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J. Biol. Chem., Vol. 277, Issue 16, 13821-13826, April 19, 2002
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From the Departments of
Received for publication, January 18, 2002, and in revised form, February 8, 2002
Human melanin-concentrating hormone (hMCH) is a
nonselective natural ligand for the human melanin-concentrating hormone
receptors: hMCH-1R and hMCH-2R. Similarly, the smaller peptide
encompassing the disulfide ring and Arg6 of hMCH,
Ac-Arg6-cyclo(S-S)(Cys7-Met8-Leu9-Gly10-Arg11-Val12-Tyr13-Arg14-Pro15-Cys16)-NH2,
Ac-hMCH(6-16)-NH2, binds to and activates equally well both human MCH receptors present in the brain. To separate the physiological functions of hMCH-1R from those of hMCH-2R, new potent
and hMCH-1R selective agonists are necessary. In the present study,
analogs of Ac-hMCH(6-16)-NH2 were prepared and tested in binding and functional assays on cells expressing the MCH receptors. In
these peptides, Arg in position 6 was replaced with various D-amino acids and/or Gly in position 10 was substituted
with various L-amino acids. Several of the new compounds
turned out to be potent agonists at hMCH-1R with improved selectivity
over hMCH-2R. For example, peptide 26 with
D-Arg in place of L-Arg in position 6 and Asn
in place of Gly in position 10, Ac-DArg6-cyclo(S-S)(Cys7-Met8-Leu9-Asn10-Arg11-Val12-Tyr13-Arg14-Pro15-Cys16)-NH2,
was a potent hMCH-1R agonist (IC50 = 0.5 nM,
EC50 = 47 nM) with more than 200-fold
selectivity with respect to hMCH-2R. Apparently, these structural
changes in positions 6 and 10 results in peptide conformations that
allow for efficient interactions with hMCH-1R but are
unfavorable for molecular recognition at hMCH-2R.
In the last couple of years, melanin-concentrating hormone
(MCH)1 emerged as an
important regulator of feeding behavior in rodents (1-7). Similarly to
neuropeptide Y, this hormone stimulates appetite in rats when injected
intracerebroventricularly, and this orexigenic effect is inhibited by
anorectic peptides such as To understand and separate the physiological functions of the MCH
receptors, selective agonists are required, because hMCH is a
nonspecific natural ligand for both hMCH-1R and hMCH-2R. Similarly, the
synthetic ligands reported in the literature do not distinguish between
the receptors (13-23).
Our previous structure-function studies on hMCH yielded a cyclic
peptide consisting of the disulfide ring and Arg6 of hMCH:
the so-called "active core" of hMCH with the four residues, Arg6, Met8, Arg11, and
Tyr13, critical for molecular recognition at hMCH-1R and
hMCH-2R (24-27). This compound, Ac-hMCH(6-16)-NH2
(Structure 2), was equipotent to the full-length hMCH at both
receptors.2
Synthesis and Biological Evaluation in Vitro of a
Selective, High Potency Peptide Agonist of Human Melanin-concentrating
Hormone Action at Human Melanin-concentrating Hormone Receptor 1*
§, Medicinal Chemistry and
¶ Obesity and Metabolic Disorders, Merck Research Laboratories,
Rahway, New Jersey 07065
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-melanocyte stimulating hormone,
glucagon-like peptide 1, and neurotensin (1-7). Additionally,
in the hypothalamus of genetically obese (ob/ob) and fasting mice, the
level of MCH messenger RNA is elevated, and the metabolic rate of mice
lacking MCH is increased. MCH (see Structure 1 for human/rat MCH) also
appears to be involved in other biological functions such as regulation
of the hypothalamic-pituitary-thyroid axis.
In humans, this 19-amino acid cyclic peptide is found in the
brain, in the lateral hypothalamus and the zona incerta, and acts
through specific receptors (8-12). At present, two receptors are known
with which hMCH interacts: hMCH-1R and hMCH-2R (13-23). These
receptors are members of the family of G-protein-coupled receptors and
their activation leads to mobilization of intracellular calcium.
Binding of hMCH to hMCH-1R results in reduction of forskolin-elevated cyclic AMP levels, but binding to hMCH-2R does not cause this effect.
