Melanocortin MC4 Research Articles

Betulin binds to melanocortin receptors and antagonizes α‐melanocyte stimulating hormone induced cAMP generation in mouse melanoma cells

Betulin is a principal component of birch bark and is known to possess a broad range of biological activities, including antiinflammatory, antiviral and anticancer actions. The present study was carried out in vitro to clarify the influence of betulin on melanocortin (MC) receptor-ergic signalling by using COS-7 cells transfected with corresponding human MC receptor DNA. The results showed that betulin binds to the human melanocortin MC1, three to five receptors with selectivity to the MC1 subtype (K(i) value 1.022 +/- 0.115 microM). Betulin binds to the MC receptors with the following potency order-MC > MC3 > MC5 > MC4. Betulin itself does not stimulate cAMP generation, however, it slightly antagonizes alpha-melanocyte-stimulating hormone (alpha-MSH)-induced cAMP accumulation in the mouse melanoma cell line B16-F1. As a water-insoluble substance, betulin was dissolved in DMSO therefore DMSO competition with the labelled ligand NDP-MSH for the binding to the MC receptors was tested in the identical experimental set-up. We found that DMSO competes for binding to all the MC receptor subtypes, at 20% concentration and above. Selectivity for one or another receptor subtype was not observed. We have demonstrated for the first time, the ability of the plant compound betulin to bind to the MC receptors. One may suggest MC receptor MC1 subtype as the essential target for the antimelanoma action of betulin and its structurally close molecules such as betulinic acid. Moreover, we have found a new non-peptide small molecule MC mimetic, that is betulin. Thus, we report a new chemical motif for the binding to the MC receptors that could be used as a template for the search of more selective MC mimetics.

Neuroprotection in focal cerebral ischemia owing to delayed treatment with melanocortins

In gerbils subjected to transient global cerebral ischemia, melanocortin peptides produce long-lasting protection with a broad time window, and through the activation of central nervous system melanocortin MC 4 receptors. Here we aimed to investigate whether melanocortins are neuroprotective also in a rat model of focal cerebral ischemia induced by intrastriatal microinjection of endothelin-1. The vasoconstrictor agent endothelin-1 caused a significant impairment in spatial learning and memory, as well as in sensory-motor orientation and limb use, associated with severe striatal morphological damage including intense neuronal death and an almost complete myelin degradation. Treatment of ischemic rats with a nanomolar dose (340 μg/kg/day i.p. for 11 days, beginning 3 h or 9 h after endothelin-1 microinjection) of the melanocortin analog [Nle 4, d-Phe 7]α-melanocyte-stimulating hormone (NDP-α-MSH) significantly reduced striatal damage, and improved subsequent functional recovery, with all scheduled NDP-α-MSH treatments. Pharmacological blockade of melanocortin MC 4 receptors prevented the protective effect of NDP-α-MSH. Our findings give evidence that melanocortins are neuroprotective, with a broad time window, also in a severe model of focal cerebral ischemia, and suggest that melanocortin MC 4 receptor agonists could produce neuroprotection in different experimental models of ischemic stroke.

Selective melanocortin MC4 receptor agonists reverse haemorrhagic shock and prevent multiple organ damage

