Novel selective melanocortin 4 receptor antagonist induces food intake after peripheral administration

We synthesized a new series of small cyclic melanocyte-stimulating hormone (MSH) analogues and screened them for binding affinity at the four MSH binding melanocortin (MC) receptors. We identified a novel substance HS131, with about 20-fold higher affinity for the MC4 receptor than the MC3 receptor. This substance proved to be antagonist for all the four MC receptors in a cAMP assay. HS131 is a six amino acid long peptide, has a molecular weight below 1000, and has only two amino acids in common with the natural MSH peptides. HS131 potently and dose dependently increased food intake after i.c.v. administration. Moreover, s.c. administration of HS131 (1.0 mg/kg) increased food intake, suggesting that HS131 may be able to pass the blood brain barrier. This cyclic low molecular weight peptidomimetic will enable studies of the functional role of the MC4 receptors by peripheral administration and it may be used as a template for further development of low molecular weight substances for the MC receptors.     

Molecular determinants of human melanocortin-4 receptor responsible for antagonist SHU9119 selective activity

The hypothalamic melanocortin-4 receptor (MC4R), a seven transmembrane G-protein-coupled receptor, plays an important role in the regulation of body weight. The synthetic melanocortin analog SHU9119 has been widely used to characterize the physiological role of MC4R in feeding behavior and energy homeostasis. Previous studies indicated that SHU9119 is an agonist at the melanocortin-1 receptor (MC1R) but an antagonist at the MC4R. However, the molecular basis of the interaction between hMC4R and SHU9119 has not been clearly defined. To gain insight into the molecular determinants of hMC4R in the selectivity of SHU9119 chimeras and mutants hMC1R and hMC4R were expressed in cell lines and pharmacologically analyzed. A region of receptor containing the third transmembrane of hMC4R was found to be required for selective SHU9119 antagonism. Further mutagenesis studies of this region of hMC4R demonstrated that the amino acid residue leucine 133 in the third transmembrane was critical for the selective antagonist activity of SHU9119. The single substitution of leucine 133 to methionine did not affect SHU9119 binding to hMC4R. However, this substitution did convert SHU9119 from an antagonist to an agonist. Conversely, exchange of Met(128) in hMC1R to Leu, the homologous residue 133 of hMC4R, displayed a reduction in SHU9119 binding affinity and potency. This report provides the details of the molecular recognition of SHU9119 antagonism at hMC4R and shows that amino acid Leu(133) of hMC4R plays a key role in melanocortin receptor subtype specificity.     

Structure activity studies of the melanocortin antagonist SHU9119 modified at the 6, 7, 8, and 9 positions

The melanocortin system is involved in the regulation of several diverse physiological pathways, including energy homeostasis. Several synthetic peptide analogs have been designed, synthesized, and pharmacologically characterized at the mouse melanocortin receptor subtypes MC1R, MC3R, MC4R, and MC5R. These peptides incorporate modifications of the melanocortin core amino acids His-Phe-Arg-Trp by using the cyclic lactam templates of the lead structures MTII and SHU9119. Analogs containing DNal(2') at position 7 resulted in partial agonist and antagonistic activities at the mMC3R while possessing full antagonistic activities at the mMC4R. Recently, the melanocortin-5 receptor (MC5R) has been demonstrated to have a role in the regulation of exocrine gland function. This study has characterized the following analogs of SHU9119 that possess antagonist activity at the MC5R: Ac-Nle-c[Asp-(1-Me)His(6)-DNal(2')(7)-Arg-Trp-Lys]-NH(2), pA(2) = 7. 1; Ac-Nle-c[Asp-(1-Me)His(6)-DNal(2')(7)-Arg-Nal(2')(9)-Lys]-NH(2), pA(2) = 7.2; and Ac-Nle-c[Asp-Trp(6)-DNal(2')(7)-Arg-Nal(2')(9)-Lys]-NH(2), pA(2) = 6. 6.     

