Pharmacological characterization of three chicken melanocortin-3 receptor mutants.

The melanocortin-3 receptor (MC3R) is a G protein-coupled receptor and potentially important in production traits. Three naturally occurring mutations (M54L, G104S, and L151R) in chicken MC3R (cMC3R) were reported previously to be associated with production traits. Here, we inserted the full-length cMC3R coding sequence into pcDNA3.1(+) and generated the 3 mutations by site-directed mutagenesis. The total and cell surface expression of the receptors was measured by flow cytometry. We analyzed the pharmacological characteristics, including binding and cyclic adenosine monophosphate (cAMP) and mitogen-activated protein kinase (MAPK) signaling, using 6 ligands ([Nle4, D-Phe7]-α-melanocyte stimulating hormone (MSH), α-, ß-, γ-, and D-Trp8-γ-MSHs, and agouti-related peptide). All mutants had similar total and cell surface expression as the wild-type (WT) cMC3R. M54L had similar pharmacological properties as the WT cMC3R. G104S did not exhibit any specific binding but had minimal response to α-, ß-, γ-, and D-Trp8-γ-MSH, although it generated 24% WT response when stimulated by NDP-MSH. Although L151R had normal binding, the responses to agonists were reduced to approximately 25% of that of the WT. In MAPK signaling, all 3 mutants showed significantly increased agonist-stimulated phosphorylation of extracellular signal-regulated protein kinases 1/2, indicating the existence of biased signaling at G104S and L151R. In summary, our studies demonstrated that although all 3 mutations are significantly associated with production traits, only G104S and L151R had severe defects in receptor pharmacology. How M54L might cause production trait differences remains to be investigated.

Polymorphisms of four candidate genes and their correlations with growth traits in blue fox (Alopex lagopus).

To improve the accuracy and genetic progress of blue fox breeding, the relationships between genetic polymorphisms and growth and reproductive traits of the blue fox were investigated. MC4R, MC3R, INHA and INHBA were selected as candidate genes for molecular evolution and statistical analyses. Single-factor variance analyses showed that the MC4R (g.267C > T, g.423C > T, and g.731C > A) and MC3R (g.677C > T) genotypes had significant impacts on body weight, chest circumference, abdominal perimeter and body mass index (BMI) (P < 0.05) in blue fox. The MC4R and MC3R combined genotypes had significant effects on the body weight and abdominal circumference. The different genotypes of INHA g.75G > A had significant effects on female fecundity, whereas the different genotypes of INHBA g.404G > T and g.467G > T and the INHA and INHBA combined genotypes had significant effects on male fecundity. The proteins encoded by the open reading frames (ORFs) of different polymorphic loci were predicted and analysed. The aims of this study were to identify genetic markers related to growth and reproduction in the blue fox and to provide an efficient, economical and accurate theoretical approach for auxiliary fox breeding.

Characterization of channel catfish (Ictalurus punctatus) melanocortin-3 receptor reveals a potential network in regulation of energy homeostasis.

The melanocortin-3 receptor (MC3R) is known to be involved in regulation of energy homeostasis, regulating feed efficiency and nutrient partitioning in mammals. Its physiological roles in non-mammalian vertebrates, especially economically important aquaculture species, are not well understood. Channel catfish (Ictalurus punctatus) is the main freshwater aquaculture species in North America. In this study, we characterized the channel catfish MC3R. The mc3r of channel catfish encoded a putative protein (ipMC3R) of 367 amino acids. We transfected HEK293T cells with ipMC3R plasmid for functional studies. Five agonists, including adrenocorticotropin, α-melanocyte stimulating hormone (α-MSH), ß-MSH, [Nle4, D-Phe7]-α-MSH, and D-Trp8-γ-MSH, were used in the pharmacological studies. Our results showed that ipMC3R bound ß-MSH with higher affinity and D-Trp8-γ-MSH with lower affinity compared with human MC3R. All agonists could stimulate ipMC3R and increase intracellular cAMP production with sub-nanomolar potencies. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation could also be triggered by ipMC3R. The ipMC3R exhibited constitutive activities in both cAMP and ERK1/2 pathways, and Agouti-related protein served as an inverse agonist at ipMC3R, potently inhibiting the high basal cAMP level. Moreover, we showed that melanocortin receptor accessory protein 2 (MRAP2) preferentially modulated ipMC3R in cAMP production rather than ERK1/2 activation. Our study will assist further investigation of the physiological roles of the ipMC3R, especially in energy homeostasis, in channel catfish.