The physiological role of hMCH-2R is less well understood than the
physiological role of hMCH-1R, but the presence of the MCH-2R messenger
RNA in the brain regions implicated in the regulation of body weight
suggests that this receptor might also be involved in the regulation of
feeding behavior (19-23).
Moreover, at hMCH-1R, an analog of Ac-hMCH(6-16)-NH2
with D-Arg in position 6 was as potent as the parent
compound, but, at hMCH-2R, this analog was a noticeably weaker agonist.
The D-Arg6 compound was the first described
peptide agonist with enhanced hMCH-1R selectivity with respect to
hMCH-2R.
The study presented here was designed to expand on the above observation and prepare potent agonists of high hMCH-1R selectivity. First, analogs of Ac-hMCH(6-16)-NH2 were synthesized in which Arg in position 6 was replaced with various D-amino acids, with anticipation that the hMCH-1R selectivity of these peptides will be improved. In the binding and calcium release assays at the human MCH receptors, most of the new compounds displayed lower affinity and potency at hMCH-2R than hMCH-1R. Further, structure-function studies on hMCH(6-16)-NH2 revealed that incorporation of various L-amino acids in place of Gly10 affects interactions of the new analogs with hMCH-2R but not with hMCH-1R.
These observations led us to design analogs of
Ac-hMCH(6-16)-NH2 modified at both positions 6 and 10. Syntheses and biological evaluation in vitro at hMCH-1R and
hMCH-2R of several cyclic peptides that are potent and selective
hMCH-1R agonists is reported here.
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EXPERIMENTAL PROCEDURES |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Peptide Synthesis, Purification, and Characterization (24)-- Elongation of peptide chains on 4-(2',4'-dimethoxyphenyl-Fmoc (N-(9-fluorenyl)methoxycarbonyl)-aminomethyl)-phenoxy resin, deprotection and cleavage of peptides from a resin, and formation of the disulfide ring in solution were performed as previously described in detail (24).3 The lyophilized crude peptides were analyzed by analytical reverse-phase high pressure liquid chromatography (HPLC) on a C18 Vydac column attached to a Waters 600E system with automatic Wisp 712 injector and 991 Photodiode Array detector. A standard gradient system of 0-100% buffer B in 30 min was used for analysis; buffer A was 0.1% trifluoroacetic acid in water, and buffer B was 0.1% trifluoroacetic acid in acetonitrile. HPLC profiles were recorded at 210 and 280 nm. Preparative separations were performed on a Waters Delta Prep 4000 system with a semipreparative C18 RP Waters column. The above-described solvent system of water and acetonitrile, in a gradient of 0-70% buffer B in 60 min, was used for separation. The chromatographically homogeneous products (purity >97%) were analyzed by electrospray mass spectrometry.
MCH-1R and MCH-2R Radioligand Filter Binding
Assays--
Membrane binding assays were performed on transiently
transfected COS-7 cells expressing human MCH-2R from the plasmid vector pCI-neo (Promega, Madison, WI), and a CHO cell line stably expressing human MCH-1R from pcDNA3.1. For transient expression, COS-7 cells were cultured in Dulbecco's modified Eagle's medium
(Invitrogen) with 10% heat-inactivated fetal calf serum. A
suspension of 7 × 106 COS-7 cells were transfected
with 20 µg of pCI-neo/MCH-2R plasmid by electroporation (26), and
cells were harvested after 60-72 h. Membranes were prepared from
transient and stable transfectants by hypotonic lysis, frozen in liquid
nitrogen, and stored at 
80 °C. A filter binding assay was
developed to measure the specific binding of
125I-[Phe13,Tyr19]hMCH.
