contemplated for use in
the presently claimed methods are
Ac-Cys-Glu-His-D-(2')Nal-Arg-Trp-Gly-Cys-Pro-Pro-Lys-Asp-NH(2) (SEQ
ID NO: 12); Ac-Cys-Nle-Arg-His-D-(2')Nal-Arg-Trp-Gly-Cys-NH(2) (SEQ
ID NO: 13); Ac-Cys-Glu-His-D-Phe
(3,4-di-Cl)-Arg-Trp-Gly-Cys-Pro-Pro-Lys-Asp-NH(2) (SEQ ID NO: 14),
Ac-Nle-c[Asp-Che-DNal(2')-Arg-Trp-Lys-NH(2) (SEQ ID NO: 15);
Ac-Nle-c[Asp-Cpe-DNal(2')-Arg-Trp-Lys-NH(2) (SEQ ID NO: 16);
cyclo(1-6)-suc-His-DPhe-Arg-Trp-Lys-NH(2) (SEQ ID NO: 17); and
Ac-DArg[Cys-Glu-His-DPhe-Arg-Trp-Cys]-NH(2) (SEQ ID NO: 18).
[0106] Peptide-based agonists and antagonists with non-naturally
occurring side chains and peptidomimetics are contemplated. See
e.g., U.S. Pat. No. 5,650,489; see also, U.S. Pat. No. 6,090,912,
especially at Section 5.5. For example, the side chains of
compounds 18-20 and the side chains in the compound class depicted
in FIG. 16 can be non-naturally occurring.
[0107] MC4R has been shown to undergo ligand-mediated receptor
internalization (Gao et al., J Pharmacol Exp Ther 2003;
307(3):870-7). Preexposure of GT1-7 cells that express endogenous
MC4R to the agonist a-melanocyte-stimulating hormone (.alpha.-MSH),
resulted in impaired cAMP formation to a second challenge of
.alpha.-MSH (Shinyama et al., Endocrinology 2003; 144(4):1301-14).
This was not seen with administration of an antagonist.
Ligand-induced internalization is triggered in G-protein coupled
receptors by phosphorylation of serine or threonine residues
between the C-terminal segment and third intracellular loop.
Phosphorylation promotes binding of beta-arrestins, which target
receptors for internalization and degradation by lysosomes. Recent
data demonstrate that the cytosolic tail of an attractin-like
protein (ALP) binds the C-terminal domain of MC4R (Yeo et al.,
Biochem. J. 2003; 376). Thus, a chaperone which increases the
stability of MC4R on the cell surface will be especially beneficial
given the short half-life of the receptor on the surface.
[0108] It is further expected that chaperone that is an agonist
that will reversibly bind to an MC4R polypeptide in the ER will not
induce receptor internalization. Similarly, where the chaperone
compound is an antagonist, it is expected that it will not inhibit
receptor activity once the receptor is at the cell surface.
Methods of Treatment
[0109] The present invention also provides a method for treating a
condition associated with reduced MC4R stability, such as obesity,
or having risk factors for developing obesity, by administering to
a subject in need of such treatment a chaperone to enhance
stability and/or activity of MC4R. The individual to be treated can
be an individual who does not exhibit a mutation in MC4R that
affects folding and processing of MC4R, but who would benefit from
increased MC4R stability on, e.g., neurons. The individual to be
treated can also have a mutation in MC4R that affects folding and
processing of the MC4R protein, and exhibits reduced MC4R stability
on neurons.
Formulation, Dosage and Administration
[0110] A specific pharmacological chaperone for MC4R, i.e., an MC4R
agonist or antagonist or other MC4R-binding compound as described
above, or as identified through the screening methods of the
invention as set forth below, is advantageously formulated in a
pharmaceutical composition together with a pharmaceutically
acceptable carrier. The chaperone may be designated as an active
ingredient or therapeutic agent for the treatment of obesity or
other disorder involving reduced MC4R cell surface expression or
transport to the cell surface.
[0111] The concentration of the active ingredient (pharmacological
chaperone) depends on the desired dosage and administration
regimen, as discussed below. Exemplary dose ranges of the active
ingredient are from about 0.01 mg/kg to about 250 mg/kg of body
weight per day; from about 1 mg/kg to about 100 mg/kg per day; or
from about 10 mg/kg to about 75 mg/kg per day.
[0112] Therapeutically effective compounds can be provided to a
subject in standard formulations, and may include any
pharmaceutically acceptable additives, such as excipients,
lubricants, diluents, flavorants, colorants, buffers, and
disintegrants. Standard formulations are well known in the art. See
e.g., Remington's Pharmaceutical Sciences, 20th edition, Mack
Publishing Company, 2000. The formulation may be produced in useful
dosage units for administration by any route that will permit the
therapeutic chaperone to cross the blood-brain barrier. Exemplary
routes include oral, parenteral, transmucosal, intranasal,
inhalation, or transdermal routes. Parenteral routes include
intravenous, intra-arteriolar, intramuscular, intradermal,
subcutaneous, intraperitoneal, intraventricular, intrathecal, and
intracranial administration.
[0113] In one embodiment, an MC4R pharmacological chaperone,
particularly those depicted in FIGS. 1-8 and 10 herein, is
formulated in a solid oral dosage form. For oral administration,
e.g., for a small molecule, the pharmaceutical composition may take
the form of a tablet or capsule prepared by conventional means with
pharmaceutically acceptable excipients such as binding agents
(e.g., pregelatinized maize starch, polyvinylpyrrolidone or
hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulfate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate.
[0114] In another embodiment, an MC4R chaperone is formulated for
parenteral administration. The chaperone may be formulated for
parenteral administration by injection, e.g., by bolus injection or
continuous infusion. Formulations for injection may be presented in
unit dosage form, e.g., in ampoules or in multi-dose containers,
with an added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0115] In addition to the formulations described previously, the
chaperone may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0116] In another embodiment, the chaperone can be delivered in a
vesicle, particularly a liposome.
[0117] In another embodiment, the chaperone can be delivered in a
controlled release manner. For example, a therapeutic agent can be
administered using intravenous infusion with a continuous pump, in
a polymer matrix such as poly-lactic/glutamic acid (PLGA), in a
pellet containing a mixture of cholesterol and the active
ingredient (SilasticR.TM.; Dow Coming, Midland, Mich.; see U.S.
Pat. No. 5,554,601), by subcutaneous implantation, or by
transdermal patch.
[0118] Combination Therapy. The pharmaceutical composition may also
include other biologically active substances in combination with
the candidate compound. Examples include but are not limited to
sibutramine, orlistat (Xenical.RTM.), leptin, neuropeptide Y,
cholecystokinin, or GLP-1.
Screening Assays for MC4R Pharmacological Chaperones
[0119] The present invention further provides a method for
identifying a candidate chaperone compound that modulates the
stability, activity, and/or cell surface localization of an MC4R
polypeptide. In one embodiment, the present invention provides a
method for identifying a chaperone for the MC4R protein, which
comprises bringing a labeled or unlabeled test compound in contact
with the MC4R protein or a fragment thereof and measuring the
amount of the test compound bound to the MC4R protein or to the
fragment thereof. This can be achieved for example as follows:
[0120] (a) contacting a first cell with a test compound for a time
period sufficient to allow the cell to respond to said contact with
the test compound;
[0121] (b) determining the conformational stability, activity,
and/or cell surface localization of a MC4R polypeptide (or a
fragment thereof comprising a ligand binding domain) in the cell
(or on the cell surface) contacted in step (a); and
[0122] (c) comparing the stability, activity, and/or cell surface
localization of the MC4R polypeptide determined in step (b) to that
of an MC4R polypeptide in a control cell that has not been
contacted with the test compound;
[0123] wherein a detectable change in the stability, activity,
and/or cell surface localization of the MC4R polypeptide in the
first cell in response to contact with the test compound compared
to the stability level of the MC4R polypeptide in the control cell
that has not been contacted with the test compound, indicates that
the test compound modulates the stability of the MC4R polypeptide
and is a candidate compound for the treatment of a disorder
associated with reduced MC4R stability or activity.
[0124] The cell can either be a host cell transformed with a
non-endogenous wild-type or mutant MC4R, or an
endogenously-MC4R-expressing cell, including mutant and wild-type
MC4Rs. Such cells include the "obesity neurons" such as GT1-7
cells, described above, those described in MacKenzie et al.,
Current Medicinal Chemistry--Immunology, Endocrine & Metabolic
Agents 2004; 4: 113-117, which endogenously express MC4R, or
transformed cells expressing normal or mutated, tagged MC4R such as
the HEK293 cells described in Blondet et al., J Biochem 2004; 135:
541-546 and below in the Examples.
[0125] In another embodiment, the present invention provides a
method for identifying a chaperone for the MC4R protein, which
comprises bringing a labeled test compound in contact with cells or
a cell membrane fraction containing the MC4R protein, and measuring
the amount of the labeled test compound bound to the cells or the
cell membrane fraction.
[0126] Numerous high-throughput screening (HTS) methods can be
employed to screen large numbers (e.g., hundreds, thousands, tens
of thousands) of test compounds simultaneously for binding to a
MC4R. A test compound can be, without limitation, a small organic
or inorganic molecule (preferred), a peptide or a polypeptide
(including an antibody, antibody fragment, or other immunospecific
molecule), an oligonucleotide molecule (such as an aptamer), a
polynucleotide molecule, or a chimera or derivative thereof. Test
compounds which are candidate chaperones that specifically bind to
an MC4R polypeptide can be identified using cell-based and/or
cell-free assays. Several methods of automated assays that have
been developed in recent years enable the screening of tens of
thousands of compounds in a short period of time (see, e.g., U.S.
Pat. Nos. 5,585,277, 5,679,582, and 6,020,141). For example, one
group reported the identification of one arylpiperazine MC4R
agonist through iterative directed screening of nonpeptidyl
G-protein-coupled receptor biased libraries (Richardson et al., J
Med Chem 2004; 47(3):744-55). Such HTS methods are particularly
useful, e.g., in microarrays.
[0127] For screening, purified classes of compounds that may be
identified include, but are not limited to, small molecules (i.e.,
organic or inorganic molecules which are less than about 2
kilodaltons (kD) in molecular weight, and, more preferably, less
than about 1 kD in molecular weight). These are components of
compound libraries.
[0128] As used herein, the term "lead compound" refers to a
molecular entity selected from a primary screen of MC4R antagonists
or agonists which may be effective on its own in stabilizing
protein conformation of wild-type or mutant MC4R protein, or which
may be modified by further development to generate an appropriate
pharmaceutical compound.
[0129] Compound libraries. Libraries of high-purity small organic
ligands and peptide agonists that have well-documented
pharmacological activities are available from Sigma-Aldrich (LOPAC
LIBRARY.TM. and LIGAND-SETS.TM.). Also available from Sigma-Aldrich
is an Aldrich Library of Rare Chemicals, which is a diverse library
of more than 100,000 small-molecule compounds, including plant
extracts and microbial culture extracts. Other compound libraries
are available from Tripos (LeadQuest.RTM.) and TimTech (including
targeted libraries for kinase modulators).