Discovery of Polypharmacological Melanocortin-3 and -4 Receptor Probes and Identification of a 100-Fold Selective nM MC3R Agonist versus a µM MC4R Partial Agonist.

The centrally expressed melanocortin-3 and melanocortin-4 receptors (MC3R and MC4R, respectively) are established targets to treat diseases of positive- and negative-energy homeostasis. We previously reported [ Doering , S. R. ; J. Med. Chem. 2017 , 60 , 4342 - 4357 ] mixture-based positional scanning approaches to identify dual MC3R agonist and MC4R antagonist tetrapeptides. Herein, 46 tetrapeptides were chosen for MC3R agonist screening selectivity profiles, synthesized, and pharmacologically characterized at the mouse melanocortin-1, -3, -4, and -5 receptors. Substitutions to the tetrapeptide template were selected solely based on MC3R agonist potency from the mixture-based screen. This study resulted in the discovery of compound 42 (Ac-Val-Gln-(pI)DPhe-DTic-NH2), a full MC3R agonist that is 100-fold selective for the MC3R over the µM MC4R partial agonist pharmacology. This compound represents a first-in-class MC3R selective agonist. This ligand will serve as a useful in vivo molecular probe for the investigation of the roles of the MC3R and MC4R in diseases of dysregulated energy homeostasis.

Pharmacology of the giant panda (Ailuropoda melanoleuca) melanocortin-3 receptor.

The melanocortin-3 receptor (MC3R) is a member of the G protein-coupled receptor superfamily that plays a critical role in controlling energy balance and metabolism. Although pharmacological characterization of MC3R has been reported previously in several other species, there is no report on the MC3R from giant panda (Ailuropoda melanoleuca). This ancient species is known as a 'living fossil' and is among the most endangered animals in the world. Giant panda survive on a specialized diet of bamboo despite possessing a typical carnivorous digestive system. We report herein the molecular cloning and pharmacological characterization of amMC3R. Homology and phylogenetic analysis showed that amMC3R was highly homologous (>85%) to several other mammalian MC3Rs. Using human MC3R (hMC3R) as a control, the binding of five agonists, [Nle4, D-Phe7]-α-melanocyte stimulating hormone (NDP-MSH), α-, ß-, γ-, and D-Trp8-γ-MSH, was investigated, as well as Gs-cAMP and pERK1/2 signaling. The results showed that amMC3R bound NDP- and D-Trp8-γ-MSH with the highest affinity, followed by α-, ß-, and γ-MSH, with the same rank order as hMC3R. When stimulated with agonists, amMC3R displayed increased intracellular cAMP and activation of pERK1/2. These data suggest that the cloned amMC3R was a functional receptor. The availability of amMC3R and knowledge of its pharmacological functions will assist further investigation of its role in controlling energy balance and metabolism.

Structural Insights into Selective Ligand-Receptor Interactions Leading to Receptor Inactivation Utilizing Selective Melanocortin 3 Receptor Antagonists.

