Human Melanin-concentrating Hormone Receptor Research Articles

Discovery, synthesis, and structure–activity relationship of 6-aminomethyl-7,8-dihydronaphthalenes as human melanin-concentrating hormone receptor 1 antagonists

Human melanin-concentrating hormone receptor 1 (hMCHR1) antagonists are promising targets for obesity treatment. We identified the tetrahydronaphthalene derivative 1a with modest binding affinity for hMCHR1 by screening an in-house G protein-coupled receptor (GPCR) ligand library. We synthesized a series of 6-aminomethyl-5,6,7,8-tetrahydronaphthalenes and evaluated their activity as hMCHR1 antagonists. Modification of the biphenylcarbonylamino group revealed that the biphenyl moiety played a crucial role in the interaction of the antagonist with the receptor. The stereoselective effect of the chiral center on binding affinity generated the novel 6-aminomethyl-7,8-dihydronaphthalene scaffold without a chiral center. Optimization of the amino group led to the identification of a potent antagonist 2s (4′-fluoro-N-[6-(1-pyrrolidinylmethyl)-7,8-dihydro-2-naphthalenyl][1,1′-biphenyl]-4-carboxamide), which significantly inhibited the nocturnal food intake in rats after oral administration. Pharmacokinetic analysis confirmed that 2s had good oral bioavailability and brain penetrance. This antagonist appears to be a viable lead compound that can be used to develop a promising therapy for obesity.

Discovery of 1,3-disubstituted-1H-pyrrole derivatives as potent Melanin-Concentrating Hormone Receptor 1 (MCH-R1) antagonists

A series of 1,3-disubstituted-1H-pyrrole-based antagonists of the human Melanin-Concentrating Hormone Receptor 1 (h-MCH-R1) are reported. High-throughput screening of the AstraZeneca compound collection yielded 1, a hit with moderate affinity towards MCH-R1. Subsequent structural manipulations and SAR analysis served to rationalize potency requirements, and 12 was identified as a novel, functional MCH-R1 antagonist with favorable pharmacokinetic properties.

Discovery of potent and stable conformationally constrained analogues of the MCH R1 antagonist SB-568849

A strategy of systematically targeting more rigid analogues of the known MCH R1 receptor antagonist, SB-568849, serendipitously uncovered a binding mode accessible to N-aryl-phthalimide ligands. Optimisation to improve the stability of this compound class led to the discovery of novel N-aryl-quinazolinones, benzotriazinones and thienopyrimidinones as selective ligands with good affinity for human melanin-concentrating hormone receptor 1.

Characterization of a neuronal cell line expressing native human melanin-concentrating hormone receptor 1 (MCHR1)

Melanin-concentrating hormone (MCH), an orexigenic neuropeptide in mammals, activates a G-protein coupled receptor, MCHR1. It is expected that antagonists of MCHR1 function will prove therapeutically useful as anti-obesity agents. Intracellular signaling by MCHR1 has been investigated primarily using non-neural cell lines expressing the recombinant receptor, in which MCHR1 has been shown to couple to Gαi/o and Gαq G-proteins. While these cell lines have been widely utilized to discover and optimize small molecule antagonists, it is unknown whether the intracellular signaling pathways in these cells accurately reflect those in neurons. Thus, we sought to develop a neurally derived cell line endogenously expressing MCHR1. IMR32, a human neuroblastoma cell line, has been shown to express MCHR1 mRNA; however, we were unable to detect either MCH-binding or MCH-stimulated Ca++-mobilization in these cells. Following transfection of IMR32 cells with a plasmid encoding human Gα16 G-protein, we isolated a cell line, I3.4.2, which responded to MCH in Ca++-mobilization assays. We found that the expression level of MCHR1 mRNA in I3.4.2 cells was 2000-fold higher than in the parent cell line. Using [125I]MCH saturation-binding to I3.4.2 cell membranes, we estimated the Bmax as 0.72pmol/mg protein and the Kd as 0.35nM. We report that Ca++-mobilization in I3.4.2 cells was insensitive to pertussis toxin (Ptx) treatment, indicating that signaling was via Gαq G-proteins. Furthermore, negative results in cAMP accumulation assays confirmed the lack of signaling via the Gαi/o G-proteins. Our results suggest that the I3.4.2 cell line may be useful for characterization of MCHR1 activity in a neural-derived cell line.