In circulatory shock, melanocortins have life-saving effects likely to be mediated by MC4 receptors. To gain direct insight into the role of melanocortin MC4 receptors in haemorrhagic shock, we investigated the effects of two novel selective MC4 receptor agonists. Severe haemorrhagic shock was produced in rats under general anaesthesia. Rats were then treated with either the non-selective agonist [Nle4, D-Phe7]-melanocyte-stimulating hormone (NDP--MSH) or with the selective MC4 agonists RO27-3225 and PG-931. Cardiovascular and respiratory functions were continuously monitored for 2 h; survival rate was recorded up to 24 h. Free radicals in blood were measured using electron spin resonance spectrometry; tissue damage was evaluated histologically 25 min or 24 h after treatment. All shocked rats treated with saline died within 30-35 min. Treatment with NDP--MSH, RO27-3225 and PG-931 produced a dose-dependent (13-108 nmol kg-1 i.v.) restoration of cardiovascular and respiratory functions, and improved survival. The three melanocortin agonists also markedly reduced circulating free radicals relative to saline-treated shocked rats. All these effects were prevented by i.p. pretreatment with the selective MC4 receptor antagonist HS024. Moreover, treatment with RO27-3225 prevented morphological and immunocytochemical changes in heart, lung, liver, and kidney, at both early (25 min) and late (24 h) intervals. Stimulation of MC4 receptors reversed haemorrhagic shock, reduced multiple organ damage and improved survival. Our findings suggest that selective MC4 receptor agonists could have a protective role against multiple organ failure following circulatory shock.

Expression of Melanocortin MC3 and MC4 Receptor mRNAs by Neuropeptide Y Neurons in the Rat Arcuate Nucleus

Neuropeptide Y (NPY) and α-melanocyte-stimulating hormone (α-MSH), two neuropeptides that are synthesized in neurons of the arcuate nucleus of the hypothalamus, exert opposite actions on food intake and body weight. NPY is orexigenic and decreases energy expenditure whereas α-MSH reduces food consumption and stimulates catabolism. α-MSH is an endogenous ligand for the central melanocortin receptors, MC3-R and MC4-R. In order to determine whether α-MSH may act directly on NPY neurons in the arcuate nucleus, we have investigated the possible occurrence of MC3-R and MC4-R mRNA in NPY-expressing cell bodies in the rat hypothalamus. Double-labeling in situ hybridization histochemistry using ³⁵S-labeled (MC3-R or MC4-R) and digoxigenin-labeled (NPY) riboprobes revealed that 38 ± 1% of the NPY mRNA-positive perikarya expressed MC3-R mRNA while only 9 ± 2% of the NPY-producing neurons contained MC4-R mRNA. The proportions of NPY neurons that express MC3-R mRNA or MC4-R mRNA were not significatively different in the anterior and posterior aspects of the arcuate nucleus. The present study shows that a large proportion of NPY neurons in the rat hypothalamus express MC3-R mRNA while a much lower number of NPY neurons express MC4-R mRNA, suggesting that melanocortins may directly modulate the activity of the hypothalamic NPY system, mainly through activation of MC3-R. These data provide additional evidence for the complex interactions between the stimulatory (NPY) and inhibitory (α-MSH) pathways controlling feeding behavior and energy homeostasis.

Broad therapeutic treatment window of [Nle 4, D-Phe 7]α-melanocyte-stimulating hormone for long-lasting protection against ischemic stroke, in Mongolian gerbils

Melanocortin peptides have been shown to produce neuroprotection in experimental ischemic stroke. The aim of the present investigation was to identify the therapeutic treatment window of melanocortins, and to determine whether these neuropeptides chronically protect against damage consequent to brain ischemia. A 10-min period of global cerebral ischemia in gerbils, induced by occluding both common carotid arteries, caused impairment in spatial learning and memory (Morris test: four sessions from 4 to 67 days after the ischemic episode), associated with neuronal death in the hippocampus. Treatment with a nanomolar dose (340 μg/kg i.p., every 12 h for 11 days) of the melanocortin analog [Nle 4, D-Phe 7]α-melanocyte-stimulating hormone (NDP-α-MSH), starting 3–18 h after the ischemic episode, reduced hippocampal damage with improvement in subsequent functional recovery. The protective effect was long-lasting (67 days, at least) with all schedules of NDP-α-MSH treatment; however, in the latest treated (18 h) gerbils, some spatial memory deficits were detected. Pharmacological blockade of melanocortin MC 4 receptors prevented the protective effects of NDP-α-MSH. Our findings indicate that, in conditions of brain ischemia, melanocortins can provide strong and long-lasting protection with a broad therapeutic treatment window, and with involvement of melanocortin MC 4 receptors, 18 h being the approximately time-limit for stroke late treatment to be effective.