[0130] Other companies that supply or have supplied compound
libraries of the type suitable for screening according to the
invention include the following: 3-Dimensional Pharmaceuticals,
Inc.; Advanced ChemTech; Abinitio PharmaSciences; Albany Molecular;
Aramed Inc.; Annovis, Inc. (formerly Bearsden Bio, Inc.); ASINEX;
AVANT Immunotherapeutics; AXYS Pharmaceuticals; Bachem; Bentley
Pharmaceuticals; Bicoll Group; Biofor Inc.; BioProspect Australia
Limited; Biosepra Inc.; Cadus Pharmaceutical Corp.; Cambridge
Research Biochemicals; Cetek Corporation; Charybdis Technologies,
Inc.; ChemBridge Corporation; ChemDiv, Inc.; ChemGenics
Pharmaceuticals Inc.; ChemOvation Ltd.; ChemStar, Ltd.; Chrysalon;
ComGenex, Inc.; Compugen Inc.; Cytokinetics; Dextra Laboratories
Ltd.; Discovery Partners International Inc.; Discovery Technologies
Ltd.; Diversa Corporation; Dovetail Technologies, Inc.; Drug
Discovery Ltd.; ECM Pharma; Galilaeus Oy; Janssen Pharmaceutica;
Jerini Bio Tools; J-Star Research; KOSAN Biosciences, Inc.; KP
Pharmaceutical Technology, Inc.; Lexicon Genetics Inc.; Libris
Discovery; MicroBotanica, Inc.; MicroChemistry Ltd.; MicroSource
Discovery Systems, Inc.; Midwest Bio-tech Inc.; Molecular Design
& Discovery; MorphoSys AG; Nanosyn, Inc.; Ontogen Corporation;
Organix, Inc.; Pharmacopeia, Inc.; Pherin Pharmaceuticals; Phytera,
Inc.; PTRL East, Inc.; REPLICor Inc.; RSP Amino Acid Analogues,
Inc.; Sanofi-Synthelab (now Sanofi-Aventis) Pharmaceuticals;
Sequitur, Inc.; Signature BioScience Inc.; Spectrum Info Ltd.;
Talon Cheminformatics Inc.; Telik, Inc.; Tera Biotechnology
Corporation; Tocris Cookson; Torrey Pines Institute for Molecular
Studies; Trega Biosciences, Inc.; and WorldMolecules/MMD.
[0131] In addition, the Institute of Chemistry and Cell Biology
(ICCB), maintained by Harvard Medical School, provides the
following chemical libraries, including natural product libraries,
for screening: Chem Bridge DiverSet E (16,320 compounds); Bionet I
(4,800 compounds); CEREP (4,800 compounds); Maybridge 1 (8,800
compounds); Maybridge 2 (704 compounds); Peakdale 1 (2,816
compounds); Peakdale 2 (352 compounds); ChemDiv Combilab and
International (28,864 compounds); Mixed Commercial Plate 1 (352
compounds); Mixed Commercial Plate 2 (320 compounds); Mixed
Commercial Plate 3 (251 compounds); Mixed Commercial Plate 4 (331
compounds); ChemBridge Microformat (50,000 compounds); Commercial
Diversity Set 1 (5,056 compounds); NCI Collections: Structural
Diversity Set, version 2 (1,900 compounds); Mechanistic Diversity
Set (879 compounds); Open Collection 1 (90,000 compounds); Open
Collection 2 (10,240 compounds); Known Bioactives Collections:
NINDS Custom Collection (1,040 compounds); ICCB Bioactives 1 (489
compounds); SpecPlus Collection (960 compounds); ICCB Discretes
Collections. The following ICCB compounds were collected
individually from chemists at the ICCB, Harvard, and other
collaborating institutions: ICCB1 (190 compounds); ICCB2 (352
compounds); ICCB3 (352 compounds); ICCB4 (352 compounds). Natural
Product Extracts: NCI Marine Extracts (352 wells); Organic
fractions--NCI Plant and Fungal Extracts (1,408 wells); Philippines
Plant Extracts 1 (200 wells); ICCB-ICG Diversity Oriented Synthesis
(DOS) Collections; DDS1 (DOS Diversity Set) (9600 wells).
[0132] There are numerous techniques available for creating more
focused compound libraries rather than large, diverse ones.
Chemical Computing Group, Inc. (Montreal) has developed software
with a new approach to high-throughput drug design. The company's
method uses high-throughput screening (HTS) experimental data to
create a probabilistic QSAR (Quantitative Structure Activity
Relationship) model, which is subsequently used to select building
blocks in a virtual combinatorial library. It is based on
statistical estimation instead of the standard regression
analysis.
[0133] In addition, ArQule, Inc. (Woburn, Mass.) also has
integrated technologies to perform high-throughput, automated
production of chemical compounds and to deliver these compounds of
known structure and high purity in sufficient quantities for lead
optimization. Its AMAP.TM. (Automated Molecular Assembly Plant)
performs high-throughput chemical syntheses for each phase of
compound discovery.
[0134] Similarly compounds are often provided on online databases
or on CD-ROM's for selective "cherry picking" of compounds. See,
e.g., AbInitio PharmaSciences; ActiMol; Aral Biosynthetics; ASDI
Biosciences; Biotechnology Corporation of America; Chembridge;
ChemDiv; Florida Center--Heterocyclic Compounds; Microsource/MSDI;
NorthStar; Peakdale; Texas Retaining Group; Zelinsky Institute;
Advanced ChemTech; Ambinter; AnalytiCon Discovery; Aurora Fine
Chemicals; Biofocus; Bionet/Key; Comgenex; Key Organics; LaboTest;
Polyphor; SPECS and Biospecs; and Bharavi Laboratories.
Microarrays
[0135] In one embodiment, HTS screening for MC4R chaperones employs
microarrays.
[0136] Protein arrays. Protein arrays are solid-phase, binding
assay systems using immobilized proteins on various surfaces that
are selected for example from glass, membranes, microtiter wells,
mass spectrometer plates, and beads or other particles. The binding
assays using these arrays are highly parallel and often
miniaturized. Their advantages are that they are rapid, can be
automated, are capable of high sensitivity, are economical in their
use of reagents, and provide an abundance of data from a single
experiment.
[0137] Automated multi-well formats are the best-developed HTS
systems. Automated 96- or 384-well plate-based screening systems
are the most widely used. The current trend in plate-based
screening systems is to reduce the volume of the reaction wells
even further, and increase the density of the wells per plate (96
wells to 384 wells to 1,536 wells per plate). The trend results in
increased throughput, dramatically decreased bioreagent costs per
compound screened, and a decrease in the number of plates that need
to be managed by automation. For a description of protein arrays
that can be used for HTS, see e.g.: U.S. Pat. Nos. 6,475,809;
6,406,921; and 6,197,599; and International Publication Nos. WO
00/04389 and WO 00/07024.
[0138] For construction of arrays, sources of MC4Rs or fragments
thereof, whether in wild-type or mutant form, can include
cell-based expression systems for recombinant proteins,
purification from natural sources, production in vitro by cell-free
translation systems, and synthetic methods for making MC4R
peptides. For capture arrays and protein function analysis, it is
often the case that MC4R polypeptides are correctly folded and
functional. This is not always the case, e.g., where recombinant
proteins are extracted from bacteria under denaturing conditions;
other methods (isolation of natural proteins, cell free synthesis)
generally retain functionality. However, arrays of denatured
proteins can still be useful in screening chaperones since the
chaperone will likely bind to the mutated protein while it is not
folded into its proper conformation.
[0139] The immobilization method used is preferably applicable to
MC4R polypeptides of different properties (e.g., wild-type, mutant,
full-length, partial-length fragments, hydrophilic, hydrophobic,
etc.), amenable to high throughput and automation, and generally
compatible with retention of chaperone-binding ability. Both
covalent or non-covalent methods of MC4R protein immobilization can
be used. Substrates for covalent attachment include, e.g., glass
slides coated with amino- or aldehyde containing silane reagents
(Telechem). In the Versalinx.TM. system (Prolinx), reversible
covalent coupling is achieved by interaction between the protein
derivatized with phenyldiboronic acid, and salicylhydroxamic acid
immobilized on the support surface. Covalent coupling methods
providing a stable linkage can be applied to a range of proteins.
Non-covalent binding of unmodified protein occurs within porous
structures such as HydroGel.TM. (PerkinElmer), based on a
3-dimensional polyacrylamide gel.
[0140] Cell-Based Arrays. Cell-based arrays combine the technique
of cell culture in conjunction with the use of fluidic devices for
measurement of cell response to test compounds in a sample of
interest, screening of samples for identifying molecules that
induce a desired effect in cultured cells, and selection and
identification of cell populations with novel and desired
characteristics. High-throughput screening (HTS) can be performed
on fixed cells using fluorescent-labeled antibodies, biological
ligands or candidate chaperones and/or nucleic acid hybridization
probes, or on live cells using multicolor fluorescent indicators
and biosensors. The choice of fixed or live cell screens depends on
the specific cell-based assay required.
[0141] There are numerous single- and multi-cell-based array
techniques known in the art. Recently-developed techniques such as
micro-patterned arrays (described, e.g., in International PCT
Publications WO 97/45730 and WO 98/38490) and microfluidic arrays
provide valuable tools for comparative cell-based analysis.
Transfected cell microarrays are a complementary technique in which
array features comprise clusters of cells overexpressing defined
cDNAs. Complementary DNAs cloned in expression vectors are printed
on microscope slides, which become living arrays after the addition
of a lipid transfection reagent and adherent mammalian cells
(Bailey et al., Drug Discov. Today 2002; 7(18 Suppl): S113-8).
Cell-based arrays are described in detail in, e.g., Beske, Drug
Discov. Today 2002; 7(18 Suppl): S131-5; Sundberg et al., Curr.
Opin. Biotechnol. 2000; 11: 47-53; Johnston et al., Drug Discov.
Today 2002; 7: 353-63; U.S. Pat. Nos. 6,406,840 and 6,103,479, and
U.S. published patent application no. 2002/0197656. For cell-based
assays specifically used to screen for modulators of ligand-gated
ion channels, see Mattheakis et al., Curr. Opin. Drug Discov.
Devel. 2001; 1: 124-34; and Baxter et al., J. Biomol. Screen. 2002;
7: 79-85.
[0142] Detectable labels. For detection of molecules such as
candidate MC4R chaperones using screening assays, a functional
assay can be used to follow unlabeled molecules as described
elsewhere herein. A molecule-of-interest (e.g., a small molecule,
an antibody, or a polynucleotide probe) or a library of same can
also be detectably labeled with an atom (e.g., a radionuclide), a
detectable molecule (e.g., fluorescein), or a complex that, due to
a physical or chemical property, serves to indicate the presence of
the molecule of interest. A molecule can also be detectably labeled
when it is covalently bound to a "reporter" molecule (e.g., a
biomolecule such as an enzyme) that acts on a substrate to produce
a detectable product. Detectable labels suitable for use in the
present invention include any composition detectable by
spectroscopic, photochemical, biochemical, immunochemical,
electrical, optical or chemical means. Labels useful in the present
invention include, but are not limited to, biotin for staining with
labeled avidin or streptavidin conjugate, magnetic beads (e.g.,
Dynabeads.TM.), fluorescent dyes (e.g., fluorescein,
fluorescein-isothiocyanate (FITC), Texas red, rhodamine, green
fluorescent protein, enhanced green fluorescent protein, lissamine,
phycoerythrin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX from
Amersham, SyBR Green I & II from Molecular Probes, and the
like), radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S, .sup.14C,
or .sup.32P), enzymes (e.g., hydrolases, particularly phosphatases
such as alkaline phosphatase, esterases and glycosidases, or
oxidoreductases, particularly peroxidases such as horse radish
peroxidase, and the like), substrates, cofactors, inhibitors,
chemiluminescent groups, chromogenic agents, and colorimetric
labels such as colloidal gold or colored glass or plastic (e.g.,
polystyrene, polypropylene, latex, etc.) beads. Examples of patents
describing the use of such labels include U.S. Pat. Nos.:
3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149;
and 4,366,241.