Hindbrain administration of NMDA receptor antagonist AP-5 increases food intake in the rat

Hindbrain administration of MK-801, a noncompetitive N-methyl-D-aspartate (NMDA) channel blocker, increases meal size, suggesting NMDA receptors in this location participate in control of food intake. However, dizocilpine (MK-801) reportedly antagonizes some non-NMDA ion channels. Therefore, to further assess hindbrain NMDA receptor participation in food intake control, we measured deprivation-induced intakes of 15% sucrose solution or rat chow after intraperitoneal injection of either saline vehicle or D(-)-2-amino-5-phosphonopentanoic acid (AP5), a competitive NMDA receptor antagonist, to the fourth ventricular, or nucleus of the solitary tract (NTS). Intraperitoneal injection of AP5 (0.05, 0.1, 1.0, 3.0, and 5.0 mg/kg) did not alter 30-min sucrose intake at any dose (10.7 +/- 0.4 ml, saline control) (11.0 +/- 0.8, 11.2 +/- 1.0, 11.2 +/- 1.0, 13.1 +/- 2.2, and 11.0 +/- 1.9 ml, AP5 doses, respectively). Fourth ventricular administration of both 0.2 mug (16.7 +/- 0.6 ml) and 0.4 mug (14.9 +/- 0.5 ml) but not 0.1 and 0.6 mug of AP5 significantly increased 60-min sucrose intake compared with saline (11.2 +/- 0.4 ml). Twenty-four hour chow intake also was increased compared with saline (AP5: 31.5 +/- 0.1 g vs. saline: 27.1 +/- 0.6 g). Furthermore, rats did not increase intake of 0.2% saccharin after fourth ventricular AP5 administration (AP5: 9.8 +/- 0.7 ml, vs. saline: 10.5 +/- 0.5 ml). Finally, NTS AP5 (20 ng/30 nl) significantly increased 30- (AP5: 17.2 +/- 0.7 ml vs. saline: 14.6 +/- 1.7 ml), and 60-min (AP5: 19.4 +/- 0.6 ml vs. saline: 15.5 +/- 1.4 ml) sucrose intake, as well as 24-h chow intake (AP5: 31.6 +/- 0.3 g vs. saline: 26.1 +/- 1.2 g). These results support the hypothesis that hindbrain NMDA receptors participate in control of food intake and suggest that this participation also may contribute to control of body weight over a 24-h period.     

Effect of centrally and peripherally administered beta-endorphin on food intake in rats

A role for beta-EP in the regulation of food intake has been suggested as a contributory factor in the obesity of some genetically obese animal models. Studies undertaken to determine whether continuous administration of beta-EP could alter food intake in normal rats are described. The present studies demonstrated that continuous subcutaneous infusion with beta-EP was ineffective in modulating food intake, but that acute intraperitoneal or intracerebroventricular administration stimulated food intake in previously food deprived or satiated animals, respectively. These results suggest that beta-EP is not involved in the long-term regulation of food intake, but under certain conditions it may play some role in the regulation of individual meals. It is speculated that the latter activity may result from the action of other appetitive regulatory hormones.     

The regulation of food intake by selective stimulation of the type 3 melanocortin receptor (MC3R)

High levels of binding sites for melanocortin peptides exist within the arcuate nucleus, and a functional response to melanocortin peptides has been demonstrated in arcuate POMC neurons. Because the MC3R is thought to function as an inhibitory autoreceptor on POMC neurons, we reasoned that peripheral injections of MC3R-specific agonists would act within the arcuate nucleus to inhibit POMC neurons and thereby stimulate feeding. We demonstrate that the peptidergic MC3R agonist, d-Trp(8)-gamma-MSH, stimulates feeding via the MC3R when injected peripherally. These data provide the first evidence that feeding can be stimulated by peripheral injection of MC3R-specific agonists.     

