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The melanocortin system has been implicated in the regulation of various physiological functions including melanogenesis, steroidogenesis, energy homeostasis, and feeding behavior. Five melanocortin receptors have been identified to date and belong to the family of G protein-coupled receptors (GPCR). Post-translational modification of the proopiomelanocortin (POMC) prohormone leads to the biosynthesis of the endogenous melanocortin agonists, including α-melanocyte stimulating hormone (α-MSH), β-MSH, γ-MSH, and adrenocorticotropic hormone (ACTH). All the melanocortin agonists derived from the POMC prohormone contain a His-Phe-Arg-Trp tetrapeptide sequence that has been implicated in eliciting the pharmacological responses at the melanocortin receptors. Herein, an alanine (Ala) positional scan is reported for the endogenous α-MSH ligand and the synthetic, more potent, NDP-MSH peptide (Ac-Ser(1)-Tyr(2)-Ser(3)-Nle(4)-Glu(5)-His(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-Lys(11)-Pro(12)-Val(13)-NH2) at the cloned mouse melanocortin receptors to test the assumption that the structure-activity relationships of one ligand would apply to the other. Several residues outside of the postulated pharmacophore altered potency at the melanocortin receptors, most notably the 1560-, 37-, and 15-fold potency loss when the Glu(5) position of α-MSH was substituted with Ala at the mMC1R, mMC3R, and mMC4R, respectively. Importantly, the altered potencies due to Ala substitutions in α-MSH did not necessarily correlate with equivalent Ala substitutions in NDP-MSH, indicating that structural modifications and corresponding biological activities in one of these melanocortin ligands may not be predictive for the other agonist.

The melanocortin system is well recognized to be involved in the regulation of food intake, body weight, and energy homeostasis. To probe the role of the MC 3 in the regulation of food intake, JRH322-18 a mixed MC 3 partial agonist/antagonist and MC 4 agonist tetrapeptide was examined in wild type (WT) and melanocortin 4 receptor (MC 4 ) knockout mice and shown to reduce food intake in both models. In the wild type mice, 2.0 nmol of JRH322-18 statistically reduced food intake 4 h post icv treatment into satiated nocturnally feeding wild type mice. The same dose in the MC 4 KO mice significantly reduced cumulative food intake 24 h post treatment. Conditioned taste aversion as well as activity studies supports that the decreased food intake was not due to visceral illness. Since these studies resulted in loss-of-function results, the SHU9119 and agouti-related protein (AGRP) melanocortin receptor antagonists were administered to wild type as well as the MC 3 and MC 4 knockout mice in anticipation of gain-of-function results. The SHU9119 ligand produced an increase in food intake in the wild type mice as anticipated, however no effect was observed in the MC 3 and MC 4 knockout mice as compared to the saline control. The AGRP ligand however, produced a significant increase in food intake in the wild type as well as the MC 3 and MC 4 knockout mice and it had a prolonged affect for several days. These data support the hypothesis that the MC 3 plays a subtle role in the regulation of food intake, however the mechanism by which this is occurring remains to be determined.

Exercise is a mechanism for maintenance of body weight in humans. Morbidly obese human patients have been shown to possess single nucleotide polymorphisms in the melanocortin-4 receptor (MC4R). MC4R knockout mice have been well characterized as a genetic model that possesses phenotypic metabolic disorders, including obesity, hyperphagia, hyperinsulinemia, and hyperleptinemia, similar to those observed in humans possessing dysfunctional hMC4Rs. Using this model, we examined the effect of voluntary exercise of MC4R knockout mice that were allowed access to a running wheel for a duration of 8 wk. Physiological parameters that were measured included body weight, body composition of fat and lean mass, food consumption, body length, and blood levels of cholesterol and nonfasted glucose, insulin, and leptin. At the termination of the experiment, hypothalamic mRNA expression levels of neuropeptide Y (NPY), agouti-related protein (AGRP), proopiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART), orexin, brain-derived neurotropic factor (BDNF), phosphatase with tensin homology (Pten), melanocortin-3 receptor (MC3R), and NPY-Y1R were determined. In addition, islet cell distribution and function in the pancreas were examined. In the exercising MC4R knockout mice, the pancreatic islet cell morphology and other physiological parameters resembled those observed in the wild-type littermate controls. Gene expression profiles identified exercise as having a significant effect on hypothalamic POMC, orexin, and MC3R levels. Genotype had a significant effect on AGRP, POMC, CART, and NPY-Y1R, with an exercise and genotype interaction effect on NPY gene expression. These data support the hypothesis that voluntary exercise can prevent the genetic predisposition of melanocortin-4 receptor-associated obesity and diabetes.—Haskell-Luevano, C., Schaub, J. W., Andreasen, A., Haskell, K. R., Moore, M. C., Koerper, L. M., Rouzaud, F., Baker, H. V., Millard, W. J., Walter, G., Litherland, S. A., Xiang, Z. Voluntary exercise prevents the obese and diabetic metabolic syndrome of the melanocortin-4 receptor knockout mouse.