[0143] Means of detecting such labels are known to those of skill
in the art. For example, radiolabels and chemiluminescent labels
can be detected using photographic film or scintillation counters;
fluorescent markers can be detected using a photo-detector to
detect emitted light (e.g., as in fluorescence-activated cell
sorting, FACS); and enzymatic labels can be detected by providing
the enzyme with a substrate and detecting, e.g., a colored reaction
product produced by the action of the enzyme on the substrate.
Stability, Localization and Activity Assays
[0144] As indicated previously, enhanced stability of MC4R can be
determined by measuring an increase in cellular MC4R polypeptide,
by determining an increase in trafficking to the cell surface,
e.g., as determined by increased cell surface expression, or by
determining increased MC4R activity. Non-limiting exemplary methods
for assessing each of the foregoing are described below.
[0145] Determining MC4R intracellular stability. Methods for
determining intracellular MC4R protein levels are well-known in the
art. Such methods include Western blotting, immunoprecipitation
followed by Western blotting (IP Western), or immunofluorescence
using a tagged MC4R protein.
[0146] Determining MC4R trafficking. Assessing trafficking of
proteins through the biosynthetic pathway can be achieved e.g.,
using pulse-chase experiments with .sup.35S-labeled receptor
protein, in conjunction with glycosidases; or by indirect or direct
immunofluorescence to determine protein modification during
trafficking. These and other methods are described for example in
Current Protocols in Cell Biology 2001; John Wiley & Sons.
[0147] Methods for detecting impaired trafficking of proteins are
well known in the art. For example, for proteins which are N-
and/or O-glycosylated in the Golgi apparatus, pulse-chase metabolic
labeling using radioactively labeled proteins, combined with
glycosidase treatment and immunoprecipitation, can be used to
detect whether the proteins are undergoing full glycosylation in
the Golgi, or whether they are being retained in the ER instead of
trafficking to the Golgi for further glycosylation.
[0148] Sensitive methods for visually detecting cellular
localization also include fluorescent microscopy using fluorescent
proteins or fluorescent antibodies. For example, MC4R proteins of
interest can be tagged with e.g., green fluorescent protein (GFP),
cyan fluorescent protein, yellow fluorescent protein, and red
fluorescent protein, followed by multicolor and time-lapse
microscopy and electron microscopy to study the fate of these
proteins in fixed cells and in living cells. For a review of the
use of fluorescent imaging in protein trafficking, see Watson et
al., Adv Drug Deliv Rev 2005; 57(1):43-61. For a description of the
use of confocal microscopy for intracellular co-localization of
proteins, see Miyashita et al., Methods Mol Biol. 2004;
261:399-410.
[0149] Fluorescence correlation spectroscopy (FCS) is an
ultrasensitive and non-invasive detection method capable of
single-molecule and real-time resolution (Vukojevic et al., Cell
Mol Life Sci 2005; 62(5): 535-50). SPFI (single-particle
fluorescence imaging) uses the high sensitivity of fluorescence to
visualize individual molecules that have been selectively labeled
with small fluorescent particles (Cherry et al., Biochem Soc Trans
2003; 31(Pt 5): 1028-31). For localization of proteins within lipid
rafts, see Latif et al., Endocrinology 2003; 144(11): 4725-8). For
a review of live cell imaging, see Hariguchi, Cell Struct Funct
2002; 27(5):333-4).
[0150] Fluorescence resonance energy transfer (FRET) microscopy is
also used to study the structure and localization of proteins under
physiological conditions (Periasamy, J Biomed Opt 2001; 6(3):
287-91).
[0151] For plasma membrane resident proteins, less sensitive assays
can be used to detect whether they are present on the membrane.
Such methods include immunohisto-chemistry of fixed cells, or
whole-cell labeling using radiolabeled ligand (e.g.,
.sup.125I).
[0152] Determining MC4R cell surface expression. Once a candidate
compound has been identified, the next step is determining whether
the candidate compound can enhance the amount of MC4R trafficked to
the cell surface. Numerous assays can be used to evaluate cell
surface receptor expression quantitatively. For example,
radioactive ligand binding assays, using e.g., .sup.125I-MSH, can
be used to determine binding to either whole cells expressing MC4R
or to cell membrane fractions. See U.S. published application
2003/0176425 for a description of one exemplary method; see also
Chhajlani, Peptides. 1996; 17(2):349-51. In addition,
immunofluorescence staining, using either labeled antibodies or
labeled MC4R (e.g., FLAG-tagged MC4R), may also be used. Another
well-known method is fluorescence-activated cell sorting (FACS),
which sorts or distinguishes populations of cells using labeled
antibodies against cell surface markers. See also, Nijenhuis et
al., supra.
[0153] Determining an Increase in MC4R Activity. MC4R activity can
be determined using, e.g., cAMP activation/accumulation assays (see
e.g., VanLeeuwen et al., J Biol Chem 2003; 18: 15935-40) or by
measuring an increase in transcription of one or more genes
activated by cAMP, or by measuring reporter gene expression by
operatively linking a reporter gene such as luciferase to a cAMP
response element (CRE) (see e.g., Lee et al., Eur J Biochem 2001;
268(3):582-91). In addition, it is also known that MC4R stimulates
TNF-.alpha. secretion in melanophores. Therefore, MC4R activity in
response to a candidate compound can be evaluated by measuring
TNF-.alpha. secretion (see e.g., Ignar et al., Peptides 2003 May;
24(5):709-16).
[0154] Lastly, melanophores provide a rapid and sensitive bioassay
for melanocortin agonists and antagonists. This method is based on
the measurement of pigment granule dispersion induced by
.alpha.-MSH, as determined by changes in optical density (Quillan
et al., PNAS U.S.A. 1995; 92: 2894; and Potenza et al., Pigment
Cell Res 1992; 5: 372).
Molecular Biology Definitions
[0155] In accordance with the present invention there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. These
techniques are generally useful for the production of recombinant
cells expressing wild-type or mutant MC4R's for use in screening
assays. Such techniques are explained fully in the literature. See,
e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A
Laboratory Manual, Second Edition (1989) Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook et
al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II
(D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed.
1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins
eds. (1985)); Transcription And Translation (B. D. Hames & S.
J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed.
(1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B.
Perbal, A Practical Guide To Molecular Cloning (1984); F. M.
Ausubel et al. (eds.), Current Protocols in Molecular Biology, John
Wiley & Sons, Inc. (1994); and later editions of each, where
available.
[0156] The term "host cell" means any cell of any organism that is
selected, modified, transformed, grown, used, or manipulated in any
way, for the production of a desired substance by the cell, for
example the expression by the cell of a gene, a DNA or RNA
sequence, a protein, or an enzyme. According to the present
invention, the host cell is modified to express a mutant or
wild-type MC4R nucleic acid and polypeptide. Host cells can further
be used for screening or other assays. Exemplary host cells for use
in the present invention are HEK293 cells, COS cells, and CHO
cells.
[0157] A "recombinant DNA molecule" is a DNA molecule that has
undergone a molecular biological manipulation.
[0158] The MC4R polynucleotides herein may be flanked by natural
regulatory (expression control) sequences, or may be associated
with heterologous sequences, including promoters, internal ribosome
entry sites (IRES) and other ribosome binding site sequences,
enhancers, response elements, suppressors, signal sequences,
polyadenylation sequences, introns, 5'- and 3'-non-coding regions,
and the like. The nucleic acids may also be modified by many means
known in the art. Non-limiting examples of such modifications
include: methylation, "caps," substitution of one or more of the
naturally occurring nucleotides with an analog, and internucleotide
modifications such as, for example, those with uncharged linkages
(e.g., methyl phosphonates, phosphotriesters, phosphoroamidates,
carbamates, etc.) and with charged linkages (e.g.,
phosphorothioates, phosphorodithioates, etc.). Polynucleotides may
contain one or more additional covalently linked moieties, such as,
for example, proteins (e.g., nucleases, toxins, antibodies, signal
peptides, poly-L-lysine, etc.), intercalators (e.g., acridine,
psoralen, etc.), chelators (e.g., metals, radioactive metals, iron,
oxidative metals, etc.), and alkylators. The polynucleotides may be
derivatized by formation of a methyl or ethyl phosphotriester or an
alkyl phosphoramidate linkage. Furthermore, the polynucleotides
herein may also be modified with a label capable of providing a
detectable signal, either directly or indirectly. Exemplary labels
include radioisotopes, fluorescent molecules, biotin, and the like.
The nucleic acids may also be altered at one or more bases by e.g.,
site-directed mutagenesis to facilitate molecular biology
associated with use of the molecules.
[0159] A "coding sequence" or a sequence "encoding" an expression
product, such as an MC4R RNA or polypeptide, is a nucleotide
sequence that, when expressed, results in the production of that
RNA or polypeptide, e.g., the MC4R nucleotide sequence encodes an
amino acid sequence for an MC4R polypeptide (protein). A coding
sequence for the protein may include a start codon (usually ATG)
and a stop codon.
[0160] The term "gene," also called a "structural gene" means a DNA
sequence that codes for or corresponds to a particular sequence of
amino acids which comprise all or part of one or more MC4R
proteins, and may or may not include regulatory DNA sequences, such
as promoter sequences, which determine for example the conditions
under which the MC4R gene is expressed.
[0161] The terms "express" and "expression," when used in the
context of producing an amino acid sequence from a nucleic acid
sequence, means allowing or causing the information in a MC4R gene
or DNA sequence to become manifest, for example producing an MC4R
protein by activating the cellular functions involved in
transcription and translation of the corresponding MC4R gene or DNA
sequence. A DNA sequence is expressed in or by a cell to form an
"expression product" such as an MC4R protein. The expression
product itself, e.g., the resulting protein, may also be said to be
"expressed" by the cell. An expression product can be characterized
as intracellular, extracellular or secreted. According to the
present invention, MC4R is expressed at the cell surface of
neurons.
[0162] The term "intracellular" means something that is inside a
cell. The term "extracellular" means something that is outside a
cell. A substance is "secreted" by a cell if it appears in
significant measure outside the cell, from somewhere on or inside
the cell.
[0163] The term "heterologous" refers to a combination of elements
not naturally occurring in combination. For example, heterologous
DNA refers to DNA not naturally located in the cell, or in a
chromosomal site of the cell. Preferably, the heterologous DNA
includes a gene foreign to the cell. A heterologous expression
regulatory element is an element operatively associated with a
different gene than the one it is operatively associated with in
nature. In the context of the present invention, a gene encoding a
protein of interest is heterologous to the vector DNA in which it
is inserted for cloning or expression, and it is heterologous to a
host cell containing such a vector, in which it is expressed, e.g.,
an E. coli cell.
[0164] The term "transformation" refers to the process by which
DNA, i.e., a nucleic acid encoding an MC4R polypeptide, is
introduced from the surrounding medium into a host cell.
[0165] The term "transduction" refers to the introduction of DNA,
i.e., a nucleic acid encoding an MC4R polypeptide, into a
prokaryotic host cell, e.g., into a prokaryotic host cell via a
bacterial virus, or bacteriophage. A prokaryotic or eukaryotic host
cell that receives and expresses introduced DNA or RNA has been
"transformed" or "transduced" and is a "transformant" or a "clone."