Dynamic regulation of hypothalamic neuropeptide gene expression and food intake by melanocortin analogues and reversal with melanocortin-4 receptor antagonist

Melanocortins are known to be involved in the inhibition of food intake and energy metabolism. Acute and chronic intracerebroventricular administration of several different analogues of alpha-MSH, such as alpha-MSH, NDP-MSH, alpha-MSH-ND, [Gln(6)]alpha-MSH-ND, and [Lys(6)]alpha-MSH-ND, which were substituted in the position of His(6) with Gln and Lys, and cyclic16k-MSH to C57J/BL6 mice resulted in a significant inhibition of both time course food intake and body weight gain, compared to the saline-administered control. However, [Gln(6)]alpha-MSH-ND(6-10), the truncated form of [Gln(6)]alpha-MSH-ND, had no inhibitory effects on food intake. In situ hybridization analysis revealed that the expression levels of AGRP and NPY in the hypothalamus were significantly and rapidly diminished while POMC expression was strongly induced by [Gln(6)]alpha-MSH-ND. Administration of JKC-363, a selective MC4R-specific antagonist, coupled with [Gln(6)]alpha-MSH-ND, specifically reversed the [Gln(6)]alpha-MSH-ND-induced inhibition of food intake, but also reversed the hypothalamic expression levels of neuropeptides such as AGRP, NPY, MCH, and POMC, which suggests [Gln(6)]alpha-MSH-ND can function as a selective MC4R agonist.     

The cholecystokinin antagonist,proglumide, increases food intake in the rat

Cholecystokinin, secreted in response to ingested food entering the duodenum, may play a role in limiting food intake. Inhibition of cholecystokinin should therefore induce an increase in food intake. Proglumide, a specific antagonist of cholecystokinin was used to block the satiety effect of a food preload in rats. A significant increase in food intake was obtained following proglumide injection, thus supporting the hypothesis that cholecystokinin, released by food in the duodenum, acts as a short-term satiety factor.     

An evaluation of central penetration from a peripherally administered oxytocin receptor selective antagonist in nonhuman primates

The physiology of the oxytocin receptor has increasingly become a focus of scientific investigation due to its connection with social behavior and psychiatric disorders with impairments in social funciton. Experimental utilization of small molecule and peptide antagonists for the oxytocin receptor has played a role in deciphering these biological and social behavior connections in rodents. Described herein is the evaluation of a potent and selective oxytocin receptor antagonist, ALS-I-41, and details to consider for its use in nonhuman primate behavioral pharmacology experiments utilizing intranasal or intramuscular administration. The central nervous system penetration and rate of metabolism of ALS-I-41 was investigated via mass spectroscopy analysis of cerebrospinal fluid and plasma in the rhesus macaque after intranasal and intramuscular administration. Positron emission tomography was also utilized with [¹⁸F] ALS-I-41 in a macaque to verify observed central nervous system (CNS) penetration and to further evaluate the effects of administration rate on CNS penetration of Sprague-Dawley rats in comparison to previous studies.     

CNS melanocortin system involvement in the regulation of food intake

Accumulating evidence indicates that the central melanocortin (MC) system plays a key role in the regulation of food intake and energy balance. This evidence includes findings that either spontaneous genetic mutations or targeted gene deletions that impair melanocortin signaling cause disrupted food intake and body-weight control. In addition, expression of the mRNA that encodes the endogenous agonists and antagonists for CNS melanocortin receptors is regulated by changes in energy balance and body-adiposity signals. Finally, administration of both natural and synthetic ligands to MC receptors produces changes in food intake. The data collectively suggest a critical role for melanocortin signaling in the control of energy balance.     

FAS inhibitor cerulenin reduces food intake and melanocortin receptor gene expression without modulating the other (an)orexigenic neuropeptides in chickens