Activation of an efferent cholinergic pathway produces strong protection against myocardial ischemia/reperfusion injury in rats*

A vagus nerve-mediated, brain cholinergic protective mechanism activated by melanocortin peptides is operative in conditions of circulatory shock; moreover, there is anatomical evidence of dual vagal-cardiac efferent pathways in rats, which could play different roles in controlling heart function. Therefore, we investigated the role and functional mechanism of such vagal efferent pathway(s) in an experimental model of ischemic heart disease. Randomized experimental study. Research laboratory. Adult Wistar rats of either sex. After bilateral cervical vagotomy (with or without pretreatment with atropine), efferent vagal fibers were electrically stimulated in rats subjected to coronary artery occlusion (5 mins) followed by reperfusion (5 mins). Other rats (intact, vagotomized, or pretreated with atropine) were treated with nanomolar doses of melanocortin peptides. Electrical stimulation of efferent vagal fibers (5 V, 2 m secs, 1-9 Hz, for the whole period of ischemia/reperfusion) strongly reduced the high incidence of severe arrhythmias and lethality, reduced the increase in free radical blood levels and left-ventricle histologic alterations, and augmented the extracellular signal-regulated kinase activation. Treatment with the melanocortin peptides adrenocorticotropin and gamma2-melanocyte-stimulating hormone (162 nmol/kg intravenously or 16.2 nmol/kg intracerebroventricularly, during coronary occlusion) produced the same protective effects of electrical stimulation and with the same muscarinic acetylcholine receptor-dependent mechanism, seemingly through brain activation (mediated by melanocortin MC3 receptors, as previously described) of such efferent vagal pathway. The present results give evidence for the identification of a protective, melanocortin-activated, efferent vagal cholinergic pathway, operative in conditions of myocardial ischemia/reperfusion. These data suggest that melanocortins and pertinent compounds able to activate such a pathway could provide the potential for development of a new class of drugs for a novel approach to management of ischemic heart disease.

Central administration of small interfering RNAs in rats: A comparison with antisense oligonucleotides

To date there are only few reports of the use of small interfering RNA (siRNA) in whole animals and most of these are restricted to systemic application of siRNAs targeting the liver. In our present studies we have investigated whether siRNAs can be used against a central target after intracerebroventricular (i.c.v.) application and compared their effects with those of antisense oligonucleotides. For this purpose we designed different siRNA and antisense oligonucleotide molecules against the rat hypothalamic melanocortin MC 4 receptor and selected the siRNA and antisense oligonucleotide with the highest efficacy in vitro. We observed that siRNA, encompassing the same gene sequence as antisense oligonucleotides, induced a stronger inhibition of melanocortin MC 4 receptor expression than antisense oligonucleotides. When fluorescence-labeled siRNA were applied i.c.v. in rats no label was detected in brain tissue in spite of the use of cell detergents to improve the delivery. In contrast to these findings the i.c.v. administered fluorescence-labeled antisense oligonucleotides reached the brain structures expressing melanocortin MC 4 receptor and were taken up by the cells in these areas. In summary it seems as if ‘naked’ antisense oligonucleotides have an advantage over ‘naked’ siRNA for experiments in vivo. The development of optimized vector systems seems to be a prerequisite before siRNA can be regarded as a suitable experimental tool for in vivo studies.

New substituted piperazines as ligands for melanocortin receptors. Correlation to the X-ray structure of "THIQ".

