Synthesis and biological evaluation of imidazole-based small molecule antagonists of the melanocortin 4 receptor (MC4-R)

A novel series of imidazole-based small molecule antagonists of the melanocortin 4 receptor (MC4-R) is reported. Members of this series have been identified, which exhibit sub-micromolar binding affinity for the MC4-R, functional potency <100nM, and good oral exposure in rat. Antagonists of the MC4-R are potentially useful in the therapeutic treatment of involuntary weight loss due to advanced age or disease (e.g. cancer or AIDS), an area of large, unmet medical need.     

Ghrelin-induced food intake and growth hormone secretion are altered in melanocortin 3 and 4 receptor knockout mice

Ghrelin stimulates food intake in part by activating hypothalamic neuropeptide Y (NPY) neurons/agouti related peptide (AGRP) neurons. We investigated the role of AGRP/melanocortin signaling in ghrelin-induced food intake by studying melanocortin 3 and 4 receptor knockout (MC3R KO and MC4R KO) mice. We also determined whether reduced ghrelin levels and/or an altered sensitivity to the GH-stimulating effects of ghrelin accompany the obesity syndromes of MC3R KO and MC4R KO mice. Compared to wild-type (WT) mice, the effects of ghrelin on food intake were reduced in MC3R KO and MC4R KO mice and circulating ghrelin levels were reduced in female MC4R KO mice. Female MC3R KO and MC4R KO mice exhibited a diminished responsiveness to the GH-releasing effects of ghrelin. Thus, deletion of the MC3R or MC4R results in a decreased sensitivity to ghrelin and verifies the involvement in the melanocortin system in ghrelin-induced food intake.     

Synthesis and SAR of potent and orally bioavailable tert-butylpyrrolidine archetype derived melanocortin subtype-4 receptor modulators

Discovery of a series of tert-butyl pyrrolidine derived, potent and orally bioavailable melanocortin receptor subtype-4 (MC4R) selective modulators is disclosed.     

The agouti-related protein decapeptide (Yc[CRFFNAFC]Y) possesses agonist activity at the murine melanocortin-1 receptor

Agouti-related protein (AGRP) is a naturally occurring antagonist of the brain melanocortin receptors (MC3R and MC4R) and is physiologically implicated as participating in feeding behavior and energy homeostasis. The human AGRP decapeptide Yc[CRFFNAFC]Y has been previously reported as binding to the human MC3 and MC4 receptors and antagonizing the MC4 receptor. We have synthesized this decapeptide and pharmacologically characterized it at the murine melanocortin receptors and found it to possess MC4R antagonist activity (pA(2) = 6.8) and, unexpectedly, MC1R agonist activity (EC(50) = 2.89 microM). This study characterizes the first AGRP-based peptide agonist at the melanocortin receptors.     

Further evidence for ancient role of ACTH peptides at melanocortin (MC) receptors; pharmacology of dogfish and lamprey peptides at dogfish MC receptors

The cloning of melanocortin (MC) receptors in distant species has provided us tools to get insight in how the ligand-receptors interactions in the MC system have evolved. We have however lacked studies on pharmacology of native ancient melanocortin peptides at the ancient MC receptors. In this paper we synthesized melanocortin peptides from both the sea lamprey (Petromyzon marinus) and spiny dogfish (Squalus acanthias) and tested them on the MC3 and MC4 receptors from spiny dogfish. The results show that both the dogfish and lamprey ACTH peptides have similar or higher affinity than the dogfish alpha-, beta- and gamma-MSH peptides to the dogfish MC3 and MC4 receptors. Moreover, both the dogfish and lamprey ACTH peptides have more than 10-fold higher affinity than alpha-MSH to the dogfish MC4 receptor. We also show that dogfish delta-MSH is able to bind to MC receptors and its potency is higher than of dogfish beta-MSH, which is considered to be its precursor. Our results provide the first evidence that native ACTH ligands from dogfish and lamprey have a preference above native MSH peptides to ancient version of the MC3 and MC4 receptors. This further strengthens the hypotheses that the ligand contributing to the first version of the melanocortin ligand-receptor system resembled ACTH.     