The DNA or RNA introduced into a host cell can come from any
source, including cells of the same genus or species as the host
cell, or cells of a different genus or species, or synthetic
sequences.
[0166] The term "recombinantly engineered cell" refers to any
prokaryotic or eukaryotic cell that has been manipulated to express
or overexpress the nucleic acid of interest, i.e., a nucleic acid
encoding an MC4R polypeptide, by any appropriate method, including
transfection, transformation or transduction. This term also
includes endogenous activation of a nucleic acid in a cell that
does not normally express that gene product or that expresses the
gene product at a sub-optimal level.
[0167] The term "transfection" means the introduction of a foreign
(i.e., extrinsic or extracellular) nucleic acid into a cell. The
"foreign" nucleic acid includes a gene, DNA or RNA sequence to a
host cell, so that the host cell will replicate the DNA and express
the introduced gene or sequence to produce a desired substance,
typically a protein or enzyme coded by the introduced gene or
sequence. The introduced gene, i.e., a nucleic acid encoding an
MC4R polypeptide, or sequence may also be called a "cloned" gene or
sequence, may include regulatory or control sequences, such as
start, stop, promoter, signal, secretion, or other sequences used
by a cell's genetic machinery. The gene or sequence may include
nonfunctional sequences or sequences with no known function. DNA
may be introduced either as an extrachromosomal element or by
chromosomal integration or a host cell that receives and expresses
introduced DNA or RNA.
[0168] Depending on the host cell used, transformation or
transfection is done using standard techniques appropriate to such
cells. The calcium treatment employing calcium chloride, as
described in section 1.82 of Sambrook et al., 1989 supra, is
generally used for bacterial cells that contain substantial
cell-wall barriers. Another method for transformation employs
polyethylene glycol/DMSO, as described in Chung and Miller (Nucleic
Acids Res. 1988, 16:3580). Yet another method is the use of the
technique termed electroporation. Alternatively, where a viral
vector is used, the host cells can be infected by the virus
containing the gene of interest.
[0169] The terms "vector," "cloning vector" and "expression vector"
mean the vehicle by which a DNA or RNA sequence (e.g., an MC4R
gene) can be introduced into a host cell, so as to transform the
host and promote expression (e.g., transcription and translation)
of the introduced sequence. Vectors include plasmids, phages,
viruses, etc.; they are well known in the art.
[0170] A "promoter sequence" is a DNA regulatory region capable of
binding RNA polymerase in a cell and initiating transcription of a
downstream (3' direction) coding sequence. For purposes of defining
the present invention, the promoter sequence is bounded at its 3'
terminus by the transcription initiation site and extends upstream
(5' direction) to include the minimum number of bases or elements
necessary to initiate transcription at levels detectable above
background. Within the promoter sequence will be found a
transcription initiation site, as well as protein binding domains
(consensus sequences) responsible for the binding of RNA
polymerase.
[0171] A coding sequence is "under the control of" or "operatively
associated with" transcriptional and translational control
sequences in a cell when RNA polymerase transcribes the coding
sequence into mRNA, which is then trans-RNA spliced (if it contains
introns) and translated into the protein encoded by the coding
sequence.
[0172] Construction of suitable vectors containing one or more of
the above listed components employs standard ligation techniques.
Isolated plasmids or DNA fragments are cleaved, tailored, and
religated in the form desired to generate the plasmids
required.
[0173] For analysis to confirm correct sequences in plasmids
constructed, the ligation mixtures are used to transform bacterial
strains, and successful transformants are selected by ampicillin or
tetracycline resistance where appropriate. Plasmids from the
transformants are prepared, analyzed by restriction endonuclease
digestion, and/or sequenced by the method of Sanger et al. (Proc.
Natl. Acad. Sci. USA 1977, 74:5463-5467) or Messing et al. (Nucleic
Acids Res. 1981, 9:309), or by the method of Maxam et al. (Methods
in Enzymology 1980, 65:499). Host cells are transformed with the
above-described expression vectors of this invention and cultured
in conventional nutrient media modified as appropriate for the
promoter utilized.
EXAMPLES
[0174] The present invention is further described by means of the
examples, presented below. The use of such examples is illustrative
only and in no way limits the scope and meaning of the invention or
of any exemplified term. Likewise, the invention is not limited to
any particular preferred embodiment described herein. Indeed, many
modifications and variations of the invention will be apparent to
those skilled in the art upon reading this specification and can be
made without departing from its spirit and scope. The invention is
therefore to be limited only by the terms of the appended claims
along with the full scope of equivalents to which the claims are
entitled.
Example 1
Generation of Cell Lines Expressing MC4R Folding Mutants
[0175] In order to determine whether some MC4R mutants result in
conformational defects of MC4R, MC4R nucleic acids containing the
various mutants are transfected into HEK-293T and COS-7 cells and
their cell surface expression and activity evaluated. Those cell
lines in which reduced or absent cell surface expression is
observed are evaluated further to determine the intracellular
presence and/or location of the MC4R polypeptide.
Methods
[0176] Generation of MC4R Mutants. A cDNA encoding a wild-type MC4R
(e.g., SEQ ID NO: 1) is modified using known techniques in the art
(e.g., PCR, site-directed mutagenesis) to generate mutant MC4R
cDNAs containing alterations in the nucleotides which result in,
e.g., one of the following MC4R mutant polypeptides: P78L, R165Q,
R165W, 1125K, C271Y, T11A, A175T, I316L, I316S, I317T, N97D, G98R,
N62S, C271R, S58C, N62S, N97D, Y157S, I102S, L106P, L250Q, Y287X,
P299H, S58C, CTCT at codon 211, and/or TGAT insertion at codon 244.
Such mutants can also be fused to a fluorescent tag, such as GFP,
as described in Blondet et al. J Biochem (Tokyo) 2004;
135(4):541-6, or FLAG-tagged, as described by VanLeeuwen et al., J.
Biol. Chem. 2003; 278: 15935-15940, or tagged-with an enzyme such
as luciferase. .
[0177] Cell Culture and Transfection. Such mutant MC4R nucleic
acids are cloned into an appropriate expression vector, e.g.,
pCDNA3.1 (Invitrogen, Carlsbad, Calif.), according to the
manufacturer's instructions. Transfection of cells is accomplished
using LipofectAMINE (Invitrogen), and permanently transfected
clonal cell lines are selected by resistance to the neomycin analog
G418.
[0178] Briefly, HEK-293T and COS-7 cells are maintained in
Dulbecco's modified Eagle's medium (with glutamine; Invitrogen)
supplemented with 10% fetal bovine serum, 100 units/ml penicillin,
and 100 .mu.g/ml streptomycin (Invitrogen). Cells are incubated at
37.degree. C. in humidified air containing 5% CO2. Cells are
generally at 70-80% confluence on the day of transfection.
[0179] GFP Tagged MC4R. Green fluorescent protein (GFP) cDNA is
available from BD Biosciences (San Jose, Calif.) or Clontech (Palo
Alto, Calif.). GFP is fused in frame to the C terminus of human
MC4R with the C-terminal termination codon removed, according to
the manufacturer's instructions. The chimeric MC4R-GFP fusion
protein construct is then transfected as above. A luciferase
construct is similarly employed.
[0180] Detection of Localization of MC4R Mutants. Binding
experiments to determine cell surface localization are performed
using conditions described previously (Yang et al., J Biol Chem
1997; 272: 23000-23010). Briefly, 2.times.10.sup.5 cpm of
.sup.125I-NDP-MSH (Amersham Biosciences, Piscataway, N.J.) is used
in combination with non-radiolabeled ligands NDP-MSH, AgRP 87-132,
or AgRP 110-117. Binding reactions are terminated by removing the
media and washing the cells twice with minimal essential medium
containing 0.2% bovine serum albumin. The cells are then lysed with
0.2 N NaOH, and the radioactivity in the lysate is quantified in an
analytical-counter. Nonspecific binding is determined by measuring
the amount of .sup.125I label bound in the presence of 10.sup.-6 M
unlabeled ligand. Additional, FACS can be used as described below
in Example 4. Specific binding is calculated by subtracting
nonspecifically bound radioactivity from total bound radioactivity.
The maximum binding (Bmax) can be calculated using the equation
Bmax=[NDP-MSH specific binding]/([NDP-MSH]/(Kd+[NDP-MSH]).
Ki=IC50/1+ligand concentration / Kd.
[0181] Where the MC4R mutant is fluorescently tagged, confocal
microscopy is used to monitor intracellular trafficking of tagged
MC4R (Blondet et al., J Biochem 2004; 135: 541-546; or Gao et al.,
J Pharmacol Exp Ther 2003; 307(3):870-7). Briefly, cells are grown
in chamber coverglasses 24 to 48 h before the experiments. After
appropriate treatments, cells are washed with cold PBS and fixed in
formalin for 20 minutes, and observed on an LSM 510 META laser
scanning microscope (Carl Zeiss, Thornwood, N.Y.). Fluorescence of
GFP is excited using a 488-nm argon/krypton laser, detected with a
band pass filter of 500 to 550 nm. Red signal is excited with a
HeNe laser at 543 nm and fluorescence is detected with a 565 to 615
band pass filter.
[0182] The digitally-acquired images are quantitated using a Scion
Image Beta 4.02. The original green fluorescence confocal images
are converted to grayscale and median filtering is performed. Each
pixel is assigned an intensity value ranging from 0 (black) to 255
(white). The cell surface and total cellular fluorescence intensity
are measured after manually selecting the corresponding area. The
subcellular distribution of MC4R-GFP is expressed as a ratio of
cell surface fluorescence intensity to total cellular fluorescence
intensity. A decrease in the ratio indicates receptor
internalization.
[0183] Using these methods, folding mutants of MC4R are identified
that would be candidates for chaperone-mediated rescue.
Example 2
Structures of Agonists and Antagonists of MC4R
[0184] Potential agonists and antagonists of MC4R were selected
based on the review of published patent and literature references
(in particular, Bednarek and Fong, Exp. Opn. Therapeutic Patents
2004; 14(3): 327-326 and WO 02/062766). Criteria used in selecting
the compounds described herein included published IC.sub.50 data,
in vivo animal data, and bioavailability data (e.g.,
pharmacokinetics), where available.
[0185] a) Synthesis of the Agonist of FIG. 1 (compound 1)
[0186] Selection of this compound, known as THIQ, was based on the
following data: TABLE-US-00002 MC4R Activity IC.sub.50 EC.sub.50
E.sub.max Name (nM) (nM) (%) PK Reference(s) THIQ/ 1.2 2.5 97 % F
14 Van der Ploeg et al (2002) compound 1 V.sub.d 3.6 L/kg PNAS 99:
11381. Cl 84 mLmin/kg Sebhat et al (2002) J Med t.sub.1/2 0.6 h
Chem 45: 4589.
[0187] Synthesis of this molecule (11-steps) was performed based on
the scheme depicted in FIG. 3.
[0188] b) Synthesis of the Agonist of FIG. 2 (compound 2)
[0189] Selection of this known compound, referred to as compound 2,
was based on the following data: TABLE-US-00003 MC4R Activity
K.sub.i EC.sub.50 E.sub.max Name (Nm) (nM) (%) PK Reference(s)
Compound 2 24 39 ND ND Richardson et al (2004) J Med Chem 47:
744.
[0190] Synthesis of this molecule (11-steps) was performed based on
the scheme depicted in FIG. 4.