Mutation analysis of the MCHR1 gene in human obesity

The importance of the melanin-concentrating hormone (MCH) system for regulation of energy homeostasis and body weight has been demonstrated in rodents. We analysed the human MCH receptor 1 gene (MCHR1) with respect to human obesity. This consisted of genomic screening of 13.4 kb encompassing the MCHR1 in extremely obese German children and adolescents and association analyses for two coding single nucleotide polymorphisms (SNPs). To confirm initial positive association results, additional association studies and transmission disequilibrium tests in further German, Danish, French and American samples were conducted. Selected SNPs were investigated using functional in vitro studies and reporter gene assays. Single-stranded conformation polymorphism analysis, re-sequencing, PCR-restriction fragment length polymorphism analyses, tetra-primer amplification refractory mutation systems, matrix-assisted laser desorption/ionization time of flight mass spectrometry and reporter gene assays were carried out as well as measuring inositol phosphate formation, inhibition of cAMP formation and activation of p42/44 MAP kinase. We identified 11 infrequent variations and two SNPs in the MCHR1 coding sequence and 18 SNPs (eight novel) in the flanking sequence. Association and transmission disequilibrium with obesity were detected for several SNPs in independent study groups of German obese children and adolescents and controls. In two German samples, encompassing 4056 and 295 individuals, trends towards association with obesity were detected. Findings in a second epidemiological German sample and in Danish, French and American samples were negative. Functional in vitro studies as well as reporter gene assays revealed no significant results. Our initial association of MCHR1 alleles/haplotype detected might be related to juvenile-onset obesity, conditional on a particular genetic and/or environmental background. Alternatively, we could not exclude the possibility that the initially detected association represented a false positive finding.

A Point Mutation in the Human Melanin Concentrating Hormone Receptor 1 Reveals an Important Domain for Cellular Trafficking

G protein-coupled receptors (GPCRs) are heptahelical integral membrane proteins that require cell surface expression to elicit their effects. The lack of appropriate expression of GPCRs may be the underlying cause of a number of inherited disorders. There is evidence that newly synthesized GPCRs must attain a specific conformation for their correct trafficking to the cell surface. In this study, we show that a single point mutation in human melanin-concentrating hormone receptor (hMCHR1) at position 255 (T255A), which is located at the junction of intracellular loop 3 and transmembrane domain 6, reduces the hMCHR1 cell surface expression level to 20% of that observed for the wild-type receptor. Most of these mutant receptors are located intracellularly, as opposed to the wild-type receptor, which is located primarily on the cell surface. Immunoprecipitation experiments show that hMCHR1-T255A has reduced glycosylation compared with the wild-type receptor and is associated with the chaperone protein, calnexin, and it colocalizes in the endoplasmic reticulum with KDEL-containing proteins. We also demonstrate that a cell-permeable small molecule antagonist of hMCHR1 can function as a pharmacological chaperone to restore cell surface expression of this and other MCHR1 mutants to wild-type levels. Once rescued, the T255A mutant couples to Gq proteins as efficiently as the wild-type receptor. These data suggest that this single mutation produces an hMCHR1 that folds incorrectly, resulting in its retention in the endoplasmic reticulum, but once rescued to the cell surface can still function normally.

Arginine Residue 155 in the Second Intracellular Loop Plays a Critical Role in Rat Melanin-Concentrating Hormone Receptor 1 Activation

Melanin-concentrating hormone (MCH) receptor 1 (MCH1R) is a class A G protein-coupled receptor. The MCH system has been linked to a variety of physiological functions, including the regulation of feeding and energy metabolism. We recently reported the importance of a dibasic motif in the membrane-proximal C-terminal region for MCH1R function. Here we reveal that an Arg residue in intracellular loop 2 of MCH1R plays a critical role in receptor function. We analyzed the roles of two distinct motifs, BBXXB and BXBB (in which B is a basic residue and X is a nonbasic residue), located in the three intracellular loops of MCH1R. Triple-substitution mutants of intracellular loops 1 and 3 could still activate calcium mobilization, albeit with lower efficacy or potency. However, mutations in intracellular loop 2 led to a complete loss of induction of signal transduction without changing the high affinity constant (Kd) value. By analyzing a series of single-substitution mutants, a point mutation of Arg155 in intracellular loop 2 was found to be responsible for the signaling pathway elicited by MCH. In addition, substitution at positions corresponding to Arg155 in human MCH receptor 2 and rat somatostatin receptor 2 also markedly abolished their ligand-induced signaling capacities, indicating that this Arg is a recognition determinant in several G protein-coupled receptors.