Contribution of melanocortin receptor exoloops to Agouti-related protein binding.

Agouti-related protein (AGRP) is an endogenous antagonist of melanocortin action that functions in the hypothalamic control of feeding behavior. Although previous studies have shown that AGRP binds three of the five known subtypes of melanocortin receptor, the receptor domains participating in binding and the molecular interactions involved are presently unknown. The present studies were designed to examine the contribution of extracytoplasmic domains of the melanocortin-4 receptor (MC4R) to AGRP binding by making chimerical receptor constructs of the human melanocortin-1 receptor (MC1R; a receptor that is not inhibited by AGRP) and the human MC4R (a receptor that is potently inhibited by AGRP). Substitutions of the extracytoplasmic NH2 terminus and the first extracytoplasmic loop (exoloop) of the MC4R with homologous domains of the MC1R had no effect on AGRP (87-132) binding affinity or inhibitory activity (the ability to inhibit melanocortin-stimulated cAMP generation). In contrast, cassette substitutions of exoloops 2 and 3 of the MC4R with the homologous exoloops of the MC1R resulted in a substantial loss of AGRP binding affinity and inhibitory activity. Conversely, the exchange of exoloops 2 and 3 of the MC1R with the homologous exoloops of the MC4R was found to confer AGRP binding and inhibitory activity to the basic structure of the MC1R. Importantly, these substitutions did not affect the ability of the alpha-melanocyte stimulating hormone analogue [Nle4,D-Phe7] melanocyte stimulating hormone to bind or activate the chimeric receptors. These data indicate that exoloops 2 and 3 of the melanocortin receptors are important for AGRP binding.     

黑皮质素受体-4调节通路的研究进展

黑皮质素系统来自阿片-促黑素细胞皮质素原,在中枢摄食行为和能量平衡代谢中起到重要作用,此系统生理功能的发挥主要通过与下丘脑神经元细胞上特定膜受体（黑皮质素受体）结合完成。黑皮质素受体（MCR）有五种亚型（MC1R-MC5R）,其中参与体重调节的受体主要是黑皮质素受体3（MC3R）和黑皮质素受体4（MC4R）。MC4R属于G蛋白耦联受体,具有七次跨膜结构。作为一种膜受体,MC4R发挥体重调节作用,一方面受外界激动剂或拮抗剂的调节;另一方面,此受体活化后会影响到细胞内的信号调节通路。研究MC4R的功能首先要了解受体的结构,本文对G蛋白耦联受体的结构进行了较详细的叙述,MC4R经信号调节通路,激活腺苷酸环化酶,增加cAMP的浓度,最终通过影响细胞内基因的转录和翻译,来调节体重和能量的消耗。     

Central administration of peptide and small molecule MC4 receptor antagonists induce hyperphagia in mice and attenuate cytokine-induced anorexia

We investigated the effect of melanocortin 4 receptor (MC4) antagonists on food intake in mice. Food intake during the light phase was significantly increased by ICV administration of mixed MC3/MC4 antagonists (AgRP and SHU9119) or MC4 selective antagonist peptide [(Cyclo (1-5)[Suc-D-Nal-Arg-Trp-Lys]NH2] (MBP10) and the small molecule antagonists THP and NBI-30. Both mixed and selective antagonists significantly reversed anorexia induced by ICV administration of the MC4 agonist (c (1-6) HfRWK-NH2) and the cytokine IL-1beta. These findings provide pharmacological evidence that the MC4 receptor mediates the effects of melanocortin agonists and antagonists on food intake in mice, and support the idea that selective small molecule MC4 antagonists may be useful as therapeutics for cachexia.     

Synthesis and characterization of trans-4-(4-chlorophenyl)pyrrolidine-3-carboxamides of piperazinecyclohexanes as ligands for the melanocortin-4 receptor

A series of trans-N-alkyl-4-(4-chlorophenyl)pyrrolidine-3-carboxamides of piperazinecyclohexanemethylamines was synthesized and characterized for binding and function at the melanocortin-4 receptor (MC4R), and several potent benzylamine derivatives were identified. Compound 18v was found to bind MC4R with potent affinity (K_i = 0.5 nM) and high selectivity over the other melanocortin subtypes and behaved as a functional antagonist (IC₅₀ = 48 nM).     