[0191] c) Synthesis of the Antagonist of FIG. 5 (compound 3)
[0192] This compound was selected based on the aMSH/MC4R data
reported by Arasasingham (J Med Chem 2003, 46: 9-11). Synthesis of
this compound was performed according to the method described
therein, summarized in FIG. 7.
[0193] d) Synthesis of the Antagonist of FIG. 6 (compound 4)
[0194] This compound was selected and synthesized based on the data
and synthetic method described in PCT International Patent
Publication WO 02/062766 to Millennium Pharmaceuticals; the
synthetic scheme is summarized in FIG. 8. The biological activity
of this compound is reported in WO 02/062766 using a scintillation
proximity assay.
[0195] Briefly, synthesis was achieved using the following
method:
[0196] 1,3-diaminopropane (Acros, 27.7 g, 0.374 mmol) is added to
thiosalicylic acid (Acros, 20 g, 0.130 mmol) in 1,2-dichlorobenzene
(Acros, 200 ml), this mixture is heated to 170.degree. C. for 4
hours. On cooling to 60.degree. C., methanol (50 ml) is added, the
reaction stirs at room temperature overnight and the resulting
yellow crystalline solid collected and washed with ether to give 9
g of pure product.
[0197] 2-Methoxy-5-nitrobenzyl bromide (Fluka, 1.86 g, 7.559 mmol)
is added to 2-(1,4,5,6-tetrahydropyrimidin-2-yl)benzenethiol (1 g,
5.201 mmol) in methanol (60 ml) at room temperature, maintained for
12 hours concentrated and ether (10 ml) added, the resulting light
yellow needles are collected and washed with ether to give 1.28 g
of pure product.
Example 3
Rescue of Misfolded MC4R Using Low Temperature or Chemical
Chaperones
[0198] Since both low temperatures (thermal rescue) and general
chemical chaperones such as DMSO are known to restore folding of
mutant proteins, cell surface expression of wild-type and various
MC4R folding mutants was evaluated in cells harboring WT and mutant
MC4R, and in those cells cultured at 30.degree. C. or with 1%
DMSO.
Methods
[0199] Cells and Transfections. HEK 293 cells were transiently
transfected with wild-type (WT) or the following hMC4R mutants
double tagged with 3HA and Venus (Enhanced Yellow Green Fluorescent
Protein; EYFP): S58C; N62S; R165W; R165Q, and P299H.
[0200] Low Temperature Assay. WT and transfected cells were
incubated at 30.degree. C. or 37.degree. C. for 12 hours prior to
evaluation of MC4R cell surface expression. The gain was determined
using the following: Gain=[(% of surface expression at 30.degree.
C.-% of surface expression at 37.degree. C.)/% of surface
expression at 37.degree. C.]*100.
[0201] Chemical Chaperone Assay: WT and transiently transfected
cells were incubated in the presence or absence of 1% DMSO for 12
hours prior to evaluation of MC4R cells surface expression.
[0202] FACS Analysis. FACS analysis was performed after
fluorescently labeling cells with primary anti-HA.11 antibody
(mouse; 1;1000 dilution) and the secondary antibody coupled to
Alexa 647 (goat anti-mouse; 1:1000 dilution) dilution. Live cells
(propidium iodide negative) were sorted into the following two
relevant populations: [0203] P4: Total YFP positive cells
(representative of total MC4R expression) [0204] P5: Percent of
cells positive for both YFP and Alexa 647 (Alexa and YFP positive
cells)/(YFP positive cells+Alexa and YFP positive cells)
(representative of cell surface expression)
Results
[0205] MC4R Cell Surface Expression. Basal surface expression of WT
cell surface expression reaches 90%. None of the mutants expressed
MC4R on the cell surface as detected by binding of
.sup.125I-labeled NDP-.alpha.-MSH (data not shown). When analyzed
by FACS, the mutants all exhibited significantly decreased surface
expression when compared to cells transfected with WT MC4R (data
not shown). About 90% of the WT 3HA-hMC4R-Venus cells exhibited
surface expression of MC4R (P5), whereas basal surface expression
on the mutants was between 12% and 18% for N62S, R165W, R165Q and
P299H. S58C exhibits about 40% surface expression.
[0206] Thermal Rescue. All five mutants exhibited a gain in cell
surface expression of MC4R (P5) in Table 2 as follows:
TABLE-US-00004 TABLE 2 Genotype % Gain WT 12 S585C 43 N62S 68 R165W
63 R165Q 93 P299H 107
[0207] Chemical chaperone: No significant enhancement of cell
surface expression was detected in the presence of 1% DMSO.
Example 4
Rescue of Misfolded MC4R Using MC4R Pharmacological Chaperones
[0208] The MC4R antagonist compounds depicted in FIG. 5 (compound
3) and FIG. 6 (HBr salt; compound 4), respectively were evaluated
for chaperone activity in cells harboring the following MC4R
mutants: S58C; N62S; R165Q; R165W; and P299H.
Methods
[0209] Pharmacological Chaperone Assay. Briefly, cells harboring
each of the above-referenced MC4R mutants (transfected as described
in Example 3) were cultured with each of the antagonist compounds
at concentrations of 1.0 .mu.M and 10 .mu.M for 12 h and evaluated
for cell surface expression of MC4R using fluorescent activated
cell sorting analysis as described above. Cell surface expression
levels are compared between treatment and basal conditions. Percent
Gain is determined according to the following: [0210] [Gain=[(% of
surface expression (with treatment)-% of surface expression (w/o
treatment))/% of surface expression (w/o treatment)]*100.
[0211] MC4R Activity Assay. Accumulation of cAMP was evaluated for
MC4R mutants S58C, N62S, R165W and P299H in response to treatment
with MC4R agonist NDP-MSH (10-7M) in the presence or absence of
each antagonist using the Catch Point cAMP Fluorescent Assay Kit
from Molecular Devices (whole cell assay; Cat. No. R8044). Controls
were untreated or treated only with NDP-MSH.
Results
[0212] Pharmacological Chaperone Rescue. As shown in Table 3,
below, both antagonists were able to increase surface expression of
MC4R mutants R165W, S58C, and R165Q at both 1.0 .mu.M and 10 .mu.M
concentrations, with a dose-dependent effect. As indicated above,
basal surface expression on the mutants was between 12% and 18% for
N62S, R165W, R165Q and P299H. S58C exhibits about 40% surface
expression. Unexpectedly, treatment with 10 .mu.M of each compound
was able to restore cell surface expression of MC4R R 165W to
levels identical to cells expressing wild-type MC4R.
[0213] For mutant N62S, no effect was seen at 1.0 .mu.M with either
of the compounds, although a significant percent gain in cell
surface expression of the mutant MC4R was observed with both
compounds at the 10 .mu.M concentration. The compound depicted in
FIG. 5 was more potent, i.e., more cell surface expression was
observed than with the compound shown in FIG. 6.
[0214] For mutant P299H, the compound shown in FIG. 6 had no effect
on restoring cell surface expression of the mutant receptor at
either 1.0 .mu.M or 10 .mu.M, and only a small effect was observed
with the compound shown in FIG. 5 at 10 .mu.M. TABLE-US-00005 TABLE
3 Compound/ % Gain concentration S58C N62S R165W R165Q P299H 4/10
.mu.M 130 212.5 675 508 38.9 4/1 .mu.M 60 6.25 150 91.7 11.11 3/10
.mu.M 130 343.75 675 508 77.78 3/1 .mu.M 77.5 25 467 300 27.78
[0215] These results demonstrate that MC4R folding mutants can be
"rescued" when contacted with low concentrations of MC4R
antagonists, which act as pharmacological chaperones to restore
cell surface expression.
[0216] Lastly, a bisaminothiazole compound described in Pedemonte
et al., J. Clin. Inves. 2005; 115: 2564-71 (FIG. 9) demonstrated a
small effect on mutants S58C (31.25% gain) and R165W (28.95%
gain).
[0217] MC4R Activity. As shown in FIG. 11, recovery of signaling
through MC4R was observed to the same extent as hMC4R WT in S58C
and R165W following treatment with both antagonists at 10 .mu.M.
Signaling capacity in MC4R mutant N62S also was restored to a
lesser extent. No signaling was restored in P299H, as expected,
since this mutant since this mutation is in a domain necessary for
G-protein coupling.
Example 5
Screening for MC4R Chaperones that Restore Stability and Activity
of Mutant MC4R or Increase Stability of Wild-type MC4R
[0218] This example describes a method for screening for MC4R
chaperone compounds for enhancing misfolded mutant or wild-type
cell surface expression and/or activity of MC4R.
Methods
[0219] Transfection of Mutant or Wild-type MC4R. Identification of
folding/trafficking MC4R mutants is achieved as described in
Example 1. Transfection of such folding mutants and/or wild-type
MC4R is achieved as described above, or by using any methods known
in the art for detecting cell surface expression of proteins,
including immunoassays such as ELISA, and FACS analysis.
[0220] Chaperone Administration. To cultures or arrays of MC4R
wild-type-expressing or folding-mutant-expressing cells are added
various concentrations of chaperone test compounds. The cellular
localization of MC4R is then determined, in addition to the
activity of the MC4R that is trafficked to the cell surface. For
example, piperazine-, piperidine-, 1,4-diazapane-, guanidine-based,
or other test compounds as described e.g., in the following are
screened: Bednarek and Fong, Exp Opn Ther Patents 2004; 14: 327-36;
Ujjainwalla et al., Bioorg. Med. Chem. Lett. 2003; 13: 4431-4435;
WO03/07949; WO03/61660; WO03/09847; WO03/09850; WO03/31410;
WO03/94918; WO03/68738; WO03/92690; WO03/93234; WO03/72056;
WO03/66597; WO03/66587; WO03/53927; WO02/67869; WO02/68387;
WO03/68738; WO02/00259; WO02/92566; WO02/81443; WO02/81430; and
WO02/80896.
[0221] Detection of Trafficking of MC4R to the Cell Surface.
Detection of cellular and/or cell surface localization of
compound-treated MC4R-expressing cells compared to untreated cells
is achieved as described above.
[0222] Detection of MC4R Activity by Measuring cAMP Accumulation.
48 h after transfection, cells are washed once with PBS and then
detached from the plate with PBS containing 0.02% EDTA (Sigma). The
detached cells are harvested by centrifugation and resuspended in
Hanks' balanced salt solution (Invitrogen) containing 0.5 mM IBMX,
2 mM HEPES, pH 7.5 (IBMX buffer). After incubation at 37.degree. C.
for 15 min to allow for IBMX uptake, 0.4 ml of cell suspension
(about 5.times.10.sup.5 cells/ml) are added to 0.1 ml of IBMX
buffer containing various concentrations of agonists (e.g.,
[Nle4-D-Phe7]-MSH (NDP-MSH), .alpha.-MSH), or other chaperone
candidates or 10 .mu.M forskolin. The cells are subsequently
incubated at 37.degree. C. for 15 min to allow for cAMP
accumulation. The activity is terminated by adding 0.5 ml of 5%
trichloroacetic acid, and cAMP released from lysed cells is assayed
by the cAMP .sup.125I, scintillation proximity assay system
(Amersham Biosciences). EC.sub.50 values are calculated with a 95%
confidence interval using GraphPad Prism software (using nonlinear
regression analysis fitted with a sigmoidal dose-response curve
with variable slope).
Example 6
Administration of Single Dose DGJ to Evaluate Safety, Tolerability,
Pharmacokinetics, and Effects on .alpha.-Galactosidase A Enzymatic
Activity
[0223] This example describes a randomized, double blind, placebo
controlled Phase I study of twice daily oral doses of DGJ to
evaluate the safety, tolerability, pharmacokinetics, and
.alpha.-Galatosidase A (.alpha.-Gal A) enzymantic activity effects
of DGJ in healthy volunteers.