SVK14 cells express an MCH binding site different from the MCH 1 or MCH 2 receptor

Melanin-concentrating hormone (MCH) is a cyclic peptide, mainly involved in the regulation of skin pigmentation in teleosts and feeding behavior in mammals. The human keratinocyte SVK14 cell line has been previously shown to express binding sites for the MCH analog [ 125 I] -[ Phe 13,3 -iodo-Tyr 19] MCH. We report here that: (1) this binding site similarly recognized [ 125 I] -[3 -iodo-Tyr 13] MCH; (2) its pharmacological profile clearly differed from those observed at the two human MCH receptor subtypes, MCH 1-R and MCH 2-R; (3) MCH did not induce any effect on second messenger systems (including cAMP, calcium, and MAP kinase signaling pathways), and (4) no mRNAs corresponding to the MCH receptors were found. In conclusion, the binding site characterized in the SVK14 cell line is distinct from the MCH 1 and MCH 2 receptors and deserves therefore further investigation.

Synthesis and biological evaluation in vitro of selective, high affinity peptide antagonists of human melanin-concentrating hormone action at human melanin-concentrating hormone receptor 1.

Human melanin-concentrating hormone (hMCH) and many of its analogues are potent but nonspecific ligands for human melanin-concentrating hormone receptors 1 and 2 (hMCH-1R and hMCH-2R). To differentiate between the physiological functions of these receptors, selective antagonists are needed. In this study, analogues of Ac-Arg(6)-cyclo(S-S)(Cys(7)-Met(8)-Leu(9)-Gly(10)-Arg(11)-Val(12)-Tyr(13)-Arg(14)-Pro(15)-Cys(16))-NH(2), a high affinity but nonselective agonist at hMCH-1R and hMCH-2R, were prepared and tested in binding and functional assays on cells expressing these receptors. In the new analogues, 5-aminovaleric acid (Ava) was incorporated in place of the Leu(9)-Gly(10) and/or Arg(14)-Pro(15) segments of the disulfide ring. Several of these compounds turned out to be high affinity antagonists selective for hMCH-1R. Moreover, even at micromolar concentrations, they were devoid of agonist potency at both hMCH receptors and not effective as hMCH-2R antagonists. For example, peptide 14, Gva(6)- cyclo(S-S)(Cys(7)-Met(8)-Leu(9)-Gly(10)-Arg(11)-Val(12)-Tyr(13)-Ava(14,15)-Cys(16))-NH(2), (Gva = 5-guanidinovaleric acid), was a full competitive hMCH-1R antagonist (IC(50) = 14 nM, K(B) = 0.9 nM) with more than 1000-fold selectivity over hMCH-2R. Examination of various compounds with Ava in positions 9,10 and/or 14,15 revealed that the Leu(9)-Gly(10) and Arg(14)-Pro(15) segments of the disulfide ring are the principal structural elements determining hMCH-1R selectivity and ability to act as a hMCH-1R antagonist.

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

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 Arg(6) of hMCH, Ac-Arg(6)-cyclo(S-S)(Cys(7)-Met(8)-Leu(9)-Gly(10)-Arg(11)-Val(12)-Tyr(13)-Arg(14)-Pro(15)-Cys(16))-NH(2), Ac-hMCH(6-16)-NH(2), 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)-NH(2) 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-dArg(6)-cyclo(S-S)(Cys(7)-Met(8)-Leu(9)-Asn(10)-Arg(11)-Val(12)-Tyr(13)-Arg(14)-Pro(15)-Cys(16))-NH(2), was a potent hMCH-1R agonist (IC(50) = 0.5 nm, EC(50) = 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.

Identification and Pharmacological Characterization of a Novel Human Melanin-concentrating Hormone Receptor, MCH-R2

Melanin-concentrating hormone (MCH) is a neuropeptide highly expressed in the brain that regulates several physiological functions mediated by receptors in the G protein-coupled receptor family. Recently an orphan receptor, SLC-1, has been identified as an MCH receptor (MCH-R1). Herein we identify and characterize a novel receptor for human MCH (MCH-R2). The receptor is composed of 340 amino acids encoded by a 1023-base pair cDNA and is 35% homologous to SLC-1. (125)I-MCH specifically bound to Chinese hamster ovary cells stably expressing MCH-R2. MCH stimulated dose-dependent increases in intracellular free Ca(2+) and inositol phosphate production in these cells but did not affect cAMP production. The pharmacological profile for mammalian MCH, [Phe(13),Tyr(19)]MCH, and salmon MCH at MCH-R2 differed compared with MCH-R1 as assessed by intracellular signaling and radioligand binding assays. The EC(50) in signaling assays and the IC(50) in radioligand binding assays of salmon MCH was an order of magnitude higher than mammalian MCH at MCH-R2. By comparison, the EC(50) and IC(50) values of salmon MCH and mammalian MCH at MCH-R1 were relatively similar. Blot hybridization revealed exclusive expression of MCH-R2 mRNA in several distinct brain regions, particularly in the cortical area, suggesting the involvement of MCH-R2 in the central regulation of MCH-mediated functions.