Systematic N-methylated derivatives of the melanocortin receptor ligand, SHU9119, lead to multiple binding and functional selectivity toward melanocortin receptors. However, the relationship between N-methylation-induced conformational changes in the peptide backbone and side chains and melanocortin receptor selectivity is still unknown. We conducted comprehensive conformational studies in solution of two selective antagonists of the third isoform of the melanocortin receptor (hMC3R), namely, Ac-Nle-c[Asp-NMe-His6-d-Nal(2')7-NMe-Arg8-Trp9-Lys]-NH2 (15) and Ac-Nle-c[Asp-His6-d-Nal(2')7-NMe-Arg8-NMe-Trp9-NMe-Lys]-NH2 (17). It is known that the pharmacophore (His6-DNal7-Arg8-Trp9) of the SHU-9119 peptides occupies a ß II-turn-like region with the turn centered about DNal7-Arg8. The analogues with hMC3R selectivity showed distinct differences in the spatial arrangement of the Trp9 side chains. In addition to our NMR studies, we also carried out molecular-level interaction studies of these two peptides at the homology model of hMC3R. Earlier chimeric human melanocortin 3 receptor studies revealed insights regarding the binding and functional sites of hMC3R selectivity. Upon docking of peptides 15 and 17 to the binding pocket of hMC3R, it was revealed that Arg8 and Trp9 side chains are involved in a majority of the interactions with the receptor. While Arg8 forms polar contacts with D154 and D158 of hMC3R, Trp9 utilizes π-π stacking interactions with F295 and F298, located on the transmembrane domain of hMC3R. It is hypothesized that as the frequency of Trp9-hMC3R interactions decrease, antagonistic activity increases. The absence of any interactions of the N-methyl groups with hMC3R suggests that their primary function is to modulate backbone conformations of the ligands.

Old drugs with new skills: fenoprofen as an allosteric enhancer at melanocortin receptor 3.

The efficiency of drug research and development has paradoxically declined over the last decades despite major scientific and technological advances, promoting new cost-effective strategies such as drug repositioning by systematic screening for new actions of known drugs. Here, we performed a screening for positive allosteric modulators (PAMs) at melanocortin (MC) receptors. The non-steroidal anti-inflammatory drug fenoprofen, but not the similar compound ibuprofen, presented PAM activity at MC3, MC4, and MC5 receptors. In a model of inflammatory arthritis, fenoprofen afforded potent inhibition while ibuprofen was nearly inactive. Fenoprofen presented anti-arthritic actions on cartilage integrity and synovitis, effects markedly attenuated in Mc3r-/- mice. Fenoprofen displayed pro-resolving properties promoting macrophage phagocytosis and efferocytosis, independently of cyclooxygenase inhibition. In conclusion, combining repositioning with advances in G-protein coupled receptor biology (allosterism) may lead to potential new therapeutics. In addition, MC3 PAMs emerged as a viable approach to the development of innovative therapeutics for joint diseases.

[THE THYROID STATUS OF RATS IMMUNIZED WITH PEPTIDES DERIVED FROM THE EXTRACELLULAR REGIONS OF THE TYPES 3 AND 4 MELANOCORTIN RECEPTORS AND THE 1B-SUBTYPE 5-HYDROXYTRYPTAMINE RECEPTOR].

The activity of the hypothalamic-pituitary-thyroid (HPT) axis is controlled by the brain neurotransmitter systems, including the melanocortin signaling system. Pharmacological inhibition of type 4 melanocortin receptor (M4R) leads to disruption of the functioning of HPT axis and to reduction of the level of thyroid hormones. At the same time, the data on how prolonged inhibition of M4R affects this axis and on its role in regulation of M3R are absent. The relationship between the thyroid status and the activity of 1B-subtype 5-hydroxytryptamine receptor (5-HT1BR) is scarcely explored. The aim of this work to study the effects of chronic inhibition of M3R, M4R and 5-HT1BR induced by immunization of rats with BSA-conjugated peptide derived from the extracellular regions of these receptors on the thyroid status and the activity of thyroid stimulating hormone (TSH)-sensitive adenylyl cyclase signaling system (ACSS) in the thyroid glarid (TG) of the immunized animals. In rats immunized with the peptides K-[TSLHL WNRSSHGLHG11-25]-A of M4R, A[PTNPYCICTTAH269-280]-A of M3R and. [QAKAEE-EVSEC(Acm)-VVNTDH189-205]-A of 5-HT1BR levels of thyroid hormones such as fT4, tT4 and tT3 were significantly reduced. In rats immunized with M4R and M3R peptides, an increase of TSH was detected whereas in the animals immunized with 5-HT1BR peptide the level of TSH, on the contrary, was reduced. In the TG of rats immunized with M4R and M3R peptides, the stimulatory effects of hormones (TSH, PA-CAP-3 8) and GppNHp on adenylyl cyclase activity were attenuated, and the changes were most pronounced in the case M4R peptide immunization. After immunization with 5-HT1BR peptide the stimulatory effects of TSH, PACAP-38 and GppNHp were retained. Thus, the main cause of thyroid hormones deficit in rats immunized with M4R and M3R peptides was the decreased sensitivity of ACSS thyrocytes to TSH, whereas in rats iimunized with 5-HT1BR peptide the deficit of thyroid hormones was associated with decreased level of TSH. Our data on the negative impact of long-term immunization of rats with BSA-conjugated peptides derived from the extracellular regions of M4R, M3R.and 5-HT1BR on their thyroid status is a strong argument in favor of participation of these receptors and intracellular signaling pathways associated with them in the regulation of HPT axis.