The melanocortin-4 receptor (MC4R) is a G-protein coupled receptor (GPCR) that is expressed in the central nervous system and has a role in regulating energy homeostasis and obesity. Up to a remarkable 6% of morbidly obese adults and children studied possess single nucleotide polymorphisms (SNPs) of the MC4R. Upon stimulation by agonist, the MC4R signals through the intracellular adenylate cyclase signal transduction pathway. Posttranslational modification of the pro-opiomelanocortin (POMC) gene transcript results in the generation of several endogenous melanocortin receptor agonists including alpha-, beta-, gamma-melanocyte stimulating hormones (MSH) and adrenocorticotropin (ACTH) ligands. The endogenous MC4R antagonist, agouti-related protein (AGRP), is expressed in the brain and is only one of two naturally occurring antagonists of GPCRs identified to date. Herein, we have generated 40 hMC4 polymorphic receptors and evaluated their cell surface expression by flow cytometry as well as pharmacologically characterized their functionality using the endogenous agonists alpha-MSH, beta-MSH, gamma2-MSH, ACTH(1-24), the antagonist hAGRP(87-132), and the synthetic agonists NDP-MSH and MTII. This is the first study in which polymorphic hMC4Rs have been pharmacologically characterized simultaneously with multiple endogenous ligands. Interestingly, at the N97D, L106P, and C271Y hMC4Rs beta-MSH was more potent than the other endogenous agonists alpha-MSH, gamma2-MSH, ACTH(1-24). The S58C and R165Q/W hMC4Rs possessed significantly reduced endogenous agonist potency (15- to 90-fold), but the synthetic ligands NDP-MSH and MTII possessed only 2-9-fold reduced potency as compared to the wild-type receptor, suggesting their potential as therapeutic ligands to treat individuals with these polymorphisms.

The melanocortin system is involved in hypothalamic regulation of energy homeostasis. The melanocortin-4 receptor (MC4R) has been linked to both obesity and reproductive dysfunction. Deletion of the MC4R from the mouse genome has resulted in phenotypes including adult onset obesity, hyperphagia, and difficulty in reproducing when homozygote parents are bred. Additionally, polymorphisms of the human MC4R have been identified in morbidly obese children and adults. Herein, we have identified that voluntary exercise, provided via the presence of a running wheel, impedes the monogenetic obesity (at 20 weeks of age running wheel housed body weight = 31 ± 1.8 g versus conventionally housed body weight = 41 ± 2.3 g, a 25% decrease in body weight p