[0224] Study Design and Duration. This study was first-in-man,
single-center, Phase I, randomized, double-blind, twice-daily dose,
placebo-controlled study to evaluate the safety, tolerability,
pharmacokinetics, and .alpha.-Gal A enzymantic activity effects of
DGJ following oral administration. The study tested two groups of 8
subjects (6 active and 2 placebo) who received a twice-daily dose
of 50 or 150 mg of DGJ or placebo administered orally for seven
consecutive days, accompanied by a seven day follow up visit.
Subjects were housed in the treatment facility from 14 hours prior
to dosing until 24 hours after dosing. Meals were controlled by
schedule and subjects remained abulatory for 4 hours post drug
administration.
[0225] Pharmacokinetic parameters were calculated for DGJ in plasma
on Day 1 and Day 7. In addition, the cumulative percentage of DGJ
excreted (12 hours post dose) in urine was calculated. .alpha.-Gal
A activity was calculated in white blood cells (WBC) before dosing
began, and again at 100 hours, 150 hours, and 336 hours into the
trial.
[0226] Study Population. Subjects were healthy,
non-institutionalized, non-smoking male volunteers between 19 and
50 years of age (inclusive) consisting of members of the community
at large.
[0227] Safety and Tolerability Assessments. Safety was determined
by evaluating vital signs, laboratory parameters (serum chemistry,
hematology, and urinalysis), ECGs, physical examination and by
recording adverse events during the Treatment Period.
[0228] Pharmacokinetic Sampling. Blood samples (10 mL each) were
collected in blood collection tubes containing EDTA before dosing
and at the following times thereafter: 0.25, 0.5, 0.75, 1, 1.5, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 hours. Blood samples were cooled
in an ice bath and centrifuged under refrigeration as soon as
possible. Plasma samples were divided into two aliquots and stored
at 20.+-.10.degree. C. pending assay. At the end of the study, all
samples were transferred to MDS Pharma Services Analytical
Laboratories (Lincoln) for analysis. The complete urine output was
collected from each subject for analysis of DGJ to determine renal
clearance for the first 12 hours after adiministration of DGJ on
days 1 and 7.
[0229] WBC .alpha.-GAL A Enzymatic Activity Sampling. Blood samples
(10 mL each) were collected in blood collection tubes containing
EDTA and WBC extracted before dosing and at the following times
thereafter: 100 hours, 150 hours, and 336 hours. Samples were
treated as described above, and WBC .alpha.-Gal A enzymatic
activity levels were determined.
[0230] Statistical Analysis. Safety data including laboratory
evaluations, physical exams, adverse events, ECG monitoring and
vital signs assessments were summarized by treatment group and
point of time of collection. Descriptive statistics (arithmetic
mean, standard deviation, median, minimum and maximum) were
calculated for quantitative safety data as well as for the
difference to baseline. Frequency counts were compiled for
classification of qualitative safety data.
[0231] Adverse events were coded using the MedDRA version 7.0
dictionary and summarized by treatment for the number of subjects
reporting the adverse event and the number of adverse events
reported. A by-subject adverse event data listing including
verbatim term, coded term, treatment group, severity, and
relationship to treatment was provided. Concomitant medications and
medical history were listed by treatment.
[0232] Pharmacokinetic parameters were summarised by treatment
group using descriptive statistics (arithmetic means, standard
deviations, coefficients of variation, sample size, minimum,
maximum and median).
Results
[0233] No placebo-treated subjects had an adverse event (AE) and no
subject presented with AEs after receiving 50 mg b.i.d. or 150 mg
b.i.d. DGJ. DGJ appeared to be safe and well tolerated by this
group of healthy male subjects as doses were administered at 50 mg
b.i.d. and 150 mg b.i.d.
[0234] Laboratory deviations from normal ranges occurred after
dosing, but none was judged clinically significant. There were no
clinically relevant mean data shifts in any parameter investigated
throughout the course of the study. No clinically relevant
abnormality occurred in any vital sign, ECG, or physical
examination parameter.
[0235] Pharmacokinetic Evaluation. The following table summarizes
the pharmacokinetic data obtained during study. TABLE-US-00006
TABLE 4 50 mg b.i.d. dose 150 mg b.i.d. dose Day 1 Day 7 Day 1 Day
7 Cmax (.mu.M) 2.3 .+-. 0.3 3.9 .+-. 0.5 11.3 .+-. 1.5 10.8 .+-.
1.4 tmax (h) 2.9 .+-. 0.4 2.5 .+-. 0.4 3.1 .+-. 0.4 2.9 .+-. 0.4
t1/2 (h) 2.5 .+-. 0.1 2.4 .+-. 0.05 Cmin (.mu.M) 0.4 .+-. 0.03 1.2
.+-. 0.1 12 h cumulative 16 .+-. 6 48 .+-. 7 42 .+-. 7 60 .+-. 5
renal excretion (%).sup.a .sup.aCumulative percentage of DGJ
excreted over the 12-hour post dose period.
[0236] The pharmacokinetics of DGJ were well characterized in all
subjects and at all dose levels. On average, peak concentrations
occurred at approximately 3 hours for all dose levels. C.sub.max of
DGJ increased in a dose-proportional manner when doses were
increased from 50 mg to 150 mg.
[0237] The mean elimination half-lives (t.sub.1/2) were comparable
at dose levels of 50 and 150 mg on Day 1 (2.5 vs. 2.4 hours).
[0238] The mean percentage of DGJ excreted over the 12-hour post
dose period was 16% and 42% at dose levels of 50 mg and 150 mg,
respectively, on Day 1, increasing to 48% and 60%, respectively, on
Day 7.
[0239] .alpha.-Galactosidase A (.alpha.-Gal A) enzymatic Activity.
The .alpha.-Gal A enzymatic activity data obtained during the study
is shown in FIG. 1. DGJ did not inhibit WBC .alpha.-Gal A enzymatic
activity in subjects at dosages of 50 mg b.i.d. or 150 mg b.i.d.
Furthermore, DGJ produced a dose-dependent trend of increased WBC
.alpha.-Gal A activity in healthy volunteers. .alpha.-Gal A
enzymatic activity was measured in WBC of subjects administered
placebo, 50 mg b.i.d., and 150 mg b.i.d. DGJ. Placebo had no effect
on WBC .alpha.-Gal A enzymatic activity. Variations in enzymatic
activity in response to placebo were not clinically significant.
Both 50 mg b.i.d. and 150 mg b.i.d. DGJ increased normalized WBC
.alpha.-Gal A enzymatic activity. In response to 50 mg b.i.d. DGJ,
WBC .alpha.-Gal A enzymatic activity increased to 120%, 130%, and
145% pre-dose levels at 100 hours, 150 hours, and 336 hours
post-dose, respectively. In response to 150 mg b.i.d. DGJ, WBC
.alpha.-Gal A enzymatic activity increased to 150%, 185%, and 185%
pre-dose levels at 100 hours, 150 hours, and 336 hours post-dose,
respectively.
[0240] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0241] Patents, patent applications, publications, product
descriptions, and protocols are cited throughout this application,
the disclosures of which are incorporated herein by reference in
their entireties for all purposes.
Sequence CWU 1
1
18 1 1000 DNA Homo sapiens 1 atggtgaact ccacccaccg tgggatgcac
acttctctgc acctctggaa ccgcagcagt 60 tacagactgc acagcaatgc
cagtgagtcc cttggaaaag gctactctga tggagggtgc 120 tacgagcaac
tttttgtctc tcctgaggtg tttgtgactc tgggtgtcat cagcttgttg 180
gagaatatct tagtgattgt ggcaatagcc aagaacaaga atctgcattc acccatgtac
240 tttttcatct gcagcttggc tgtggctgat atgctggtga gcgtttcaaa
tggatcagaa 300 accattgtca tcaccctatt aaacagtaca gatacggatg
cacagagttt cacagtgaat 360 attgataatg tcattgactc ggtgatctgt
agctccttgc ttgcatccat ttgcagcctg 420 ctttcaattg cagtggacag
gtactttact atcttctatg ctctccagta ccataacatt 480 atgacagtta
agcgggttgg gatcatcata agttgtatct gggcagcttg cacggtttca 540
ggcattttgt tcatcattta ctcagatagt agtgctgtca tcatctgcct catcaccatg
600 ttcttcacca tgctggctct catggcttct ctctatgtcc acatgttcct
gatggccagg 660 cttcacatta agaggattgc tgtcctcccc ggcactggtg