Functions of the DRY motif and intracellular loop 2 of human melanocortin 3 receptor.

The melanocortin 3 receptor (MC3R) regulates several physiological functions, including feed efficiency, nutrient partitioning, fasting response, natriuresis, and immune reactions. Naturally occurring mutations in the MC3R gene have been shown to be associated with increased adiposity and lung diseases such as tuberculosis and cystic fibrosis. The DRY motif at the cytoplasmic end of transmembrane domain 3 (TM3) and the second intracellular loop 2 (ICL2) are known to be important for receptor function in several G protein-coupled receptors (GPCRs). To gain a better understanding of the functions of this domain in MC3R, we performed alanine-scanning mutagenesis on 18 residues. We showed that alanine mutation of 11 residues reduced the maximal binding and maximal cAMP production stimulated by agonists. Mutation of two residues did not change maximal binding but resulted in impaired signaling in the Gs-cAMP pathway. Mutation of five residues impaired signaling in the ERK1/2 pathway. We have also shown that alanine mutants of seven residues that were defective in the cAMP pathway were not defective in the ERK1/2 pathway, demonstrating biased signaling. In summary, we demonstrated that the cytoplasmic end of TM3 and the ICL2 were critical for MC3R function. We also reported for the first time biased signaling in MC3R.

Development of potent selective competitive-antagonists of the melanocortin type 2 receptor.

Cushing's disease, a hypercortisolemic state induced by an ACTH overexpressing pituitary adenoma, causes increased morbidity and mortality. Selective antagonism of the melanocortin type 2 receptor (MC2R) may be a novel treatment modality. Five structurally related peptides with modified HFRW sites but intact putative MC2R binding sites were tested for antagonistic activity at MC1R, MC2R/MRAP, MC3R, MC4R and MC5R. Two of these peptides (GPS1573 and GPS1574) dose-dependently antagonized ACTH-stimulated MC2R activity (IC50s of 66±23 nM and 260±1 nM, respectively). GPS1573 and 1574 suppressed the Rmax but not EC50 of ACTH on MC2R, indicating non-competitive antagonism. These peptides did not antagonize α-MSH stimulation of MC1R and antagonized MC3, 4 and 5R at markedly lower potency. GP1573 and GPS1574 antagonize MC4R with IC50s of 950 nM and 3.7 µM, respectively. In conclusion, two peptide antagonists were developed with selectivity for MC2R, forming a platform for development of a medical treatment for Cushing's disease.

Effect of peptides corresponding to extracellular domains of serotonin 1B/1D receptors and melanocortin 3 and 4 receptors on hormonal regulation of adenylate cyclase in rat brain.

The ligand-recognizing part of G protein-coupled receptors consists of their extracellular loops and N-terminal domain. Identification of these sites is essential for receptor mapping and for the development and testing of new hormone system regulators. The peptides corresponding by their structure to extracellular loop 2 of serotonin 1B/1D receptor (peptide 1), extracellular loop 3 of melanocortin 3 receptor (peptide 2), and N-terminal domain of melanocortin 4 (peptide 3) were synthesized by the solid-phase method. In synaptosomal membranes isolated from rat brain, peptide 1 (10(-5)-10(-4) M) attenuated the effects of 5-nonyloxytryptamine (selective agonist of serotonin 1B/1D receptor) and to a lesser extent serotonin and 5-methoxy-N,N-dimethyltryptamine acting on all the subtypes of serotonin receptor 1. Peptide 2 (10(-5)-10(-4) M) significantly reduced the adenylate cyclase-stimulating effect of γ-melanocyte-stimulating hormone (agonist of melanocortin receptor 3), but had no effect on the adenylate cyclase effect of THIQ (agonist melanocortin receptor 4). Peptide 3 reduced the adenylate cyclase-stimulating effects of THIQ and α-melanocyte-stimulating hormone (non-selective agonist of melanocortin receptors 3 and 4), but did not modulate the effect of γ-melanocyte-stimulating hormone. The effect of peptide 3 was weaker: it was observed at peptide 3 concentration of 10(-4) M. Peptides 1-3 did no change the adenylate cyclase-modulating effects of hormones acting through non-homologous receptors. Thus, the synthesized peptides specifically inhibited the regulatory effects of hormones acting through homologous receptors. This suggests that the corresponding extracellular domains are involved in ligand recognition and binding and determine functional activity of the receptor.