Scintilation proximity assays were carried out using wheat germ
agglutinin-polyvinyltoluene beads (Amersham Biosciences), in 96-well
OptiPlates (Packard, Meriden, CT). Each well contained 0.5-10 µg of
membrane protein and 200 µl of binding buffer (50 mM
Tris, pH 7.4, 10 mM MgCl2, 2 mM
EDTA, 12% glycerol, 0.1% bovine serum albumin). Binding buffer
contained 50 mM Tris, pH 7.4, 10 mM
MgCl2, 2 mM EDTA, and protease inhibitors: 200 µg/ml bacitracin (Sigma), 1 µM phosphoramidon
(Peninsula Laboratories), Assays were optimized with respect to
membrane preparations: for CHO/MCH-1R membranes, 1 µg of
membranes/well yielded a 10× specific binding window and for COS
MCH-2R membranes, 8 µg of membrane protein yielded a window of
~8×. Specific binding is defined as the difference between total
binding and nonspecific binding conducted in the presence of 500 nM unlabeled hMCH. In 96-well dishes, the membranes were
combined with peptide at various dilutions and the radioligand 125I-[Phe13,Tyr19]hMCH at 0.3 nM final concentration and incubated at room temperature for 1 h. The membrane-bound counts were collected by filter
harvesting through a Filtermate harvester (Packard Instruments) and
washing with binding buffer as described above with added 0.04% Tween detergent, dried, scintillant added, and the plates were read in a
TopCount (Packard). IC50 calculations were performed using Prism 3.0 (GraphPad Software, San Diego, CA). The IC50
values were measured in three different experiments.
Aequorin Bioluminescence Functional Assays (24, 26, 27)--
For
the functional receptor activation assays, stable cell lines expressing
either the MCH-1R or the MCH-2R and the aequorin reporter protein were
used. The assays were performed on a Luminoskan RT luminometer
(Labsystems Inc., Gaithersburg, MD) controlled by custom software
written for a PC-compatible computer. 293AEQ17/MCH-1R(or MCH-2R) cells
were cultured for 72 h, and the apo-aequorin in the cells was
charged for 1 h with coelenterazine (10 µM) under reducing conditions (300 M reduced glutathione) in ECB
buffer (140 mM NaCl, 20 mM KCl, 20 mM HEPES-NaOH, pH 7.4, 5 mM glucose, 1 mM MgCl2, 1 mM CaCl2,
0.1 mg/ml bovine serum albumin). The cells were harvested, washed once
in ECB medium, and resuspended to 500,000 cells/ml. 100 µl of cell
suspension (corresponding to 5 × 104 cells) was then
injected into the test plate containing the hMCH peptides, and the
integrated light emission was recorded over 30 s, in 0.5-s units.
20 µl of lysis buffer (0.1% final Triton X-100 concentration) was
then injected and the integrated light emission recorded over 10 s, in 0.5-s units. The "fractional response" values for each well
were calculated by taking the ratio of the integrated response to the
initial challenge to the total integrated luminescence including the
Triton X-100 lysis response. The EC50 values were measured
in three different experiments.
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RESULTS |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Analogs of hMCH listed in Tables I-III were prepared by solid-phase syntheses as described under "Experimental Procedures." They were evaluated for their respective binding affinities for cloned human MCH receptor 1 and 2 in the competition binding assays with 125I-[Phe13,Tyr19]hMCH as the radiolabeled ligand (28) and, also, for their ability to stimulate inositol trisphosphate-coupled mobilization of intracellular calcium in human HEK-293 cells expressing hMCH-1R and hMCH-2R (24, 29, 30).
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Binding and functional data for analogs of Ac-hMCH(6-16)-NH2 modified in position 6 are compiled in Table I. Omission of Ac and the amino group of Arg6, through incorporation of 5-guanidino-valeric acid (des-amino-arginine) in position 6, resulted in compound 1, which was not a fully effective agonist at hMCH-1R (29% activation at 10 µM concentration), but was a full agonist at hMCH-2R of potency similar to that of the parent compound. In contrast, at hMCH-1R, analog 2 without the Ac group but with D-Arg in position 6 was equipotent to Ac-hMCH(6-16)-NH2 but, at hMCH-2R, this des-acetyl-peptide was an ~10-fold weaker agonist.
In compounds 3-10, D-enantiomers of hydrophobic amino acids, Ala, Nle, Pro, and Phe, and hydrophilic amino acids, Asn, Ser, Glu and Lys, were incorporated in position 6 of hMCH(6-16)-NH2. The new peptides were efficient binders to hMCH-1R, but their signal transduction efficacies at this receptor were more than 4-fold lower than that of Ac-hMCH(6-16)-NH2. These compounds poorly activated the second hMCH receptor, thus showing the enhanced selectivity for hMCH-1R. Replacement of Arg6 with D-enantiomer of citrulline, yielded analog 11 of binding affinity for hMCH-1R diminished ~20-fold and of agonist potency at both MCH receptors significantly reduced (>50-fold).