ccatccgcca aggtgccaat 720 atgaagggag cgattacctt gaccatcctg
attggcgtct ttgttgtctg ctgggcccca 780 ttcttcctcc acttaatatt
ctacatctct tgtcctcaga atccatattg tgtgtgcttc 840 atgtctcact
ttaacttgta tctcatactg atcatgtgta attcaatcat cgatcctctg 900
atttatgcac tccggagtca agaactgagg aaaaccttca aagagatcat ctgttgctat
960 cccctgggag gcctttgtga cttgtctagc agatattaaa 1000 2 332 PRT Homo
sapiens 2 Met Val Asn Ser Thr His Arg Gly Met His Thr Ser Leu His
Leu Trp 1 5 10 15 Asn Arg Ser Ser Tyr Arg Leu His Ser Asn Ala Ser
Glu Ser Leu Gly 20 25 30 Lys Gly Tyr Ser Asp Gly Gly Cys Tyr Glu
Gln Leu Phe Val Ser Pro 35 40 45 Glu Val Phe Val Thr Leu Gly Val
Ile Ser Leu Leu Glu Asn Ile Leu 50 55 60 Val Ile Val Ala Ile Ala
Lys Asn Lys Asn Leu His Ser Pro Met Tyr 65 70 75 80 Phe Phe Ile Cys
Ser Leu Ala Val Ala Asp Met Leu Val Ser Val Ser 85 90 95 Asn Gly
Ser Glu Thr Ile Val Ile Thr Leu Leu Asn Ser Thr Asp Thr 100 105 110
Asp Ala Gln Ser Phe Thr Val Asn Ile Asp Asn Val Ile Asp Ser Val 115
120 125 Ile Cys Ser Ser Leu Leu Ala Ser Ile Cys Ser Leu Leu Ser Ile
Ala 130 135 140 Val Asp Arg Tyr Phe Thr Ile Phe Tyr Ala Leu Gln Tyr
His Asn Ile 145 150 155 160 Met Thr Val Lys Arg Val Gly Ile Ile Ile
Ser Cys Ile Trp Ala Ala 165 170 175 Cys Thr Val Ser Gly Ile Leu Phe
Ile Ile Tyr Ser Asp Ser Ser Ala 180 185 190 Val Ile Ile Cys Leu Ile
Thr Met Phe Phe Thr Met Leu Ala Leu Met 195 200 205 Ala Ser Leu Tyr
Val His Met Phe Leu Met Ala Arg Leu His Ile Lys 210 215 220 Arg Ile
Ala Val Leu Pro Gly Thr Gly Ala Ile Arg Gln Gly Ala Asn 225 230 235
240 Met Lys Gly Ala Ile Thr Leu Thr Ile Leu Ile Gly Val Phe Val Val
245 250 255 Cys Trp Ala Pro Phe Phe Leu His Leu Ile Phe Tyr Ile Ser
Cys Pro 260 265 270 Gln Asn Pro Tyr Cys Val Cys Phe Met Ser His Phe
Asn Leu Tyr Leu 275 280 285 Ile Leu Ile Met Cys Asn Ser Ile Ile Asp
Pro Leu Ile Tyr Ala Leu 290 295 300 Arg Ser Gln Glu Leu Arg Lys Thr
Phe Lys Glu Ile Ile Cys Cys Tyr 305 310 315 320 Pro Leu Gly Gly Leu
Cys Asp Leu Ser Ser Arg Tyr 325 330 3 999 DNA Homo sapiens 3
atggtgaact ccacccaccg tgggatgcac acttctctgc acctctggaa ccgcagcagt
60 tacagactgc acagcaatgc cagtgagtcc cttggaaaag gctactctga
tggagggtgc 120 tacgagcaac tttttgtctc tcctgaggtg tttgtgactc
tgggtgtcat cagcttgttg 180 gagaatatct tagtgattgt ggcaatagcc
aagaacaaga atctgcattc acccatgtac 240 tttttcatct gcagcttggc
tgtggctgat atgctggtga gcgtttcaaa tggatcagaa 300 accattatca
tcaccctatt aaacagtaca gatacggatg cacagagttt cacagtgaat 360
attgataatg tcattgactc ggtgatctgt agctccttgc ttgcatccat ttgcagcctg
420 ctttcaattg cagtggacag gtactttact atcttctatg ctctccagta
ccataacatt 480 atgacagtta agcgggttgg gatcatcata agttgtatct
gggcagcttg cacggtttca 540 ggcattttgt tcatcattta ctcagatagt
agtgctgtca tcatctgcct catcaccatg 600 ttcttcacca tgctggctct
catggcttct ctctatgtcc acatgttcct gatggccagg 660 cttcacatta
agaggattgc tgtcctcccc ggcactggtg ccatccgcca aggtgccaat 720
atgaagggag cgattacctt gaccatcctg attggcgtct ttgttgtctg ctgggcccca
780 ttcttcctcc acttaatatt ctacatctct tgtcctcaga atccatattg
tgtgtgcttc 840 atgtctcact ttaacttgta tctcatactg atcatgtgta
attcaatcat cgatcctctg 900 atttatgcac tccggagtca agaactgagg
aaaaccttca aagagatcat ctgttgctat 960 cccctgggag gcctttgtga
cttgtctagc agatattaa 999 4 332 PRT Homo sapiens 4 Met Val Asn Ser
Thr His Arg Gly Met His Thr Ser Leu His Leu Trp 1 5 10 15 Asn Arg
Ser Ser Tyr Arg Leu His Ser Asn Ala Ser Glu Ser Leu Gly 20 25 30
Lys Gly Tyr Ser Asp Gly Gly Cys Tyr Glu Gln Leu Phe Val Ser Pro 35
40 45 Glu Val Phe Val Thr Leu Gly Val Ile Ser Leu Leu Glu Asn Ile
Leu 50 55 60 Val Ile Val Ala Ile Ala Lys Asn Lys Asn Leu His Ser
Pro Met Tyr 65 70 75 80 Phe Phe Ile Cys Ser Leu Ala Val Ala Asp Met
Leu Val Ser Val Ser 85 90 95 Asn Gly Ser Glu Thr Ile Ile Ile Thr
Leu Leu Asn Ser Thr Asp Thr 100 105 110 Asp Ala Gln Ser Phe Thr Val
Asn Ile Asp Asn Val Ile Asp Ser Val 115 120 125 Ile Cys Ser Ser Leu
Leu Ala Ser Ile Cys Ser Leu Leu Ser Ile Ala 130 135 140 Val Asp Arg
Tyr Phe Thr Ile Phe Tyr Ala Leu Gln Tyr His Asn Ile 145 150 155 160
Met Thr Val Lys Arg Val Gly Ile Ile Ile Ser Cys Ile Trp Ala Ala 165
170 175 Cys Thr Val Ser Gly Ile Leu Phe Ile Ile Tyr Ser Asp Ser Ser
Ala 180 185 190 Val Ile Ile Cys Leu Ile Thr Met Phe Phe Thr Met Leu
Ala Leu Met 195 200 205 Ala Ser Leu Tyr Val His Met Phe Leu Met Ala
Arg Leu His Ile Lys 210 215 220 Arg Ile Ala Val Leu Pro Gly Thr Gly
Ala Ile Arg Gln Gly Ala Asn 225 230 235 240 Met Lys Gly Ala Ile Thr
Leu Thr Ile Leu Ile Gly Val Phe Val Val 245 250 255 Cys Trp Ala Pro
Phe Phe Leu His Leu Ile Phe Tyr Ile Ser Cys Pro 260 265 270 Gln Asn
Pro Tyr Cys Val Cys Phe Met Ser His Phe Asn Leu Tyr Leu 275 280 285
Ile Leu Ile Met Cys Asn Ser Ile Ile Asp Pro Leu Ile Tyr Ala Leu 290
295 300 Arg Ser Gln Glu Leu Arg Lys Thr Phe Lys Glu Ile Ile Cys Cys
Tyr 305 310 315 320 Pro Leu Gly Gly Leu Cys Asp Leu Ser Ser Arg Tyr
325 330 5 1888 DNA Rattus norvegicus 5 atgctcggga agctcaactt
ctgagaggct gcgctgtgag tgtgggcgcg cagatgcaga 60 ggcggctccc
agctctccag cgactctcag gaaaaggact ctgaaaagac cccgagtgaa 120
tactacggct aaagggaaag ccacaaaaaa cgaactgcag actggtcagc cgagagtgag
180 ctttcagtag cgccagcttc taaagaaatg atgagcaaag ctgaacccag
aagagaccaa 240 caactccttt gcaagctccg ctgcttctga ccctgttcac
cgcaggcgcc aactgcagcc 300 ttccaacttc tacaggcaga caggctggga
gaaaaaccac tcggggcttc cctgacctag 360 gaggttggac cacttcaagg
aggattcgaa tccagctgct gcaggaagat gaactccacc 420 caccaccatg
gcatgtatac ttccctccac ctctggaacc gcagcagcca cgggctgcac 480
ggcaatgcca gcgagtctct ggggaagggg cactcagacg gaggatgcta tgagcaactt
540 tttgtctccc ccgaggtgtt tgtgactctg ggtgtcataa gcctgttgga
gaacattcta 600 gtgatcgtgg cgatagccaa gaacaagaac ctgcactcac
ccatgtactt tttcatctgt 660 agtctggctg tggcggacat gctggtgagc
gtttcgaacg ggtcagaaac catcgtcatc 720 accctgctaa acagtacgga
cacggacgcc cagagcttca ccgtgaatat tgataatgtc 780 attgactctg
tgatctgtag ctccttgctc gcatccattt gcagcctgct ttccattgca 840
gtggacaggt atttcactat cttttacgcg ctccagtacc ataacattat gacggttagg
900 cgggtcggga tcatcatcag ttgtatctgg gcagcttgca cagtatcggg
cgttcttttt 960 atcatttact cggacagcag cgctgtcatc atctgcctca
ttaccatgtt cttcaccatg 1020 ctggttctca tggcctctct ctatgtccac
atgttcctga tggcgaggct tcacattaag 1080 aggatcgctg tcctcccggg
cacgggtacc atccgacagg gtgccaacat gaagggcgca 1140 attaccttga
ccattctgat tggagtgttt gttgtctgct gggccccgtt tttcctccat 1200
ttactgttct acatctcttg tcctcagaat ccatactgcg tgtgcttcat gtctcatttt
1260 aacttgtatc tcatactgat catgtgtaac gctgtcatcg accctctcat
ttatgccctg 1320 cggagtcaag aactgaggaa aaccttcaaa gagatcatct
gtttctaccc cctgggaggc 1380 atctgtgagt tacctggcag gtattaagtg
gggacagagt gcatactagg tagagacctg 1440 cagaatttgt cactcaggca
caacctgagc agtgtacttc ccaacagctg cctctactgt 1500 atagtgcttt
ggttggaaaa tatctactgt ataaaatgta agtttatgac ttttgacgtg 1560
gggaaaaagt ctcaacgtgt tatgtttatt gaccttactt tttttgtgtg taaactgctt
1620 atttatgttc tacagcgtgg gcgctatgga gttccataaa agaaaaagac
acccttatta 1680 aaactttgac agtgtttctt tccatgttat ttatcaagag
tcaacccttg ttctttctgt 1740 ggtagcagaa atcagagcct tctgaaaagc
tgtttccatt gcatcacccc cacagcacag 1800 cagaagcctg attccactgt
ttatggggaa atatttaaac actggatgct cgatcattta 1860 atgagtcagc
tctactcgtg aaatttca 1888 6 332 PRT Rattus norvegicus 6 Met Asn Ser
Thr His His His Gly Met Tyr Thr Ser Leu His Leu Trp 1 5 10 15 Asn
Arg Ser Ser His Gly Leu His Gly Asn Ala Ser Glu Ser Leu Gly 20 25
30 Lys Gly His Ser Asp Gly Gly Cys Tyr Glu Gln Leu Phe Val Ser Pro
35 40 45 Glu Val Phe Val Thr Leu Gly Val Ile Ser Leu Leu Glu Asn
Ile Leu 50 55 60 Val Ile Val Ala Ile Ala Lys Asn Lys Asn Leu His
Ser Pro Met Tyr 65 70 75 80 Phe Phe Ile Cys Ser Leu Ala Val Ala Asp
Met Leu Val Ser Val Ser 85 90 95 Asn Gly Ser Glu Thr Ile Val Ile
Thr Leu Leu Asn Ser Thr Asp Thr 100 105 110 Asp Ala Gln Ser Phe Thr
Val Asn Ile Asp Asn Val Ile Asp Ser Val 115 120 125 Ile Cys Ser Ser
Leu Leu Ala Ser Ile Cys Ser Leu Leu Ser Ile Ala 130 135 140 Val Asp
Arg Tyr Phe Thr Ile Phe Tyr Ala Leu Gln Tyr His Asn Ile 145 150 155