[The influence of immunization of rats with BSA-conjugated peptide 269-280 of type 3 melanocortin receptor on the metabolic parameters and thyroid functions].

One of the approaches to study the role of the brain hormonal signaling systems in the regulation of biochemical and physiological processes is their shutdown using the antibodies generated to peptides corresponding to extracellular regions of receptors. The brain type 3 melanocortin receptors (M3R) play an important role in the central regulation of the metabolism and the endocrine system. However, the influence of prolonged inhibition of M3R on energy metabolism, insulin resistance, and thyroid gland (TG) function is practically not studied. The aim of the study was to investigate the influence of prolonged repeated immunization of male rats with the BSA-conjugated peptide Ala-[Pro-Thr-Asn-Pro-Tyr-Cys-Ile-Cys-Thr-Thr-Ala-His269-280]-Ala (A[269- 280]A) corresponding to the third extracellular loop of M3R on their metabolic parameters and functional activity of TG. 9 months after the first immunization, the weight of rats was reduced and after 12-13 months was significantly lower than in controls. The weight of abdominal and brown adipose tissues, on the contrary, increased. At the same timeline there was an increase in the fasting glucose and insulin levels, and increase of the HOMA-IR index (by 75%) indicating that immunized animals develop insulin resistance. The rats have increased glucose utilization due to an increase of insulin production by pancreatic ß-cells. 12 months after the first immunization, the increase of the triglycerides level (by 74%) and the ratio of LDL- and HDL-cholesterol (by 36%) were revealed. 13 months after the start of immunization, the levels of free and total thyroxine and total triiodothyronine significantly decreased. In the TG plasma membranes of immunized rats the weakening adenylyl cyclase stimulating effect of thyroid-stimulating hormone was detected. Thus, long-term decrease in the bra- in M3R activity due to repeated immunization of rats with BSA-conjugated peptide A[269-280]A induces the disturbances of the peripheral metabolism and TG function.

Functions of the third intracellular loop of the human melanocortin-3 receptor.

The melanocortin-3 receptor (MC3R) is a G protein-coupled receptor involved in regulating energy metabolism, cardiovascular function, and inflammation. To gain a better understanding of the structure-function relationship of the MC3R, we used alanine-scanning mutagenesis to investigate the functions of residues 247-273 in the third intracellular loop (ICL3) of the human MC3R (hMC3R). Ligand binding and signaling parameters of the mutants were measured. The results showed that six mutants at the N terminus of ICL3 had decreased receptor occupancy (an estimate of relative binding capacity) whereas six mutants at the C terminus of ICL3 had increased receptor occupancy. M247A, R252A, H254A, and K256A had decreased maximal responses (M247A also had increased EC50) whereas F250A, P262A, and H272A had increased maximal responses. None of the mutants was constitutively active. The binding and signaling properties of the other mutants were not significantly different from that of the wild type hMC3R. In summary, we presented detailed functional data on the functions of the residues in ICL3 of hMC3R, providing important constraints for modeling ligand binding and G protein coupling/activation in the hMC3R.

Structural insight into the role of the human melanocortin 3 receptor cysteine residues on receptor function.