Cerulenin, a natural fatty acid synthase (FAS) inhibitor, and its synthetic analog C75 are hypothesized to alter the metabolism of neurons in the hypothalamus that regulate ingestive behavior to cause a profound decrease of food intake and an increase in metabolic rate, leading to body weight loss. The bulk of data exclusively originates from mammals (rodents); however, such effects are currently lacking in nonmammalian species. We have, therefore, addressed this issue in broiler chickens because this species is selected for high growth rate and high food intake and is prone to obesity. First, we demonstrate that FAS messenger and protein are expressed in the hypothalamus of chickens. FAS immunoreactivity was detected in a number of brain regions, including the nucleus paraventricularis magnocellularis and the nucleus infundibuli hypothalami, the avian equivalent of the mammalian arcuate nucleus, suggesting that FAS may be involved in the regulation of food intake. Second, we show that hypothalamic FAS gene expression was significantly (P < 0.05) decreased by overnight fasting similar to that in liver, indicating that hypothalamic FAS gene is regulated by energy status in chickens. Finally, to investigate the physiological consequences of in vivo inhibition of fatty acid synthesis on food intake, we administered cerulenin by intravenous injections (15 mg/kg) to 2-wk-old broiler chickens. Cerulenin administration significantly reduced food intake by 23 to 34% (P < 0.05 to P < 0.0001) and downregulated FAS and melanocortin receptors 1, 4, and 5 gene expression (P < 0.05). However, the known orexigenic (neuropeptide Y, agouti gene-related peptide, orexin, and orexin receptor) and anorexigenic (pro-opiomelanocortin and corticotropin-releasing hormone) neuropeptide mRNA levels remained unchanged after cerulenin treatment. These results suggest that the catabolic effect of cerulenin in chickens may be mediated through the melanocortin system rather than the other neuropeptides known to be involved in food intake regulation.     

A potent and selective nonpeptide antagonist of the melanocortin-4 receptor induces food intake in satiated mice

Optimization on a series of piperazinebenzylamines resulted in analogues with low nanomolar binding at the human MC4 receptor but weak affinity (Ki > 500 nM) at the MC3 receptor. Compound 14c was identified to be a potent MC4R antagonist (Ki = 3.2 nM) with a selectivity of 240-fold over MC3R. It proved to be an insurmountable antagonist in a cAMP assay. Compound 14c potently stimulated food intake in satiated mice when given by intracerebroventricular administration.     

Potency of L-364,718 as an antagonist of the behavioral effects of peripherally administered cholecystokinin

A new antagonist of the peripheral cholecystokinin receptor, L-364,718, was found to block the reductions in food intake and exploratory activity induced by intraperitoneal administration of cholecystokinin octapeptide sulfate. L-364,718 significantly reversed the cholecystokinin-induced reduction in feeding at doses of 10 micrograms/kg - 10 mg/kg i.p. L-364,718 significantly reversed the cholecystokinin-induced reduction in exploratory activity at doses of 500 ng/kg - 10 mg/kg i.p. The time course of antagonist activity of L-364,718 was immediate to 90 minutes after intraperitoneal administration. L-364,718 had no significant effect on food intake or exploratory activity when administered alone, over the dose range of 100 ng/kg-10 mg/kg i.p. This compound appears to be at least one hundred times more potent than proglumide or benzotript as an antagonist of the behavioral effects of peripherally administered cholecystokinin.     

Divergence of melanocortin pathways in the control of food intake and energy expenditure

Activation of melanocortin-4-receptors (MC4Rs) reduces body fat stores by decreasing food intake and increasing energy expenditure. MC4Rs are expressed in multiple CNS sites, any number of which could mediate these effects. To identify the functionally relevant sites of MC4R expression, we generated a loxP-modified, null Mc4r allele (loxTB Mc4r) that can be reactivated by Cre-recombinase. Mice homozygous for the loxTB Mc4r allele do not express MC4Rs and are markedly obese. Restoration of MC4R expression in the paraventricular hypothalamus (PVH) and a subpopulation of amygdala neurons, using Sim1-Cre transgenic mice, prevented 60% of the obesity. Of note, increased food intake, typical of Mc4r null mice, was completely rescued while reduced energy expenditure was unaffected. These findings demonstrate that MC4Rs in the PVH and/or the amygdala control food intake but that MC4Rs elsewhere control energy expenditure. Disassociation of food intake and energy expenditure reveals unexpected divergence in melanocortin pathways controlling energy balance.     