The agouti-related protein (AGRP) is an endogenous antagonist of the brain melanocortin receptors (MC3R and MC4R) and is believed to be involved in feeding behavior and energy homeostasis. Previous results identified that the human AGRP decapeptide Yc[CRFFNAFC]Y binds to the MC3R and MC4R and acts as an antagonist at the MC4R but not at the MC3R. We have synthesized the amidated version of this decapeptide as well as performed elongation studies at both the N-and C-terminus of the monocyclic hAGRP(109-118) peptide. This study was designed to identify monocyclic peptide fragments of the hAGRP(86-132) to determine the minimal active monocyclic sequence necessary for antagonism at the MC3R. For binding studies, radiolabeled 125I-NDP-MSH versus 125I-hAGRP(86-132) were utilized to determine if there were differences in the ability of the AGRP fragments prepared herein to competitively displace the 125I-NDP-MSH versus AGRP(86-132) radiolabel. The binding IC(50) values of radiolabeled hAGRP(86-132) versus NDP-MSH were identical within experimental error, supporting the hypothesis that AGRP and NDP-MSH interact with overlapping binding epitopes at the MC3R and MC4R. The most notable results include identification of the TAYc[CRFFNAFC]YAR-NH(2) (pA(2)=6.1, K(i)=790nM, mMC3R) and the Yc[CRFFNAFC]YARKL-NH(2) (pA(2)=6.2, K(i)=630nM, mMC3R) peptides as the minimal monocyclic AGRP-based fragments possessing antagonist pharmacology at the MC3R. Interestingly, extension of the AGRP(109-118) decapeptide at both the N- and C-terminus by two amino acids conferred detectable mMC3R antagonism, while retaining high nanomolar MC4R antagonist and micromolar MC1R agonist pharmacological properties. These data support the hypothesis that elongation of the hAGRP(109-118) decapeptide results in antagonism at the MC3R while retaining MC1R agonist activity and MC4R antagonist activity.

The melanocortin system is implicated in multiple physiological pathways including pigmentation, inflammation, erectile function, feeding behavior, energy homeostasis, weight homeostasis, and exocrine gland function, just to list a few. The endogenous agonists for the melanocortin receptors include the gene transcripts derived from the proopiomelanocortin gene and include the core tetrapeptide His-Phe-Arg-Trp sequence postulated to be important for melanocortin receptor selectivity and stimulation. Posttranslational processing of the proopiomelanocortin derived agonists results in the N-terminal acetylation and C-terminal amidation of alpha-melanocyte stimulation hormone (alpha-MSH). In this study we generated 25 N-terminally "capped" tetrapeptides containing the core sequence X-His-D-Phe-Arg-Trp-NH(2) and pharmacologically characterized them at the mouse melanocortin MC(1) receptor, melanocortin MC(3) receptor, melanocortin MC(4) receptor, and melanocortin MC(5) receptor. The N-terminal "capping" groups consisted of linear, cyclic, or aromatic moieties and all resulted in full agonist activity at the melanocortin receptors examined in this study. Increasing aliphatic chain length increased potency of the tetrapeptide derivatives, with the addition of octanoyl capping group resulting in 70- to 110-fold increased tetrapeptide potency at the melanocortin MC(1) receptor (EC(50)=0.4 nM), melanocortin MC(3) receptor (EC(50)=4.0 nM), and melanocortin MC(4) receptor (EC(50)=0.4 nM) while only enhancing potency at the melanocortin MC(5) receptor (EC(50)=0.8 nM) by 8-fold, compared to the tetrapeptide His-D-Phe-Arg-Trp-NH(2). This octanoyl derivative surprisingly resulted in a 14-fold greater potency than alpha-MSH (EC(50)=5.4 nM) at the mouse melanocortin MC(4) receptor implicated in feeding behavior and obesity. The 3,3,3-triphenylpropionyl derivative resulted in greater than 14 microM agonist potencies at the melanocortin MC(1) receptor, melanocortin MC(3) receptor, and melanocortin MC(4) receptor and possessed a 140 nM agonist EC(50) value at the melanocortin MC(5) receptor. This 3,3,3-triphenylpropionyl-His-D-Phe-Arg-Trp-NH(2) peptide is a 100-fold selective agonist for the melanocortin MC(5) receptor, versus the other melanocortin receptors studied herein, and is the first melanocortin MC(5) receptor selective tetrapeptide derivative reported to date with nanomolar potency.

The melanocortin pathway is an important participant in obesity and energy homeostasis. The centrally located melanocortin-3 and melanocortin-4 receptors (MC3R, MC4R) are involved in the metabolic and food intake aspects of energy homeostasis and are stimulated by melanocortin agonists such as α-melanocyte stimulation hormone (α-MSH). The melanocortin agonists contain the putative message sequence “His-Phe-Arg-Trp”, and it has been well documented that inversion of chirality of the Phe to DPhe results in a dramatic increase in melanocortin receptor potency. Herein, we report a tetrapeptide library based on the template Ac-His-DPhe-Arg-Trp-NH2, consisting of 17 members that have been modified at the His6 position (α-MSH numbering) and pharmacologically characterized for agonist activity at the mouse melanocortin receptors MC1R, MC3R, MC4R, and MC5R. These studies provide further experimental evidence that the His6 position can determine MC4R versus MC3R agonist selectivity and that chemically nonreactive s...

The melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor (GPCR) that is expressed in the central nervous system and has a role in regulating feeding behavior, obesity, energy homeostasis, male erectile response, and blood pressure. Since the report of the MC4R knockout mouse in 1997, the field has been searching for links between this genetic biomarker and human obesity and type 2 diabetes. More then 80 single nucleotide polymorphisms (SNPs) have been identified from human patients, both obese and nonobese controls. Many significant studies have been performed examining the pharmacological characteristics of these hMC4R SNPs in attempts to identify a molecular defects/insights that might link a genetic factor to the obese phenotype observed in patients possessing these mutations. Our laboratory has previously reported the pharmacological characterization of 40 of these polymorphic hMC4 receptors with multiple endogenous and synthetic ligands. The goal of the current study is to perform a similar comprehensive side-by-side characterization of 30 additional human hMC4R with single nucleotide polymorphisms using multiple endogenous agonists [alpha-, beta-, and gamma(2)-melanocyte stimulating hormones (MSH) and adrenocorticotropin (ACTH)], the antagonist agouti-related protein hAGRP(87-132), and synthetic agonists [NDP-MSH, MTII, and the tetrapeptide Ac-His-dPhe-Arg-Trp-NH(2) (JRH887-9)]. These in vitro data, in some cases, provide a putative molecular link between dysfunctional hMC4R's and human obesity. These 30 hMC4R SNPs include R7H, R18H, R18L, S36Y, P48S, V50M, F51L, E61K, I69T, D90N, S94R, G98R, I121T, A154D, Y157S, W174C, G181D, F202L, A219 V, I226T, G231S, G238D, N240S, C271R, S295P, P299L, E308K, I317V, L325F, and 750DelGA. All but the N240S hMC4R were identified in obese patients. Additionally, we have characterized a double I102T/V103I hMC4R. In addition to the pharmacological characterization, the hMC4R variants were evaluated for cell surface expression by flow cytometry. The F51L, I69T, and A219V hMC4Rs possessed full agonist activity and significantly decreased endogenous agonist ligand potency. At the E61K, D90N, Y157S, and C271R hMC4Rs, all agonist ligands examined were only partially efficacious in generating a maximal signaling response (partial agonists) and possessed significantly decreased endogenous agonist ligand potency. Only the A219V, G238D, and S295P hMC4Rs possessed significantly decreased AGRP(87-132) antagonist potency. These data provide new information for use in GPCR computational development as well as insights into MC4R structure ad function.

Background Melanocortin-4-receptor (MC4R) mutations represent the most frequent genetic cause of non-syndromic early onset obesity. Children carrying MC4R mutations seem to show a particular phenotype characterized by early onset, severe obesity and high stature. To verify whether MC4R mutations are associated with this particular phenotype in the Italian pediatric population, we decided to screen the MC4R gene in a group of obese children selected on the basis of their phenotype. Methods To perform this study, a multicentric approach was designed. Particularly, to be enrolled in the study subjects needed to meet the following criteria: Body mass index ≥ 3 deviation scores according to age and sex, familiar history of obesity (at least one parent obese), obesity onset before the 10 years old, height ≥ 2 deviation scores. The coding region of MC4R gene was screened in 240 obese children (mean age 8.3 ± 3.1, mean BMI 30.8 ± 5.4) and in 200 controls (mean age 8.1 ± 2.8; mean BMI 14.2 ± 2.5). Results Three mutations have been found in five obese children. The S127L (C380T), found in three unrelated children, had been described and functionally characterized previously. The Q307X (C919T) and the Y332H (T994C) mutations were found in two patients. Functional studies showed that only Q307X impaired protein function. Conclusion The low prevalence of MC4R mutations (1.6%) in this group of obese children selected according to the obesity degree, the tall stature and the family history of obesity was similar to the prevalence observed in previous screenings performed in obese adults and in not phenotypically selected obese children.