In analog 12, the Ac-Arg6 segment of Ac-hMCH(6-16)-NH2 was omitted and 3-mercaptopropionic acid (des-aminocysteine) was used instead of Cys in position 7 to form the disulfide ring. This peptide showed ~40-fold lower binding affinity and activity at hMCH-1R than Ac-hMCH(6-16)-NH2. Similarly to the other peptides modified at position 6, compound 12 was a very weak ligand for hMCH-2R (IC50 and EC50 > 3000 nM).
The disulfide cycle of Ac-hMCH(6-16)-NH2 encompasses Gly in position 10. This residue is known to facilitate formation of reversed turns in peptide chains. To test the effect of conformational changes in position 10 on biological activity, analogs of Ac-hMCH(6-16)-NH2 were synthesized and tested in which various L-amino acids were incorporated in place of Gly10; their binding and functional data are compiled in Table II.
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Replacement of Gly10 with hydrophobic L-amino acids which possess long, branch, or aromatic side chains yielded compounds 13-18 (Ala10, Leu10, Nle10, Cha10, Phe10, and (2')Nal10 analogs). These peptides bound to hMCH-1R almost as efficiently as the parent compound but, at hMCH-2R, analogs with Leu, Cha, and Nal(2') in position 10 were 2-20-fold weaker binders (compounds 14, 16, and 18). At hMCH-1R, signal transduction efficacies of peptides 13-15 and 17 were similar to that of Ac-hMCH(6-16)-NH2, whereas, at hMCH-2R, these analogs were 2-10 times less potent. The Cha10 analog with the bulky branched side chain in position 10, compound 16, activated poorly both receptors. Additionally, incorporation of the conformationally constraining Pro in place of Gly10 was deleterious to agonism at both hMCH receptors; the Pro10 analog was virtually inactive at micromolar concentrations (peptide 19).
In compounds 20-25, ionic residues were incorporated in position 10. Peptides with Lys, Asn, Ser, and Cit in this position were high affinity binders to hMCH-1R, but their affinities for hMCH-2R were 20-700 times lower than that of Ac-hMCH(6-16)NH2. At both hMCH receptors, the Asn10, Ser10, and Cit10 analogs were 2-50-fold weaker agonists than the parent compound, but the Lys10 analog was even less potent (more than 250 times). Peptide 25 with an acidic residue in position 10, the Glu10 analog, was virtually inactive at hMCH-1R and hMCH-2R.
The cyclic peptides listed in Table III, compounds 26-31, were designed to incorporate changes in the structure of Ac-hMCH(6-16)NH2, which were reported above as favorable for hMCH-1R selectivity. Hence, in compounds 26-31, amino acid residues in both positions 6 and 10 were replaced: Arg6 with D-Arg or D-Cit, and Gly10 with Asn or Gln. The new peptides turned out to be poor ligands for hMCH-2R, IC50 > 1000 nM, EC50 > 1300 nM. However, the D-Arg6,Asn10 analog, peptide 26, activated hMCH-1R almost as efficiently as Ac-hMCH(6-16)NH2 and, thus, was more than 200-fold selective with respect to hMCH-2R. Fig. 1 depicts binding and functional agonism of compound 26 (hMCH-1R selective agonist) and the parent peptide, Ac-hMCH(6-16)-NH2 (nonselective agonist at hMCH-1R and hMCH-2R). Rather conservative replacement of Asn in position 10 with Gln turned out to be detrimental to potency at hMCH-1R. The D-Arg6,Gln10 analog was ~50-fold weaker agonist at hMCH-1R (EC50 = 2300 nM) than the D-Arg6,Asn10 peptide.
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In peptides 28 and 29, the critical for molecular recognition Met8 of peptide 26 was replaced with the isosteric Nle. The new peptides were more than 5-fold less potent at hMCH-1R than compound 26.