PYY(3-36) into the arcuate nucleus inhibits food deprivation-induced increases in food hoarding and intake

Central administration of neuropeptide Y (NPY) increases food intake in laboratory rats and mice, as well as food foraging and hoarding in Siberian hamsters. The NPY-Y1 and Y5 receptors (Rs) within the hypothalamus appear sufficient to account for these increases in ingestive behaviors. Stimulation of NPY-Y2Rs in the Arcuate nucleus (Arc) has an anorexigenic effect as shown by central or peripheral administration of its natural ligand peptide YY (3-36) and pharmacological NPY-Y2R antagonism by BIIE0246 increases food intake. Both effects on food intake by NPY-Y2R agonism and antagonism are relatively short-lived lasting ∼4 h. The role of NPY-Y2Rs in appetitive ingestive behaviors (food foraging/hoarding) is untested, however. Therefore, Siberians hamsters, a natural food hoarder, were housed in a semi-natural burrow/foraging system that had (a) foraging requirement (10 revolutions/pellet), no free food (true foraging group), (b) no running wheel access, free food (general malaise control) or (c) running wheel access, free food (exercise control). We microinjected BIIE0246 (antagonist) and PYY_(3-36) (agonist) into the Arc to test the role of NPY-Y2Rs there on ingestive behaviors. Food foraging, hoarding, and intake were not affected by Arc BIIE0246 microinjection in fed hamsters 1, 2, 4, and 24 h post injection. Stimulation of NPY-Y2Rs by PYY_(3-36) inhibited food intake at 0–1 and 1–2 h and food hoarding at 1–2 h without causing general malaise or affecting foraging. Collectively, these results implicate a sufficiency, but not necessity, of the Arc NPY-Y2R in the inhibition of food intake and food hoarding by Siberian hamsters.     

Intraventricular (i3vt) ghrelin increases food intake in fatty Zucker rats

Ghrelin is an orexigenic peptide secreted from the stomach and also made in the brain. Ghrelin receptors are expressed on hypothalamic cells important in appetite and energy balance. We determined that intra-3rd-ventricular (i3vt) ghrelin dose-dependently increases acute (1 and 2 h) food intake in lean and fatty Zucker rats (0, 0.01, 0.1 and 1.0 nmol ghrelin). The percentage increase of food intake in fatty Zucker rats was significantly greater than that in lean rats. Fatty Zucker rats had 4.5 times more ghrelin receptor mRNA in the hypothalamus than lean Zucker rats, suggesting a possible mechanism for the increased sensitivity.     

The first selective agonist for the neuropeptide YY5 receptor increases food intake in rats

The first Y(5) receptor-selective analog of neuropeptide Y (NPY), [Ala(31),Aib(32)]NPY, has been developed and biologically characterized. Using competition binding assays on cell lines that express different Y receptors, we determined the affinity of this analog to be 6 nm at the human Y(5) receptor, >500 nm at the Y(1) and Y(2) receptors, and >1000 nm at the Y(4) receptor. Activity studies performed in vitro using a cAMP enzyme immunoassay, and in vivo using food intake studies in rats, showed that the peptide acted as an agonist. Further peptides obtained by the combination of the Ala(31)-Aib(32) motif with chimeric peptides containing segments of NPY and pancreatic polypeptide displayed the same selectivity and even higher affinity (up to 0.2 nm) for the Y(5) receptor. In vivo administration of the new Y(5) receptor-selective agonists significantly stimulated feeding in rats. The NMR solution structures of NPY and [Ala(31),Aib(32)]NPY showed a different conformation in the C-terminal region, where the alpha-helix of NPY was substituted by a more flexible, 3(10)-helical turn structure.     