A series of piperazine analogues of the melanocortin 4 receptor (MC4R) specific small-molecule agonist "THIQ" was synthesized and characterized structurally and pharmacologically. First, several THIQ imitations lacking the triazole moiety were prepared. Syntheses included acylation of 4-phenylpiperazine or 4-cyclohexylpiperazine. In two cases the tertiary amine function was replaced by the corresponding N-oxide. To obtain more complex structures, a 4-substituted piperazine ring was formed by alkylation of the primary amino group of cyclohexane-derived amino alcohols with N,N-bis(2-chloroethyl)benzylamine. The hydroxylic group of the intermediate was first activated with methanesulfonyl chloride, and the sulfonic ester formed in situ was introduced into the reaction with the sodium salt of 1,2,4-triazole. In one case (i.e., preparation of 23c) introduction of the 1,2,4-triazole moiety was performed at a carbon of the cyclohexane ring. In addition, this intermediate contained a piperazine moiety connected via its nitrogen atom to a cyclohexane ring carbon neighboring the reaction center. As established in NMR and X-ray investigations herein, this substitution proceeded with retention of the initial trans configuration of 1,2-disubstituted cyclohexane. To obtain pure enantiomers of 23c, its precursor 21c was subjected to chiral chromatography on a Chirobiotic V column. The derivatives (R,R)-21cand (S,S)-21c obtained were introduced into further syntheses steps, giving (R,R)-23c and (S,S)-23c, respectively. Melanocortin MC(1,3-5) receptor binding studies showed that all tested piperazine derivatives were active. Several compounds showed clear selectivity for MC4R, with submicromolar affinities being obtained. Among them, one substance, (R,R)-23c, displayed a biphasic curve in displacement of [125I]NDP-MSH on MC4R [K(i)high = 1 nM and K(i)low = 260 nM]. This biphasic competition curve was similarly biphasic to the competition curve obtained herein using THIQ. An X-ray study performed on crystals of the THIQ sulfate salt revealed two closely related conformations, which resemble the shape of the letter "Y", where piperidine and 4-chlorophenyl groups are situated close to each other, but the 1,2,3,4-tetrahydroisoquinoline residue is remote, the triazole function being highly exposed to the environment. The crystals of the dinitrate salt of (R,R)-23c showed a different conformation, where parts of the molecule are spread out almost symmetrically around the central section. Molecular modeling, based on the THIQ crystal structure and the functional similarity of THIQ and (R,R)-23c, allowed us to suggest a possible "bioactive" conformation of (R,R)-23c that is similar to the crystal conformation of THIQ.

Adrenocorticotropin reverses hemorrhagic shock in anesthetized rats through the rapid activation of a vagal anti-inflammatory pathway.

Several melanocortin peptides have a prompt and sustained resuscitating effect in conditions of hemorrhagic shock. The transcription nuclear factor kappaB (NF-kappaB) triggers a potentially lethal systemic inflammatory response, with marked production of tumor necrosis factor-alpha (TNF-alpha), in hemorrhagic shock. Here we investigated whether the hemorrhagic shock reversal produced by the melanocortin ACTH-(1-24) (adrenocorticotropin) depends on the activation of the recently recognized, vagus nerve-mediated, brain "cholinergic anti-inflammatory pathway". Anesthetized rats were stepwise bled until mean arterial pressure (MAP) stabilized at 20-25 mm Hg. The severe hypovolemia was incompatible with survival, and all saline-treated animals died within 30 min. In rats intravenously (i.v.) treated with ACTH-(1-24), neural efferent activity along vagus nerve (monitored by means of a standard system for extracellular recordings) was markedly increased, and the restoration of cardiovascular and respiratory functions was associated with blunted NF-kappaB activity and with decreased TNF-alpha mRNA liver content and TNF-alpha plasma levels. Bilateral cervical vagotomy, pretreatment with the melanocortin MC(4) receptor antagonist HS014, atropine sulfate or chlorisondamine, but not with atropine methylbromide, prevented the life-saving effect of ACTH-(1-24) and the associated effects on NF-kappaB activity and TNF-alpha levels. HS014 and atropine sulfate prevented, too, the ACTH-(1-24)-induced increase in neural efferent vagal activity, and accelerated the evolution of shock in saline-treated rats. The present data show, for the first time, that the melanocortin ACTH-(1-24) suppresses the NF-kappaB-dependent systemic inflammatory response triggered by hemorrhage, and reverses shock condition, by brain activation (in real-time) of the "cholinergic anti-inflammatory pathway", this pathway seeming to be melanocortin-dependent.