The tetrapeptide sequence His-Phe-Arg-Trp, derived from melanocyte-stimulating hormone (alphaMSH) and its analogs, causes a decrease in food intake and elevates energy utilization upon binding to the melanocortin-4 receptor (MC4R). To utilize this sequence as an effective agent for treating obesity, we improved its metabolic stability and intestinal permeability by synthesizing a library of backbone cyclic peptidomimetic derivatives. One analog, peptide 1 (BL3020-1), was selected according to its selectivity in activating the MC4R, its favorable transcellular penetration through enterocytes and its enhanced intestinal metabolic stability. This peptide was detected in the brain following oral administration to rats. A single oral dose of 0.5 mg/kg in mice led to reduced food consumption (up to 48% vs the control group) that lasted for 5 h. Repetitive once daily oral dosing (0.5 mg/kg/day) for 12 days reduced weight gain. Backbone cyclization was shown to produce a potential drug lead for treating obesity.

The melanocortin pathway, specifically the melanocortin-4 receptor and the cognate endogenous agonist and antagonist ligands, have been strongly implicated in the regulation of energy homeostasis and satiety. Genetic studies of morbidly obese human patients and normal weight control patients have resulted in the discovery of over 70 human melanocortin-4 receptor (MC4R) polymorphisms observed as both heterozygous and homozygous forms. A number of laboratories have been studying these hMC4R polymorphisms attempting to understand the molecular mechanism(s) that might explain the obese human phenotype. Herein, we have studied 13 polymorphic hMC4Rs that have been identified to possess statistically significant decreased endogenous agonist potency with synthetic peptides and small molecules attempting to identify ligands that can pharmacologically rescue the hMC4R polymorphic agonist response. The ligands examined in this study include NDP-MSH, MTII, Ac-His-DPhe-Arg-Trp-NH2 (JRH887-9), Ac-Anc-DPhe-Arg-Trp-NH2 (amino-2-naphtylcarboxylic acid, Anc, JRH420-12), Ac-His-(pI)DPhe-Arg- Trp-NH2 (JRH322-18), chimeric AGRP-melanocortin based ligands (Tyr-c(Cys-His-DPhe-Arg-Trp-Asn- Ala-Phe-Cys)-Tyr-NH2, AMW3-130 and Ac-mini-(His-DPhe-Arg-Trp)-hAGRP-NH 2, AMW3-106), and the small molecules JB25 and THIQ. The hMC4R polymorphisms included in this study are S58C, N97D, I102S, L106P, S127L, T150I, R165Q, R165W, L250Q, G252S, C271Y, Y287Stop, and I301T. These studies resulted in the NDP-MSH, MTII, AMW3-130, THIQ, and AMW3-106 ligands possessing nanomolar to subnanomolar agonist potency at the hMC4R polymorphisms examined in this study. Thus, these ligands could generically rescue the potency and stimulatory response of the abnormally functioning hMC4Rs studied and may provide tools to further clarify the molecular mechanism(s) involving these receptor modifications.

The Melanocortin-4 Receptor is a G-protein coupled receptor that has been physiologically linked to participate in the regulation of energy homeostasis. The Melanocortin-4 Receptor is stimulated by endogenous melanocortin agonists derived from the pro-opiomelanocortin gene transcript and antagonized by the endogenous antagonist agouti-related protein. Central administration of melanocortin agonists has been demonstrated to decrease food intake and conversely, treatment with antagonists resulted in increased food intake. Deletion of the Melanocortin-4 Receptor gene from the mouse genome results in an obese and hyperphagic phenotype. Polymorphisms of the human Melanocortin-4-Receptor have been found in severely obese individuals, suggesting that Melanocortin-4 Receptor malfunction might be involved in human obesity and obesity-associated diabetes. Herein, we have performed experiments to understand the molecular mechanisms associated with the L250Q human Melanocortin-4-Receptor polymorphism discovered in an extremely obese woman. This L250Q human Melanocortin-4-Receptor has been pharmacologically characterized to result in a constitutively active receptor. The fact that a constitutively active human Melanocortin-4-Receptor mutation was found in an obese person is a physiologic contradiction, as chronic activation of the human Melanocortin-4-Receptor and subsequently high cyclic adenosine monophosphate levels should theoretically result in a normal or lean phenotype. In this study, we demonstrated that agouti-related protein acts as an inverse agonist at this constitutively active receptor, and we propose a mechanism by which agouti-related protein might contribute to the obese phenotype in the L250Q patient. In addition, using receptor mutagenesis, pharmacology, and computer modeling approaches, we investigated the molecular mechanism by which modification of the L250 residue results in constitutive activation of the human Melanocortin-4-Receptor.