A comparative study of the central effects of melanocortin peptides on food intake in broiler and layer chicks

Broiler chicks eat more food than layer chicks. However, the causes of the difference in food intake in the neonatal period between these strains are not clear. In this study, we examined the involvement of proopiomelanocortin (POMC)-derived melanocortin peptides α-, β- and γ-melanocyte-stimulating hormones (MSHs) in the difference in food intake between broiler and layer chicks. First, we compared the hypothalamic mRNA levels of POMC between these strains and found that there was no significant difference in these levels between broiler and layer chicks. Next, we examined the effects of central administration of MSHs on food intake in these strains. Central administration of α-MSH significantly suppressed food intake in both strains. Central administration of β-MSH significantly suppressed food intake in layer chicks, but not in broiler chicks, while central administration of γ-MSH did not influence food intake in either strain. It is therefore likely that the absence of the anorexigenic effect of β-MSH might be related to the increased food intake in broiler chicks.     

Opioid receptor involvement in the effect of AgRP- (83-132) on food intake and food selection

Agouti-related peptide (AgRP) is a receptor antagonist of central nervous system (CNS) melanocortin receptors and appears to have an important role in the control of food intake since exogenous CNS administration in rats and overexpression in mice result in profound hyperphagia and weight gain. Given that AgRP is heavily colocalized with neuropeptide Y (NPY) and that orexigenic effects of NPY depend on activity at opioid receptors, we hypothesized that AgRP's food-intake effects are also mediated by opioid receptors. Subthreshold doses of the opioid receptor antagonist naloxone blocked AgRP-induced intake when given simultaneously but not 24 h after AgRP injection. Opioids not only influence food intake but food selection as well. Hence, we tested AgRP's effect to alter food choice between matched diets with differing dietary fat content. AgRP selectively enhanced intake of the high-fat but not the low-fat diet. Additionally, AgRP selectively increased chow intake in rats given ad libitum access to a 20% sucrose solution and standard rat chow. The current results indicate that AgRP influences not only caloric intake but food selection as well and that the early effects of AgRP depend critically on an interaction with opioid receptors.     

1H-NMR studies on a potent and selective antagonist at human melanocortin receptor 4 (hMC-4R)

Melanocortin receptor 4 (MC-4R) is involved in the regulation of energy balance and body weight, and recognizes alpha-, beta-, and gamma-melanocyte stimulating hormones (alpha-, beta-, gamma-MSH). In the search for compounds that regulate food intake and body weight, two synthetic lactam-derivative ligands of alpha-MSH were discovered, MTII and SHU9119. MTII is an agonist and reduces food intake in rats, whereas SHU9119 is an antagonist, and increases food intake and body weight in rats. MTII and SHU9119 are nonselective compounds to MC-4R. To enhance the potency and selectivity at the human MC-4R (hMC-4R), we recently synthesized analogs of SHU9119 (M. A. Bednarek, T. MacNeil, R. N. Kalyani, R. Tang, Van der L. H. T. Ploeg, and D. H. Weinberg, Journal of Medicinal Chemistry, 2001, Vol. 44, pp. 401-409), wherein compound 1 was the most selective for hMC-4R. Replacement of D-Nal by L-Nal in compound 1 made compound 2 weakly active. Comparison of the structures by NMR and molecular modeling of compounds 1 and 2 vs SHU9119 and MTII indicated that, even though they existed as an average of several conformations in solution, there were distinctions in their structures. The gamma-methylene protons of Arg in compound 1 were nonequivalent and shielded probably by the aromatic ring of Nal. The NHi-NHi+1 NOE cross peaks and the temperature coefficients of the amide protons around the "essential core" Nal/Phe7-Arg8-Trp9, required for high affinity and high selectivity at hMC-4R, were indicative of a possible turn structure for these compounds but with differences in their NOE strengths and temperature coefficient values. Molecular modeling of these compounds based on their NMR data showed that the essential core appeared as a "V" shape with two different orientations, one for compound 1 and some of the conformers of SHU9119 and MTII, and the other for compound 2 and some other conformers of SHU9119 and MTII. The remaining conformers of SHU9119 and MTII, which did not map to compound 1 or 2, suggested that they were outside of the hMC-4R binding envelop. These observations may lead to conjectures as to why compound 1 is highly active and selective toward hMC-4R.