Accelerating sensory recovery after sciatic nerve crush: non-selective versus melanocortin MC 4 receptor-selective peptides

Melanocortin receptor ligands accelerate functional recovery after peripheral nerve crush. It is not known which mechanism is involved or via which melanocortin receptor this effect occurs, albeit indirect evidence favours the melanocortin MC 4 receptor. To test whether the melanocortin MC 4 receptor is involved in the effects of melanocortins on functional recovery, we used melanocortin compounds that distinguish the melanocortin MC 4 receptor from the melanocortin MC 1, MC 3 and MC 5 receptors on basis of selectivity and agonist/antagonist profile. Activation and binding studies indicated that the previously described peptides JK1 (Ac-Nle-Gly-Lys- D-Phe-Arg-Trp-Gly-NH 2) and [ D-Tyr 4]melanotan-II ([ D-Tyr 4]MTII. Ac-Nle-c[Asp-His- D-Tyr-Arg-Trp-Lys]NH 2) are selective for the rat melanocortin MC 4 receptor as compared to the rat melanocortin MC 3 and MC 5 receptors, but are also potent on the melanocortin MC 1 receptor. Both peptides did not accelerate sensory recovery in rats with a sciatic nerve crush, whereas the non-selective melanocortin agonist melanotan-II (MTII, Ac-Nle-c[Asp-His- D-Phe-Arg-Trp-Lys]NH 2) was effective. The melanocortin MC 3/MC 4 receptor antagonist SHU9119 (Ac-Nle-c[Asp-His- D-Nal(2)-Arg-Trp-Lys]NH 2) also enhanced sensory recovery. This effect was probably not due to interaction with the melanocortin MC 4 receptor, since JK46 (Ac-Gly-Lys-His- D-Nal(2)-Arg-Trp-Gly-NH 2), a selective melanocortin MC 4 receptor antagonist, was ineffective. Taken together, these data suggest that melanocortins do not accelerate sensory recovery via interaction with the melanocortin MC 4 receptor. From the known melanocortin receptors, only the involvement of the melanocortin MC 5 receptor in acceleration of recovery could not be excluded.

Phenylguanidines as selective nonpeptide melanocortin-5 receptor antagonists.

A series of phenylguanidine analogues represented by 10, 12, and 21 were synthesized and found to have high binding affinities for the human melanocortin-5 receptor. Their binding affinities for three other melanocortin receptor subtypes, MC1, MC3, and MC4, were low. Selected compounds were also tested for their functional activity and exhibited inhibition of alpha-MSH-stimulated cAMP production in cells expressing the human MC5 receptor. Compound 10 had a K(i) value of 2.1 nM in the binding assay and an IC(50) of 72 nM in the functional assay. Some analogues such as 13 from this series possessed weak agonist activity at the human MC4 receptor.

Proopiomelanocortin and melanocortin receptors in the adult rat retino-tectal system and their regulation after optic nerve transection

The aim of this study was to characterise the expression of the melanocortin system in the normal and injured rat visual system. Using real-time polymerase chain reaction and immunohistochemistry, we detected melanocortin MC 3, MC 4 and MC 5 receptors and proopiomelanocortin in adult retina and superior colliculus. Melanocortin MC 4 receptor mRNA was the most abundant receptor. Melanocortin MC 3, MC 4 and MC 5 receptors were localised to the ganglion cell and inner nuclear layers and the melanocortin MC 3 and MC 4 receptors were localised to retinal ganglion cells. Transection of the optic nerve leads to ganglion cell death and both melanocortin receptor and proopiomelanocortin expression decreased in superior colliculus after transection whereas the expression was unchanged or even increased in the retina. α-Melanocyte-stimulating hormone elicited neurite outgrowth from embryonic retinal explants. Together, these data implicate a role for the melanocortin system in the adult rat retina and that melanocortins can stimulate neurite growth from retinal neurons.