Identification and characterization of a second melanin-concentrating hormone receptor, MCH-2R.

Melanin-concentrating hormone (MCH) is a 19-aa cyclic neuropeptide originally isolated from chum salmon pituitaries. Besides its effects on the aggregation of melanophores in fish several lines of evidence suggest that in mammals MCH functions as a regulator of energy homeostasis. Recently, several groups reported the identification of an orphan G protein-coupled receptor as a receptor for MCH (MCH-1R). We hereby report the identification of a second human MCH receptor termed MCH-2R, which shares about 38% amino acid identity with MCH-1R. MCH-2R displayed high-affinity MCH binding, resulting in inositol phosphate turnover and release of intracellular calcium in mammalian cells. In contrast to MCH-1R, MCH-2R signaling is not sensitive to pertussis toxin and MCH-2R cannot reduce forskolin-stimulated cAMP production, suggesting an exclusive G(alpha)q coupling of the MCH-2R in cell-based systems. Northern blot and in situ hybridization analysis of human and monkey tissue shows that expression of MCH-2R mRNA is restricted to several regions of the brain, including the arcuate nucleus and the ventral medial hypothalamus, areas implicated in regulation of body weight. In addition, the human MCH-2R gene was mapped to the long arm of chromosome 6 at band 6q16.2-16.3, a region reported to be associated with cytogenetic abnormalities of obese patients. The characterization of a second mammalian G protein-coupled receptor for MCH potentially indicates that the control of energy homeostasis in mammals by the MCH neuropeptide system may be more complex than initially anticipated.

125I]-S36057: a new and highly potent radioligand for the melanin-concentrating hormone receptor.

Shortened, more stable and weakly hydrophobic analogues of melanin-concentrating hormone (MCH) were searched as candidates for radioiodination. Starting from the dodecapeptide MCH(6 - 17), we found that: (1) substitution of Tyr(13) by a Phe residue; (2) addition of a 3-iodo-Tyr residue at the N-terminus; and (3) addition of a hydrophilic spacer 8-amino-3,6-dioxyoctanoyl between the 3-iodo-Tyr and MCH(6 - 17) (compound S36057), led to an agonist more potent than MCH itself in stimulating [35S]-GTPgammaS binding at membranes from HEK293 cells stably expressing the human MCH receptor. Specific binding of [125I]-S36057 was found in HEK293 and CHO cell lines stably expressing the human MCH receptor. This radioligand recognized a similar number of binding sites (ca. 800 fmol mg(-1)) than [125I]-[3-iodo Tyr(13)]-MCH. However, the K(D) for [125I]-S36057 obtained from saturation studies (0.037 nM) or from binding kinetics (0.046 nM) was at least 10 fold higher to that of [125I]-[3-iodo Tyr(13)]-MCH (0.46 nM). Affinities determined for a series of MCH analogues were similar with both radioligands, S36057 being the most potent compound tested (K(i)=0.053 nM). Finally, [125I]-S36057 also potently labelled the MCH receptor in membranes from whole rat brain (K(D) 0.044 nM, B(max)=11 fmol mg(-1)). In conclusion, [125I]-S36057 is a more potent and more stable radioligand than [125I]-[3-iodo Tyr(13)]-MCH that will represent a reliable tool for binding assays in the search of novel MCH ligands. It should also provide great help for autoradiographic studies of the MCH receptor distribution in the central nervous system.