Omission of the guanidine group in position 14 in compound
26, through the replacement of Arg with Ala, yielded peptide 30, which was 5-fold weaker agonist at hMCH-1R than the parent compound. Also unfavorable to molecular recognition at hMCH-1R
was replacement of D-Arg in position 6 of compound
26 with D-citrulline; the
D-Cit6,Asn10 analog (compound
31) was a weak hMCH-1R agonist of EC50 = 1500 nM.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Previous structure-function studies (24) on a short analog of hMCH, the Ac-hMCH(6-16)-NH2 peptide, indicated that chirality of Arg6 determines, to some extent, the receptor selectivity of peptide agonists; the D-Arg6 analog was ~4 times more potent at hMCH-1R than hMCH-2R. We concluded (24) that, for maximum agonist activity at hMCH-1R, the side chain of Arg6 does not need to be in a preferred orientation but, for hMCH-2R activation, the side chain orientation provided by the chiral center of L-Arg6 is desired. These conclusions were supported in the present study through testing in binding and functional assays at hMCH-1R and hMCH-2R of several Ac-hMCH(6-16)-NH2 analogs with various D-amino acid residues in position 6. Similarly to the D-Arg6 peptide, these compounds displayed enhanced selectivity for hMCH-1R. At hMCH-2R, peptides with hydrophobic, hydrophilic, basic, or acidic side chains in position 6 were significantly weaker binders and agonists than Ac-hMCH(6-16)-NH2. In contrast, at hMCH-1R, their binding affinities were similar to those of Ac-hMCH(6-16)-NH2 and its D-Arg6 analog, but their agonist potencies were noticeably lower. In fact, none of the new compounds was more potent at, and selective for, hMC-1R than the D-Arg6 analog. This confirmed yet again that the guanidine group in the side chain of residue 6 is essential for maximum activity at hMCH-1R (24-27), and that its orientation allowed by the D-chiral center contributes to hMCH-1R selectivity.
The disulfide ring of the Ac-hMCH(6-16)-NH2 peptide
encompasses two residues, Gly and Pro, known to facilitate formation of bends in peptide chains. Although conformationally constrained pyrrolidine ring of the Pro residue imposes steric restrictions on the
peptide backbone, the flexible chain of the Gly residue is able to
adopt various conformations. To stabilize some of the low energy
conformers of biological significance, Gly is frequently replaced with
sterically constraining amino acids such as -amino acids
(L or D) or ,-di-alkylated amino acids.
These replacements are usually done with anticipation that changes
observed in peptide-receptor recognition pattern can indicate
conformational requirements necessary for ligand-receptor interactions
and aid to the design of new potent and selective agonist and antagonists.
In the present study, the effect on biological activity of Ac-hMCH(6-16)-NH2 of conformational changes in position 10 was explored through analogs in which Gly10 was replaced with various L-amino acids. Peptides with hydrophobic, linear, or branched side chains in position 10 (the Ala10, Leu10, or Nle10 analogs), and with the hydrophobic aromatic side chain in the same position (the Phe10 analog), were slightly less potent at both receptors than the parent compound, suggesting that their biologically active conformations are rather similar. However, further decrease of conformational freedom in analogs with bulky Cha or (2')Nal in position 10 affected significantly the interactions with hMCH-2R and hMCH-1R. Thus, the side chains of Cha10 and (2')Nal10 unfavorably alter the conformation of the peptide backbone and/or hinder formation of ligand-receptor complexes.
The steric constrain introduced by Pro in position 10 was deleterious
to binding and activity at both receptors. Inability of the
Pro10 analog to activate hMCH-1R and hMCH-2R even at
micromolar concentrations could also be attributed to the lack of free
NH in position 10 available for H-bond formation. However, the only
slightly lower agonist potency of a further analog lacking free NH
in position 10, the N-Me-Gly10
peptide,3 did not support this assumption. This suggested
that potential H-bonds involving NH of Gly10 do not
contribute significantly to the stabilization of the biologically active conformers and/or peptide-receptor complexes.