Appetite regulation: from the gut to the hypothalamus.

these proportions had increased to 26% and 22%, respectively, with 55% of women and 66% of men being overweight (BMI > 25 kg/m 2 ; Health Survey for England, 2001), reflecting a worldwide trend which is most marked in, but not restricted to, the developed world. Most of us in affluent countries live in a privileged land of plenty where high calorie foods are easily available and in which we have a limited need for exercise. The rising prevalence of obesity in children is of particular concern (Chinn & Rona, 2001).

Melanocortin receptor agonists and antagonists modulate nociceptive sensitivity in the mouse formalin test

A number of studies suggest the involvement of melanocortins in nociception, and although the mechanism through which this occurs is still unknown, experimental evidence would suggest an involvement of melanocortin MC 4 receptors. We investigated the effect of melanocortin receptor agonist and antagonists on nociceptive behaviour induced by formalin in the mouse. The intrathecal injection of the melanocortin receptor agonist MTII ([Ac-Nle 4,Asp 5, d-Phe 7,Lys 10]cyclo-α-MSH-(4–10) amide) (5 nmol; P<0.05) significantly increased nociception in both phases of the formalin test, whereas the synthetic melanocortin receptor antagonists, SHU9119 ([Ac-Nle 4,Asp 5, d-2-Nal 7,Lys 10]cyclo-α-MSH-(4–10) amide) (5 nmol), HS014 ([Ac-Cys 11, d-2-Nal 14,Cys 18]β-MSH-(11–22)amide) (5 nmol), and JKC-363 (cyclic [Mpr 11, d-Nal 14,Cys 18,Asp 22-NH 2]β-MSH-11–22)) (5 nmol), and the endogenous receptor antagonist Agouti-related protein (AgRP) (1.5 nmol) were effective in reducing nociception in the late phase of the formalin test (50–60% of reduction in licking/flinching response; P<0.05). The present findings further support the involvement of the melanocortin system in the control of nociception. Moreover, considering that melanocortin MC 4 receptors are the only melanocortin subtype receptors present in the spinal cord, we can assume that the activity of the peptides in the formalin model is mediated through melanocortin MC 4 receptors.

Further evidence that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC 3 receptors

In rats subjected to myocardial ischemia/reperfusion, melanocortin peptides, including γ 1-melanocyte-stimulating hormone (γ 1-MSH), are able to exert a protective effect by stimulating brain melanocortin MC 3 receptors. A non-melanocortin receptor belonging to a group of receptors for Phe-Met-Arg-Phe-NH 2 (FMRFamide)-like peptides may be involved in some of the cardiovascular effects of the γ-MSHs. FMRFamide-like peptides and γ 1-/γ 2-MSH share, among other things, the C-terminal Arg-Phe sequence, which seems to be essential for cardiovascular effects in normal animals. So we aimed to further investigate which receptor and which structure are involved in the protective effects of melanocortins in anesthetized rats subjected to myocardial ischemia by ligature of the left anterior descending coronary artery (5 min), followed by reperfusion. In saline-treated rats, reperfusion induced, within a few seconds, a high incidence of ventricular tachycardia and ventricular fibrillation, and a high percentage of death within the 5 min of observation period. Reperfusion was associated with a massive increase in free radical blood levels and with an abrupt and marked fall in systemic arterial pressure. The i.v. treatment (162 nmol/kg) during the ischemic period with the adrenocorticotropin fragment 1–24 [ACTH-(1–24): the reference protective melanocortin which binds all melanocortin receptors], as well as with both the melanocortin MC 3 receptor agonists γ 2-MSH and [ d-Trp 8]γ 2-MSH, reduced the incidence of ventricular tachycardia, ventricular fibrillation and death, the increase in free radical blood levels and the fall in arterial pressure. On the contrary, γ 2-MSH-(6–12) (a fragment unable to bind melanocortin receptors) was ineffective. Such protective effect was prevented by the melanocortin MC 3/MC 4 receptor antagonist SHU 9119. In normal (i.e., not subjected to myocardial ischemia/reperfusion) rats, the same i.v. dose (162 nmol/kg) of γ 2-MSH, [ d-Trp 8]γ 2-MSH and γ 2-MSH-(6–12) provoked a prompt and transient increase in arterial pressure; on the other hand, ACTH-(1–24), which lacks the C-terminal Arg-Phe sequence, decreased arterial pressure, but only at higher doses. Heart rate of normal rats was not affected by any of the assayed peptides. The present data confirm and extend our previous findings that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC 3 receptors. Moreover, they further support the notion that, in normal rats, cardiovascular effects of γ-MSHs are mediated by receptors for FMRFamide-like peptides, for whose activation, but not for that of melanocortin MC 3 receptors, the C-terminal Arg-Phe structure being relevant.