Melanocortin-4 receptor-mediated inhibition of apoptosis in immortalized hypothalamic neurons via mitogen-activated protein kinase

The melanocortin-4 receptor (MC4R) is a seven transmembrane member of the melanocortin receptor family. The GT1-1 cell line exhibits endogenous expression of MC4R. In this study, GT1-1 cells were used to study MC4R signaling pathways and to examine the effects of melanocortin receptor agonist NDP-MSH on apoptosis. MC4R mRNA expression was demonstrated by RT-PCR. Functional melanocortin receptor expression was implied by specific binding of NDP-MSH and cAMP production. NDP-MSH-stimulated GnRH release in a dose-dependent manner. Serum deprivation-induced apoptosis in GT1-1 cells, and the NDP-MSH inhibited this effect. The melanocortin receptor antagonist SHU9119 blocked the antiapoptotic actions of NDP-MSH, and the MAP kinase inhibitor PD98059 significantly attenuated the antiapoptotic effect. NDP-MSH-stimulated ERK1/2 phosphorylation in a dose-dependent manner. ERK1/2 phosphorylation could be abolished by SHU9119. In GT1-1 cells, melanocortin receptor activation causes ERK1/2 phosphorylation. In these cells, MC4R activation is also associated with antiapoptotic effects.     

Agouti-related protein has an inhibitory paracrine role in the rat adrenal gland

alpha-Melanocyte-stimulating-hormone (alpha-MSH) is an agonist at the melanocortin 3 receptor (MC3-R) and melanocortin 4 receptor (MC4-R). alpha-MSH stimulates corticosterone release from rat adrenal glomerulosa cells in vitro. Agouti-related protein (AgRP) an endogenous antagonist at the MC3-R and MC4-R, is expressed in the adrenal gland. We investigated the expression of the MC3-R and MC4-R and the role of AgRP in the adrenal gland. MC3-R and MC4-R expression was detected in rat adrenal gland using RT-PCR. The effect of AgRP on alpha-MSH-induced corticosterone release was investigated using dispersed rat adrenal glomerulosa cells. AgRP administered alone did not affect corticosterone release, but co-administration of AgRP and alpha-MSH attenuated alpha-MSH-induced corticosterone release. To investigate glucocorticoid feedback, adrenal AgRP expression was compared in rats treated with dexamethasone to controls. AgRP mRNA was increased in rats treated with dexamethasone treatment compared to controls. Our findings demonstrate that adrenal AgRP mRNA is regulated by glucocorticoids. AgRP acting via the MC3-R or MC4-R may have an inhibitory paracrine role, blocking alpha-MSH-induced corticosterone secretion.     

Anxiety-like behavior induced by IL-1beta is modulated by alpha-MSH through central melanocortin-4 receptors

The proinflammatory cytokine interleukin-1beta (IL-1beta) influences neuroendocrine activity and produces other effects, including fever and behavioral changes such as anxiety. The melanocortin neuropeptides, such as alpha-melanocyte-stimulating hormone (alpha-MSH), antagonize many actions of IL-1, including fever, anorexia and hypothalamic-pituitary-adrenal (HPA) axis activation through specific melanocortin receptors (MC-R) in the central nervous system. The objective of the present study was to establish the effect of MSH peptides on IL-1beta-induced anxiety-like behavior and the melanocortin receptors involved. We evaluated the effects of intracerebroventricular (i.c.v.) administration of IL-1beta (30 ng) and melanocortin receptor agonists: alpha-MSH, an MC3/MC4-R agonist (0.2 microg) or gamma-MSH, an MC3-R agonist (2 microg) or HS014, an MC4-R antagonist (2 microg), on an elevated plus-maze (EPM) test. Injection of IL-1beta induced an anxiogenic-like response, as indicated by reduced open arms entries and time spent on open arms. The administration of alpha-MSH reversed IL-1beta-induced anxiety with co-administration of HS014 inhibiting the effect of alpha-MSH. However, the associated treatment with gamma-MSH did not affect the anxiety response to IL-1beta. These data suggest that alpha-MSH, through central MC4-R can modulate the anxiety-like behavior induced by IL-1beta.     