Melanocortin-3 receptor (MC3R), expressed in the hypothalamus and limbic systems of the brain, as well as by peripheral sites, plays an important role in the regulation of energy homeostasis and other physiological functions. Past work shows that MC3R-deficiency resulted in fat mass increase, feeding efficiency increase, hyperleptinemia and mild hyperinsulinemia in mice and human. MC3R belongs to G-protein coupled receptor (GPCR) family and many studies indicate that some cysteine residues in GPCR play key roles in maintaining receptor tertiary structure and function. In this study, we examined the role of cysteine residues in MC3R on receptor function. Human MC3R (hMC3R) has eighteen cysteine residues where they are located in the extracellular loops (ELs), the transmembrane domains (TMs) and the intracellular loops (ILs). We replaced these cysteines with serine and expressed these receptors in HEK-293 cells which lack endogenous MC3R. Our results indicate that five cysteines in eighteen of the hMC3R are important for hMC3R function. Mutations, C305S, C311S, and C313S in EL3, resulted in significant decrease in receptor expression and receptor function while two other mutations C115S and C162S in TM3 significantly decreased NDP-MSH binding affinity and potency. These results suggest that extracellular cysteine residue 305, 311 and 313 are crucial for receptor expression and the transmembrane cysteine residue, C115 and 162 are important for ligand binding and signaling. These findings provide important insights into the importance of cysteine residues of hMC3R on receptor tertiary structure and function.

Physiological roles of the melanocortin MC3 receptor.

The melanocortin MC(3) receptor remains the most enigmatic of the melanocortin receptors with regard to its physiological functions. The receptor is expressed both in the CNS and in multiple tissues in the periphery. It appears to be an inhibitory autoreceptor on proopiomelanocortin neurons, yet global deletion of the receptor causes an obesity syndrome. Knockout of the receptor increases adipose mass without a readily measurable increase in food intake or decrease in energy expenditure. And finally, no melanocortin MC(3) receptor null humans have been identified and associations between variant alleles of the melanocortin MC(3) receptor and diseases remain controversial, so the physiological role of the receptor in humans remains to be determined.

Antibodies raised against different extracellular loops of the melanocortin-3 receptor affect energy balance and autonomic function in rats.

Melanocortin receptors (MCR) play an important role in the regulation of energy balance and autonomic function. In the present studies, we used active immunization against peptide sequences from the first and the third extracellular loop (EL1 and EL3) of the MC3R to generate selective antibodies (Abs) against this MCR subtype in rats. Immunization with the EL1 peptide resulted in Abs that enhanced the effects of the endogenous ligand α-melanocyte-stimulating hormone (α-MSH), whereas immunization with the EL3 peptide resulted in Abs acting as non-competitive antagonists. The phenotype of immunized rats chronically instrumented with telemetry transducers was studied under four different conditions: a high-fat diet was followed by standard lab chow, by fasting, and finally by an intraperitoneal injection of lipopolysaccharide (LPS). Under high-fat diet, food intake and body weight were higher in the EL3 than in the EL1 or the control group. Blood pressure was increased in EL3 rats and locomotor activity was reduced. Plasma concentrations of triglycerides, insulin, and leptin tended to rise in the EL3 group. After switching to standard lab chow, the EL1 group showed a small significant increase in blood pressure that was more pronounced and associated with an increase in heart rate during food restriction. No differences between the EL1 or the EL3 group were observed after LPS injection. These results show that immunization against the MC3R resulted in the production of Abs with positive or negative allosteric properties. The presence of such Abs induced small changes in metabolic and cardiovascular parameters.

γ2-Melanocyte stimulation hormone (γ2-MSH) truncation studies results in the cautionary note that γ2-MSH is not selective for the mouse MC3R over the mouse MC5R.