Involvement of the melanocortin MC 4 receptor in stress-related behavior in rodents

The melanocortin subtype 4 (MC 4) receptor has been postulated to be involved in stress and stress-related behavior. We made use of melanocortin MC 4 receptor agonists and antagonist to investigate the relationship between the melanocortin MC 4 receptor and stress related disorders. The nonspecific melanocortin receptor agonist α-melanocyte stimulating hormone (α-MSH) and the melanocortin MC 4 receptor agonist, Ac-[Nle 4,Asp 5, d-Phe 7,Lys 10]α-MSH-(4-10)-NH2 (MT II) dose-dependently and significantly reduced the number of licking periods in the rat Vogel conflict test, suggesting that stimulation of the melanocortin MC 4 receptor causes anxiogenic-like activity in rats. We synthesized a peptidemimetic melanocortin MC 4 receptor selective antagonist, Ac- d-2Nal-Arg-2Nal-NH2 (MCL0020), which has high affinity for the melanocortin MC 4 receptor with IC 50 values of 11.63±1.48 nM, in contrast, the affinities for melanocortin MC 1 and MC 3 receptors were negligible. In addition, MCL0020 significantly attenuated the cAMP formation induced by α-MSH in COS-1 cells expressing the melanocortin MC 4 receptor without affecting basal cAMP contents. Thus, we considered MCL0020 to be a selective melanocrotin MC 4 receptor antagonist among melanocortin receptors. Restraint stress significantly reduced food intake in rats, and i.c.v. administration of MCL0020 dose-dependently and significantly attenuated restraint stress-induced anorexia without affecting food intake. Swim stress induced reduction in the time spent in the light area in the mouse light/dark exploration test, and MCL0020 significantly prevented it. Taken together our findings suggest that the melanocortin MC 4 receptor might be related to stress-induced changes in behavior, and blockade of the melanocortin MC 4 receptor may prevent stress-induced disorders such as anxiety.

Melanocortin tetrapeptides modified at the N-terminus, His, Phe, Arg, and Trp positions.

The endogenous melanocortin agonists all contain the conserved His-Phe-Arg-Trp sequence proposed to be important for melanocortin receptor selectivity and stimulation. We have generated peptide libraries consisting of over 100 peptides modified at the N-terminus and at each of the four amino acid positions. These peptides were characterized at the mouse melanocortin MC1, MC3, MC4, and MC5 receptors for agonist or antagonist functional activity. The results from these studies include the identification of a nM MC4 versus MC3 receptor selective (>4700-fold) agonist (JRH 420-12), a nM MC4 receptor agonist that is a nM MC3 receptor antagonist (JRH 322-18), a nM MC5 receptor selective (>100-fold) agonist versus the MC1, MC3, and MC4 receptors (FFM 1-60), and side-chain substitutions that may be utilized for non-peptide design considerations.