Structure-Activity Relationship Studies of Melanin-concentrating Hormone (MCH)-related Peptide Ligands at SLC-1, the Human MCH Receptor

Melanin-concentrating hormone (MCH) is a cyclic nonadecapeptide involved in the regulation of feeding behavior, which acts through a G protein-coupled receptor (SLC-1) inhibiting adenylcyclase activity. In this study, 57 analogues of MCH were investigated on the recently cloned human MCH receptor stably expressed in HEK293 cells, on both the inhibition of forskolin-stimulated cAMP production and guanosine-5'-O-(3-[(35)S]thiotriphosphate ([(35)S]- GTPgammaS) binding. The dodecapeptide MCH-(6-17) (MCH ring between Cys(7) and Cys(16), with a single extra amino acid at the N terminus (Arg(6)) and at the C terminus (Trp(17))) was found to be the minimal sequence required for a full and potent agonistic response on cAMP formation and [(35)S]- GTPgammaS binding. We Ala-scanned this dodecapeptide and found that only 3 of 8 amino acids of the ring, namely Met(8), Arg(11), and Tyr(13), were essential to elicit full and potent responses in both tests. Deletions inside the ring led either to inactivity or to poor antagonists with potencies in the micromolar range. Cys(7) and Cys(16) were substituted by Asp and Lys or one of their analogues, in an attempt to replace the disulfide bridge by an amide bond. However, those modifications were deleterious for agonistic activity. In [(35)S]- GTPgammaS binding, these compounds behaved as weak antagonists (K(B) 1-4 microm). Finally, substitution in MCH-(6-17) of 6 out of 12 amino acids by non-natural residues and concomitant replacement of the disulfide bond by an amide bond led to three compounds with potent antagonistic properties (K(B) = 0.1-0.2 microm). Exploitation of these structure-activity relationships should open the way to the design of short and stable MCH peptide antagonists.

Molecular Cloning and Functional Characterization of MCH2, a Novel Human MCH Receptor

Melanin-concentrating hormone (MCH) is involved in the regulation of feeding and energy homeostasis. Recently, a 353-amino acid splice variant form of the human orphan receptor SLC-1 () (hereafter referred to as MCH(1)) was identified as an MCH receptor. This report describes the cloning and functional characterization of a novel second human MCH receptor, which we designate MCH(2), initially identified in a genomic survey sequence as being homologous to MCH(1) receptors. Using this sequence, a full-length cDNA was generated with an open reading frame of 1023 base pairs, encoding a polypeptide of 340 amino acids, with 38% identity to MCH(1) and with many of the structural features conserved in G protein-coupled receptors. This newly discovered receptor belongs to class 1 (rhodopsin-like) of the G protein-coupled receptor superfamily. HEK293 cells transfected with MCH(2) receptors responded to nanomolar concentrations of MCH with an increase in intracellular Ca(2+) levels and increased cellular extrusion of protons. In addition, fluorescently labeled MCH bound with nanomolar affinity to these cells. The tissue localization of MCH(2) receptor mRNA, as determined by quantitative reverse transcription-polymerase chain reaction, was similar to that of MCH(1) in that both receptors are expressed predominantly in the brain. The discovery of a novel MCH receptor represents a new potential drug target and will allow the further elucidation of MCH-mediated responses.

Molecular characterization of the melanin-concentrating hormone/receptor complex: identification of critical residues involved in binding and activation.

A molecular model of the human melanin-concentrating hormone (MCH) peptide was constructed and docked into a helical, bacteriorhodopsin-based model of the recently identified human MCH receptor. From this hormone-receptor complex, potential sites of agonist-receptor interaction were identified, and site-directed mutagenesis was used to substitute residues predicted to reside within the receptor binding pocket. Substitution of Asp(123)(3.32) in the third transmembrane domain of the receptor resulted in a loss of detectable (125)I-MCH binding and of MCH-stimulated Ca(2+) flux; cell surface expression of the mutant receptor was not affected. Arg(11) and Arg(14) of the MCH ligand were identified as potential sites of interaction with Asp(123)(3.32). [Ala(14)]-MCH was equipotent to native MCH in its ability to bind to and activate the wild-type MCH receptor, whereas [Ala(11)]-MCH displayed a 3000-fold reduction in binding affinity and a complete loss of measurable functional activity. Furthermore, [Lys(11)]-MCH and [D-Arg(11)]-MCH displayed reduced affinity for the receptor. [Lys(11)]-MCH was observed to be a partial agonist, eliciting approximately 67% of the native peptide's activity in a Ca(2+) flux assay, and [D-Arg(11)]-MCH was determined to be a functional antagonist with a K(b) valve of 15.8 microM. These data provide evidence that a basic moiety with specific stereochemical requirements at this site is needed for receptor activation. We conclude that both Asp(123)(3.32) in the MCH receptor and Arg(11) in the MCH peptide are required for the formation of the MCH peptide/receptor complex and propose that they form a direct interaction that is critical for receptor function.