160 Met Thr Val Arg Arg Val Gly Ile Ile Ile Ser Cys Ile Trp Ala Ala
165 170 175 Cys Thr Val Ser Gly Val Leu Phe Ile Ile Tyr Ser Asp Ser
Ser Ala 180 185 190 Val Ile Ile Cys Leu Ile Thr Met Phe Phe Thr Met
Leu Val Leu Met 195 200 205 Ala Ser Leu Tyr Val His Met Phe Leu Met
Ala Arg Leu His Ile Lys 210 215 220 Arg Ile Ala Val Leu Pro Gly Thr
Gly Thr Ile Arg Gln Gly Ala Asn 225 230 235 240 Met Lys Gly Ala Ile
Thr Leu Thr Ile Leu Ile Gly Val Phe Val Val 245 250 255 Cys Trp Ala
Pro Phe Phe Leu His Leu Leu Phe Tyr Ile Ser Cys Pro 260 265 270 Gln
Asn Pro Tyr Cys Val Cys Phe Met Ser His Phe Asn Leu Tyr Leu 275 280
285 Ile Leu Ile Met Cys Asn Ala Val Ile Asp Pro Leu Ile Tyr Ala Leu
290 295 300 Arg Ser Gln Glu Leu Arg Lys Thr Phe Lys Glu Ile Ile Cys
Phe Tyr 305 310 315 320 Pro Leu Gly Gly Ile Cys Glu Leu Pro Gly Arg
Tyr 325 330 7 2769 DNA Mus musculus 7 gctggctcac aaagatgctc
aggaagctga acttctgaga ggctgcggtg tgagtgtggg 60 cgcgcagatg
cagacgcggc tcccagcagt acagcgagtc tcagggaaaa ggactctgaa 120
aagaccccga gtgaatacta aagtgaaagc cgcactgaga gagagagaaa aaaaagcaaa
180 cagcagactg gtcaaccgag aatgagcatt cagaagcacc agcttctaaa
gagacgatga 240 tctgagccga acccagaaga gaccaacaac tcctttgcga
gttccgctgc ttctgaccct 300 gctcctagca ggcgccaagc gcagcctccc
aacttctaca ggcatacaga ctgggagaga 360 atcactcgga gcttccctga
cccaggaggt tggatcagtt caaggaggac tcaaatccag 420 ctgctgcagg
aagatgaact ccacccacca ccatggcatg tatacttccc tccacctctg 480
gaaccgcagc agctacgggc tgcacggcaa tgccagcgag tcgctgggga agggccaccc
540 ggacggagga tgctatgagc aactttttgt ttcccccgag gtgtttgtga
ctctgggtgt 600 cataagcctg ttggagaaca ttctagtgat cgtggcgata
gccaagaaca agaacctgca 660 ctcacccatg tactttttca tctgtagcct
ggctgtggca gatatgctgg tgagcgtttc 720 gaatgggtcg gaaaccatcg
tcattaccct gttaaacagt acggatacgg atgcccagag 780 cttcaccgtg
aacattgata atgtcattga ctctgtgatc tgtagctcct tgctcgcatc 840
catttgcagc ctgctttcca ttgcggtgga caggtatttc actatctttt acgcgctcca
900 gtaccataac atcatgacgg ttaggcgggt cgggatcatc ataagttgta
tctgggcagc 960 ttgcactgtg tcaggcgtcc tcttcatcat ttactcggac
agcagcgctg tcatcatctg 1020 cctcatttcc atgttcttca ctatgctagt
tctcatggcc tctctctatg tccacatgtt 1080 cctgatggcg aggcttcaca
ttaagaggat tgctgtcctc ccaggcacag ggaccatccg 1140 ccagggtacc
aacatgaagg gggcgattac cttgaccatc ctgattggag tctttgttgt 1200
ctgctgggcc ccgttctttc tccatttact gttctacatc tcttgccctc agaatccata
1260 ctgcgtgtgc ttcatgtctc attttaattt gtatctcata ctgatcatgt
gtaacgccgt 1320 catcgaccct ctcatttatg ccctccggag tcaagaactg
aggaaaactt tcaaagagat 1380 catctgtttc tatcctctgg gaggcatctg
tgagttgtct agcaggtatt aagtggggga 1440 cagagtgcaa actaggtaga
tacctgcaga ctttgtcact ctggcccgat ctgagcagtg 1500 tacttcccaa
cagctgcctc ttctgtgtaa tgctttggtt gaaaatatct actgtataaa 1560
tgtaagtttg tgacttttga catggaaaaa aaagtctcaa cgtgttatgt ttattgacac
1620 gctatttttt ttgtttgtaa aatgcttatt tatgttctat atagtgtggg
cgttatgaat 1680 tgacatgaaa gaaaaacaga cacccttatt aaaactttga
cagtgtttct ttcctgttat 1740 ttatcaaggt tccacacttg ttctttctgt
agtggccgaa atcagaacct tattaaacgt 1800 gttctcagct gttctcatgt
attagcccca cagtactgca gaggcactga ccccactgtt 1860 tatggggaaa
tatttaaaca ctacatgctt gatcattaaa atgagtcagc tctcttagtg 1920
aaatttcgag caatcgaata aaagcttgcc tattatcctt gctgtccaaa tacactgatg
1980 cttcttttta agtaaaggaa agagaaaggg ggaagaagca gctactgagg
agaaagtgag 2040 atttctgtca catgcatttc tccaagaagg aatggttcat
tgcccgagac tcagagttca 2100 cacaggcaag tcagctgtgg taggggaaat
gcccacttaa tagattaaag atattataat 2160 agataataat agataaaata
gattaataga taaaatagat accaatctta atagattaaa 2220 gtgtcctgtt
aaatataaac tgtccacacc atgctgaaat ttcctatgcc aaatgatacc 2280
ccaccataac agaatgattt ctttctggct tcttaccagg gatctggttc ctacagaaag
2340 gtctagaaca gctccctctg cacttagagg tccagcgttc atttcatctt
agagttaata 2400 gtgagttgtg ctatctttca tgtggcgggg gacttgttgt
tcactttctg attacttttt 2460 gagctggaat ataagtgctg aagatcaagt
gatttaattc ccaagccaaa tccacatcac 2520 aaaacatttt gggacagggt
ttgtaaatat ctaaagtgtg gagccctgtg gtgcttgcac 2580 ataacgagat
ggaaagagaa cacaaatggg gtcctggaag gtacagtaaa acaccctgct 2640
gttcttagtc atgtcttggg atggggaatg cttgttttct ccaaactaat accaaaggtg
2700 tggccactga gcaaccaaat ctatgctttc tagtctgtgt atactttgaa
ataaaaggga 2760 taaaaacct 2769 8 332 PRT Mus musculus 8 Met Asn Ser
Thr His His His Gly Met Tyr Thr Ser Leu His Leu Trp 1 5 10 15 Asn
Arg Ser Ser Tyr Gly Leu His Ser Asn Ala Ser Glu Ser Leu Gly 20 25
30 Lys Gly His Pro Asp Gly Gly Cys Tyr Glu Gln Leu Phe Val Ser Pro
35 40 45 Glu Val Phe Val Thr Leu Gly Val Ile Ser Leu Leu Glu Asn
Ile Leu 50 55 60 Val Ile Val Ala Ile Ala Lys Asn Lys Asn Leu His
Ser Pro Met Tyr 65 70 75 80 Phe Phe Ile Cys Ser Leu Ala Val Ala Asp
Met Leu Val Ser Val Ser 85 90 95 Asn Gly Ser Glu Thr Ile Val Ile
Thr Leu Leu Asn Ser Thr Asp Thr 100 105 110 Asp Ala Gln Ser Phe Thr
Val Asn Ile Asp Asn Val Ile Asp Ser Val 115 120 125 Ile Cys Ser Ser
Leu Leu Ala Ser Ile Cys Ser Leu Leu Ser Ile Ala 130 135 140 Val Asp
Arg Tyr Phe Thr Ile Phe Tyr Ala Leu Gln Tyr His Asn Ile 145 150 155
160 Met Thr Val Arg Arg Val Gly Ile Ile Ile Ser Cys Ile Trp Ala Ala
165 170 175 Cys Thr Val Ser Gly Val Leu Phe Ile Ile Tyr Ser Asp Ser
Ser Ala 180 185 190 Val Ile Ile Cys Leu Ile Ser Met Phe Phe Thr Met
Leu Val Leu Met 195 200 205 Ala Ser Leu Tyr Val His Met Phe Leu Met
Ala Arg Leu His Ile Lys 210 215 220 Arg Ile Ala Val Leu Pro Gly Thr
Gly Thr Ile Arg Gln Gly Thr Asn 225 230 235 240 Met Lys Gly Ala Ile
Thr Leu Thr Ile Leu Ile Gly Val Phe Val Val 245 250 255 Cys Trp Ala
Pro Phe Phe Leu His Leu Leu Phe Tyr Ile Ser Cys Pro 260 265 270 Gln
Asn Pro Tyr Cys Val Cys Phe Met Ser His Phe Asn Leu Tyr Leu 275 280
285 Ile Leu Ile Met Cys Asn Ala Val Ile Asp Pro Leu Ile Tyr Ala Leu
290 295 300 Arg Ser Gln Glu Leu Arg Lys Thr Phe Lys Glu Ile Ile Cys
Phe Tyr 305 310 315 320 Pro Leu Gly Gly Ile Cys Glu Leu Ser
Ser Arg Tyr 325 330 9 7 PRT Artificial Sequence Synthetic peptide
MOD_RES (1)..(1) ACETYLATION, Nle MISC_FEATURE (4)..(4) D amino
acid MOD_RES (7)..(7) AMIDATION 9 Xaa Gly Lys Phe Arg Trp Gly 1 5
10 7 PRT Artificial Sequence Synthetic peptide MOD_RES (1)..(1)
ACETYLATION, Nle MISC_FEATURE (4)..(4) beta-naphthylalanine (2)
MOD_RES (4)..(4) AMIDATION 10 Xaa Gly Lys Xaa Arg Trp Gly 1 5 11 10
PRT Artificial Sequence Synthetic peptide, cyclic MOD_RES (1)..(4)
ACETYLATION, Nle MOD_RES (2)..(4) Nle MISC_FEATURE (7)..(7)
beta-naphthylalanine (2') MOD_RES (10)..(10) AMIDATION 11 Xaa Xaa
Xaa Xaa Asp His Xaa Arg Trp Lys 1 5 10 12 12 PRT Artificial
Sequence Synthetic peptide MOD_RES (1)..(1) ACETYLATION
MISC_FEATURE (4)..(4) D-(2') beta-naphthylalanine MOD_RES
(12)..(12) AMIDATION 12 Cys Glu His Xaa Arg Trp Gly Cys Pro Pro Lys
Asp 1 5 10 13 9 PRT Artificial Sequence Synthetic peptide MOD_RES
(1)..(1) ACETYLATION MOD_RES (2)..(2) Nle MISC_FEATURE (5)..(5)
D-(2') beta-naphthylalanine MOD_RES (9)..(9) AMIDATION 13 Cys Xaa
Arg His Xaa Arg Trp Gly Cys 1 5 14 12 PRT Artificial Sequence
Synthetic peptide MOD_RES (1)..(1) ACETYLATION MISC_FEATURE
(4)..(4) D-Phe (3,4-di-Cl) MOD_RES (12)..(12) AMIDATION 14 Cys Glu
His Phe Arg Trp Gly Cys Pro Pro Lys Asp 1 5 10 15 7 PRT Artificial
Sequence Synthetic peptide, cyclic MOD_RES (1)..(1) ACETYLATION,
Nle MISC_FEATURE (2)..(2) c [Asp MISC_FEATURE (3)..(3) Che
MISC_FEATURE (4)..(4) DNal(2') MOD_RES (7)..(7) AMIDATION 15 Xaa
Asp Xaa Xaa Arg Trp Lys 1 5 16 7 PRT Artificial Sequence Synthetic
peptide MOD_RES (1)..(1) ACETYLATION, Nle MISC_FEATURE (2)..(2)
c[Asp MISC_FEATURE (3)..(3) Cpe MISC_FEATURE (4)..(4) DNal (2')
MOD_RES (7)..(7) AMIDATION 16 Xaa Asp Xaa Xaa Arg Trp Lys 1 5 17 5
PRT Artificial Sequence Synthetic petide MOD_RES (1)..(1) BLOCKED,
cyclo (1,6) - suc MISC_FEATURE (2)..(2) D amino acid MOD_RES
(5)..(5) AMIDATION 17 His Phe Arg Trp Lys 1 5 18 8 PRT Artificial
Sequence Synthetic peptide MOD_RES (1)..(1) ACETYLATION, DArg
MISC_FEATURE (5)..(5) D amino acid MOD_RES (8)..(8) AMIDATION 18
Arg Cys Glu His Phe Arg Trp Cys 1 5
* * * * *