Characterization of aliphatic, cyclic, and aromatic N-terminally “capped” His- d-Phe-Arg-Trp-NH 2 tetrapeptides at the melanocortin receptors

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 α-melanocyte stimulation hormone (α-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 α-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 μM 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.

Metal Ion-mediated Agonism and Agonist Enhancement in Melanocortin MC1 and MC4 Receptors

An endogenous metal-ion site in the melanocortin MC1 and MC4 receptors was characterized mainly in transiently transfected COS-7 cells. ZnCl(2) alone stimulated signaling through the Gs pathway with a potency of 11 and 13 microm and an efficacy of 50 and 20% of that of alpha-melanocortin stimulating hormone (alpha-MSH) in the MC1 and MC4 receptors, respectively. In the presence of peptide agonist, Zn(II) acted as an enhancer on both receptors, because it shifted the dose-response curves to the left: most pronounced was a 6-fold increase in alpha-MSH potency on the MC1 receptor. The effect of the metal ion appeared to be additive, because the maximal cAMP response for alpha-MSH in the presence of Zn(II) was 60% above the maximal response for the peptide alone. The affinity of Zn(II) could be increased through binding of the metal ion in complex with small hydrophobic chelators. The binding affinities and profiles were similar for a number of the 2,2'-bipyridine and 1,10-phenanthroline analogs in complex with Zn(II) in the MC1 and MC4 receptors. However, the potencies and efficacies of the metal-ion complexes were very different in the two receptors, and close to full agonism was obtained in the MC1 receptor. Metal ion-chelator complexes having antagonistic properties were also found. An initial attempt to map the metal-ion binding site in the MC1 receptor indicated that Cys(271) in extracellular loop 3 and possibly Asp(119) at the extracellular end of TM-III, which are both conserved among all MC receptors, are parts of the site. It is concluded that the function of the MC1 and MC4 receptors can be positively modulated by metal ions acting both as partial agonists and as potentiators for other agonists, including the endogenous peptide ligand alpha-MSH at Zn(II) concentrations that could be physiological. Furthermore, the metal ion-chelator complexes may serve as leads in the development of novel melanocortin receptor modulators.

Activation of melanocortin MC 4 receptors increases erectile activity in rats ex copula

Melanocortin peptide agonists, α-melanocyte stimulating hormone (α-MSH) and melanotan-II, stimulate erectile activity in a variety of species, including man. Since neither peptide discriminates amongst melanocortin receptors, it is not clear which subtype mediates these pro-erectile effects. Here, we present data that melanocortin-induced erectogenesis is mediated by melanocortin MC 4 receptors. Systemic administration of a melanocortin MC 4 receptor agonist ( N-[(3 R)-1,2,3,4-tetrahydroisoquinolinium-3-ylcarbonyl]-(1 R)-1-(4-chlorobenzyl)-2-[4-cyclohexyl-4-(1 H-1,2,4-triazol-1ylmethyl)piperidin-1-yl]-2-oxoethylamine; THIQ) with high selectivity over other melanocortin receptors enhanced intracavernosal pressure and stimulated erectile activity in rats ex copula. THIQ dose-dependently (1–5 mg/kg, i.v.) increased the total number of erections, to an extent comparable or greater than that produced by apomorphine (0.025 mg/kg, s.c.). Central administration of THIQ (20 μg, intracerebroventricular (i.c.v.)) increased the number of reflexive penile erections; whereas administration of both a nonselective endogenous melanocortin MC 4 receptor antagonist (agouti-related protein (AgRP), 5.5. μg, i.c.v.) and a melanocortin MC 4 receptor preferring antagonist (MPB10, 1 mg/kg, i.v.) blocked THIQ-induced erectogenesis. These pro-erectile effects were also attenuated by systemic or central administration of an oxytocin antagonist (L-368899, 1 mg/kg, i.v.). Thus, melanocortin MC 4 receptor activation is sufficient for erectogenesis and these effects may involve oxytocinergic pathways.