Modified melanocortin tetrapeptide Ac-His-dPhe-Arg-Trp-NH at the arginine side chain with ureas and thioureas.

The Ac-His-dPhe-Arg-Trp-NH2 tetrapeptide is a nonselective melanocortin agonist and replacement of Arg in the tetrapeptide with acidic, basic or neutral amino acids results in reduced potency at the melanocortin receptor (MCR) isoforms (MC1R and MC3-5R). To determine the importance of the positive charge and the guanidine moiety for melanocortin activity, a series of urea- and thiourea-substituted tetrapeptides were designed. Replacement of Arg with Lys or ornithine reduced agonist activity at the mouse mMC1 and mMC3-5 receptors, thus supporting the hypothesis that the guanidine moiety is important for receptor potency, particularly at the MC3-5 receptors. The Arg side chain-modified tetrapeptides examined in this study include substituted phenyl, naphthyl, and aliphatic urea and thiourea residues using a Lys side-chain template. These ligands elicit full-agonist pharmacology at the mouse MCRs examined in this study.     

Synthesis and structure-activity relationships of novel dipeptides and reduced dipeptides as ligands for melanocortin subtype-4 receptor

A series of benzylic piperazines (e.g., 4 and 5) attached to an 'address element', the dipeptide H-D-Tic-D-p-Cl-Phe-OH, 3 has been identified as ligands for the melanocortin subtype-4 receptor (MC4R). We describe herein the structure-activity relationship (SAR) studies on the N-terminal residue of the 'address element'. Several novel dipeptides and reduced dipeptides with high MC4R binding affinities and selectivity emerged from this SAR study.     

Redundancy of a functional melanocortin 1 receptor in the anti-inflammatory actions of melanocortin peptides: studies in the recessive yellow (e/e) mouse suggest an important role for melanocortin 3 receptor

The issue of which melanocortin receptor (MC-R) is responsible for the anti-inflammatory effects of melanocortin peptides is still a matter of debate. Here we have addressed this aspect using a dual pharmacological and genetic approach, taking advantage of the recent characterization of more selective agonists/antagonists at MC1 and MC3-R as well as of the existence of a naturally defective MC1-R mouse strain, the recessive yellow (e/e) mouse. RT-PCR and ultrastructural analyses showed the presence of MC3-R mRNA and protein in peritoneal macrophages (M phi) collected from recessive yellow (e/e) mice and wild-type mice. This receptor was functional as Mphi incubation (30 min) with melanocortin peptides led to accumulation of cAMP, an effect abrogated by the MC3/4-R antagonist SHU9119, but not by the selective MC4-R antagonist HS024. In vitro M phi activation, determined as release of the CXC chemokine KC and IL-1 beta, was inhibited by the more selective MC3-R agonist gamma(2)-melanocyte stimulating hormone but not by the selective MC1-R agonist MS05. Systemic treatment of mice with a panel of melanocortin peptides inhibited IL-1 beta release and PMN accumulation elicited by urate crystals in the murine peritoneal cavity. MS05 failed to inhibit any of the inflammatory parameters either in wild-type or recessive yellow (e/e) mice. SHU9119 prevented the inhibitory actions of gamma(2)-melanocyte stimulating hormone both in vitro and in vivo while HS024 was inactive in vivo. In conclusion, agonism at MC3-R expressed on peritoneal M phi leads to inhibition of experimental nonimmune peritonitis in both wild-type and recessive yellow (e/e) mice.     

Discovery of potent,selective, and orally bioavailable 3H-spiro[isobenzofuran-1,4′-piperidine] based melanocortin subtype-4 receptor agonists

Design, synthesis, and SAR of a series of 3H-spiro[isobenzofuran-1,4′-piperidine] based compounds as potent, selective and orally bioavailable melanocortin subtype-4 receptor (MC4R) agonists are disclosed.