The melanocortin pathway consists of endogenous agonists, antagonists, G-protein coupled receptors, and ancillary proteins that mediate the function of the endogenous antagonists. The melanocortin-4 receptor (MC4R) is involved in the regulation of obesity and the melanocortin-2 receptor (MC2R) is involved in the regulation of steroidogenesis. Herein, we present the effects of voluntary exercise on the MC4R knockout mice in terms of bypassing the morbid obesity and hyperphagia phenotypes associated with this genetic obesity model. Additionally, a systematic truncation study of the adrenocorticotropin hormone (ACTH 1-24) has been performed and characterized at the cloned MC2R.

The agouti-related protein (AGRP) is an endogenous antagonist of the centrally expressed melanocortin receptors. The melanocortin-4 receptor (MC4R) is involved in energy homeostasis, food intake, sexual function, and obesity. The endogenous hAGRP protein is 132 amino acids in length, possesses five disulfide bridges at the C-terminus of the molecule, and is expressed in the hypothalamus of the brain. We have previously reported that a monocyclic hAGRP(103-122) peptide is an antagonist at the melanocortin receptors expressed in the brain. Stereochemical inversion from the endogenous l- to d-isomers of single or multiple amino acid modifications in this monocyclic truncated hAGRP sequence resulted in molecules that are converted from melanocortin receptor antagonists into melanocortin receptor agonists. The Asp-Pro-Ala-Ala-Thr-Ala-Tyr-cyclo[Cys-Arg-DPhe-DPhe-Asn-Ala-Phe-Cys]-Tyr-Ala-Arg-Lys-Leu peptide resulted in a 60 nM melanocortin-1 receptor agonist that is 100-fold selective versus the mMC4R, 1000-fold selective versus the mMC3R, and ca. 180-fold selective versus the mMC5R. In attempts to identify putative ligand-receptor interactions that may be participating in the agonist induced stimulation of the MC4R, selected ligands were docked into a homology molecular model of the mMC4R. These modeling studies have putatively identified hAGRP ligand DArg111-mMC4RAsn115 (TM3) and the hAGRP DPhe113-mMC4RPhe176 (TM4) interactions as important for agonist activity.

Agouti-related protein (AGRP) is one of only two known endogenous antagonists of G-protein coupled receptors (GPCRs). Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis, regulation of feeding behavior, and obesity. Alpha-melanocyte stimulating hormone (alpha-MSH) is one of the known endogenous agonists for these receptors. It has been hypothesized that the Arg-Phe-Phe (111-113) human AGRP amino acids may be mimicking the melanocortin agonist Phe-Arg-Trp (7-9) residue interactions with the melanocortin receptors that are important for both receptor molecular recognition and stimulation. To test this hypothesis, we generated thirteen chimeric peptide ligands based upon the melanocortin agonist peptides NDP-MSH (Ac-Ser-Tyr-Ser-Nle4-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2) and MTII (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2). In these chimeric ligands, the agonist DPhe-Arg-Trp amino acids were replaced by the AGRP Arg-Phe-Phe residues, and resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3-5Rs), supporting the hypothesis that the AGRP antagonist ligand Arg-Phe-Phe residues mimic the agonist Phe-Arg-Trp amino acids. Interestingly, the Ac-Ser-Tyr-Ser-Nle4-Glu-His-Arg-DPhe-Phe-Gly-Lys-Pro-Val-NH2 peptide possessed 7 nM mMC1R agonist potency, and is 850-fold selective for the mMC1R versus the mMC3R, 2300-fold selective for the mMC1R versus the mMC4R, and 60-fold selective for the MC1R versus the mMC5R, resulting in the discovery of a new peptide template for the design of melanocortin receptor selective ligands.