The melanocortin system has been implicated in a multitude of physiological pathways including obesity, satiety, energy homeostasis, sexual behavior, pigmentation, sodium regulation, hypertension, and many others. Based upon studies of the endogenous melanocortin receptor agonists at the cloned human melanocortin receptor proteins, it was concluded that the γ-MSH related agonist ligands are selective for the MC3 versus the MC4 and MC5 receptors. In attempts to understand and identify the specific amino acids of γ2-MSH important for MC3R selectivity, we have performed N- and C-terminal truncation studies and pharmacologically characterized twenty-eight ligands at the mouse MC1 and MC3-5 melanocortin receptors. The C-terminal Trp-Asp9-Arg¹°-Phe¹¹ residues are important for nM potency at the mMC3R and the Arg7-Trp8 residues are important for mMC5R nM potency. We observed the unanticipated results that several of the C-terminal truncated analogs possessed nM agonist potency at the mMC3 and mMC5Rs which lead us to perform a comparative side-by-side study of the mouse and human MC5R. These data resulted in µM γ2-MSH analog potency at the hMC5R, consistent with previous reports, however at the mMC5R, nM γ2-MSH analog potency was observed. Thus, these data support the hypothesis of important species specific differences in γ-MSH related ligand potency at the rodent versus human MC5R subtype that is critical for the interpretation of in vivo rodent physiological studies. These results prompted us to examine the affects of a peripherally administered melanocortin agonist on hypothalamic gene expression levels of the MC3R, MC4R, and MC5R. The super potent non-selective NDP-MSH agonist was administered i.p. and resulted in significantly decreased levels of mMC3R and mMC5R hypothalamic mRNA versus saline control. These data provide for the first time data demonstrating peripherally administered NDP-MSH can modify hypothalamic melanocortin receptor expression levels.

Novel binding motif of ACTH analogues at the melanocortin receptors.

The melanocortin receptor (MCR) subtype family is a member of the GPCR superfamily, and each of them has a different pharmacological profile with regard to the relative potency of the endogenous and synthetic melanocortin peptides. Alpha-MSH and ACTH are endogenous nonselective agonists for MC1R, MC3R, MC4R, and MC5R. In this study, we examined the role of Phe(7) in ACTH on human (h) MC1R, MC3R, and MC4R binding and signaling. Our results indicate that substitution of Phe(7) with d-Nal(2')(7) in ACTH1-24 yields a pharmacological profile different from that for substitution of Phe(7) with d-Nal(2')(7) in MSH in hMC1R, hMC3R, and hMC4R. N-d-Nal(2')(7)-ACTH1-24 is an agonist at hMC3R and hMC4R which did not change the peptide from an agonist to an antagonist at hMC3R and hMC4R. Further experiments indicate that N-d-Nal(2')(7)-ACTH1-17 is the minimal peptide required for hMC3R and hMC4R activation. Single-amino acid substitution studies of d-Nal(2')(7)-ACTH1-17 indicate that amino acid residues 15-17 in N-d-Nal(2')(7)-ACTH1-17 are crucial for hMC3R and hMC4R activation. Substitutions of these amino acid residues reduced or abolished agonist activity at hMC3R and hMC4R. Conformational studies revealed a new beta-turn (Arg(8)-Trp(9)-Gly(10)-Lys(11)) in N-d-Nal(2')(7)-ACTH1-17, compared to the beta-turn-like structure at NDP-alpha-MSH (His(6)-d-Phe(7)-Arg(8)-Trp(9)). Our results suggest that NDP-alpha-MSH and N-d-Nal(2')(7)-ACTH1-17 do not share the same binding site; the highly basic C-terminal fragment (Lys(15)-Lys(16)-Arg(17)) of N-d-Nal(2')(7)-ACTH1-17 induced a new beta-turn, and this shift contributed the selective agonist activity at hMC3R and hMC4R.

Melanotropins as drugs for the treatment of obesity and other feeding disorders: potential and problems.

Current biological and pharmacological evidence suggests that the melanocortin 4 and melanocortin 3 receptors which are seven transmembrane G-protein coupled receptors (GPCRs) are involved in various aspects of energy balance and feeding behaviors in animals including humans. The natural endogenous ligands for these receptors are products of the gene pro-opiomelanocortin (POMC), and include alpha-melanocyte stimulating hormone, gamma-melanocyte stimulating hormone and perhaps other modified products of POMC. Thus well designed agonists and antagonists of these ligands might serve as drugs for the treatment of feeding disorders. However, these melanotropin peptides also can have other biological activities that involve the MC3R and MC4R, and these other biological properties will need to be modulated in ligands that are likely to be useful drugs for feeding disorders. Current progress in these areas with special emphasis on the MC3R will be discussed along with possible new directions that might be fruitful in these important aspects of contemporary biology and medicine.