The effect of conformational changes in position 10 on biological activity of Ac-hMCH(6-16)-NH2 was further studied in conjunction with potential ionic interactions between the residues in position 10 and the MCH receptors. Evaluation of the Arg10, Lys10, and Glu10 analogs of Ac-hMCH(6-16)-NH2 in binding and functional assays revealed that both basic and acidic side chains in position 10 are deleterious to agonism at the MCH receptors. These side chains might be repelled by identical charges in the side chains of the receptors or might form salt bridges and new H-bonds unfavorable to stabilization of biologically active conformations or ligand-receptor complexes. At hMCH-1R, binding and agonist activities of compounds with the hydrophilic side chains of Asn and Ser in position 10 were similar to, or slightly lower than, those of Ac-hMCH(6-16)-NH2. This seems to suggest that, in the ligand-receptor complexes, these hydrophilic side chains are not in direct contact with the receptors but are facing the outside environment. In contrast, the side chain of Asn10 appears to affect significantly interactions with hMCH-2R; this analog was ~40 times less active at hMCH-2R than the parent compound. As a consequence of these differences in molecular recognition pattern of compounds with L-amino acids in place of Gly10, the Asn10 analog emerged as the most selective high affinity ligand for hMCH-1R.
Subsequently, the cyclic peptide designed to accommodate structural
changes at both positions 6 and 10 of Ac-hMCH(6-16)-NH2, described above as the most favorable for high potency and selectivity at hMCH-1R, was evaluated. The new compound with
D-Arg6 and Asn10, analog
26 (Structure 3), was a potent high affinity agonist at
hMCH-1R (IC50 = 0.5 nM, EC50 = 47 nM) but a poor ligand for hMCH-2R (see also Fig. 1).
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To gain further insight into the conformational properties of our new hMCH-1R selective agonist, several analogs of compound 26 were synthesized and evaluated in binding and functional assays at both hMCH-1R and hMCH-2R. Similarly to the parent peptide, these compounds were virtually inactive at hMCH-2R and, rather unexpectedly, their binding affinities and agonist potencies were noticeably lower at hMCH-1R than those of Ac-hMCH(6-16)-NH2. Replacement of Asn10 in position 10 of compound 26 with Gln showed that the side chain of Gln in position 10, merely one carbon atom longer than the side chain of Asn, significantly disturbs molecular recognition at hMCH-1R. Additionally, potency and selectivity of compound 26 at hMCH-1R were affected by incorporation of Nle in place of Met8 (compound 28), or by the simultaneous omission of the N-terminal acetyl group (compound 29). The observed drop of potency was larger than previously reported (24) for other analogs of Ac-hMCH(6-16)-NH2 with Nle8. Not unexpectedly, substitution of Arg14 with Ala in compound 26 yielded peptide 28, that was only 4-fold less potent at hMCH-1R than the parent compound, thus confirming that the Arg14 side chain is not essential for ligand-receptor interactions (24-27).
Our present study yielded a cyclic peptide that is a potent and selective agonist at hMCH-1R. The new compound is an analog of the nonselective Ac-hMCH(6-16)-NH2 agonist with D-Arg instead of L-Arg in position 6 and Asn in place of Gly in position 10. These alterations render the parent molecule virtually inactive at hMCH-2R even at micromolar concentrations but do not reduce its binding affinity and potency at hMCH-1R.
In summary, we report here a potent agonist at hMCH-1R, which shows
more than 200-fold selectivity with respect to hMCH-2R This compound
should be useful in elucidation of the physiological role of the human
MCH-1R. The insight gained in this study should also be helpful in the
design of new agonists; we are continuing our investigation in this direction.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Medicinal Chemistry, Merck Research Laboratories, R50G-141, Rahway, NJ 07065. Tel.: 732-594-4798; Fax: 732-594-8080; E-mail: maria_bednarek@merck.com.
Published, JBC Papers in Press, February 11, 2002, DOI 10.1074/jbc.M200563200
2 Throughout this report, the numbering of the amino acid residues in hMCH has been retained for all cyclic analogs of this neuropeptide.
3 M. A. Bednarek, D. L. Hreniuk, C. Tan, O. C. Palyha, D. J. MacNeil, L. H. Y. Van der Ploeg, A. D. Howard, and S. D. Feighner, manuscript submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: MCH, melanin-concentrating hormone; CHO, Chinese hamster ovary; Gva, 5-guanidinivaleric acid; hMCH, human melanin-concentrating hormone; hMCH-1R, human melanin-concentrating hormone receptor 1; hMCH-2R, human melanin-concentrating hormone receptor 2; Mpr, 3-mercaptopropionic acid; Cit, citrulline; HPLC, high performance liquid chromatography.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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