Human MC4 Research Articles

Characterizing the body morphology of the first metacarpal in the Homininae using 3D geometric morphometrics

AbstractObjectivesThe morphological characteristics of the thumb are of particular interest due to its fundamental role in enhanced manipulation. Despite its possible importance regarding this issue, the body of the first metacarcapal (MC1) has not been fully characterized using morphometrics. This could provide further insights into its anatomy, as well as its relationship with manipulative capabilities. Hence, this study quantifies the shape of the MC1's body in the extant Homininae and some fossil hominins to provide a better characterization of its morphology.Materials and methodsThe sample includes MC1s of modern humans (n = 42), gorillas (n = 27), and chimpanzees (n = 30), as well as Homo neanderthalensis, Homo naledi, and Australopithecus sediba. 3D geometric morphometrics were used to quantify the shape of MC1's body.ResultsThe results show a clear distinction among the three extant genera. H. neanderthalensis mostly falls within the modern human range of variation. H. naledi varies slightly from modern humans, although also showing some unique trait combination, whereas A. sediba varies to an even greater extent. When classified using a discriminant analysis, the three fossils are categorized within the Homo group.ConclusionThe modern human MC1 is characterized by a distinct suite of traits, not present to the same extent in the great apes, that are consistent with an ability to use forceful precision grip. This morphology was also found to align very closely with that of H. neanderthalensis. H. naledi shows a number of human‐like adaptations, while A. sediba presents a mix of both derived and more primitive traits.

AB0068 NOVEL CHONDROPROTECTIVE AND ANTI-INFLAMMATORY EFFECTS OF THE SELECTIVE HUMAN MELANOCORTIN MC3 RECEPTOR AGONIST PG-990 ON SNAP ACTIVATED CHONDROCYTES

Background:Osteoarthritis (OA) is a degenerative joint disease that affects over 250 million people worldwide [1] with treatments focussing on the symptoms rather than the cause of the pathology [2, 3]. Thus, this degenerative joint disease requires novel treatment options [3, 4].Therefore, the melanocortin system [4] could provide a novel avenue to explore given its ability to exert anti-inflammatory effects and chondroprotection [5], although the receptor subtype involved is unclear.Objectives:This study aims to assess the chondroprotective and anti-inflammatory effects of the selective human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride and the selective human MC3 receptor agonist PG-990 on S-Nitroso-N-acetyl-DL-penicillamine (SNAP) activated chondrocytes.Methods:The human chondrocytic cell-line C-20/A4 was seeded at 25.0 x 106viable cells/ml (5 μl droplet was transferred into individual wells of a 96-well plate). Micromass cultures [6] were stimulated with SNAP (1.0 mM) and after 2h treated with Dexamethasone (1.0 μM), selective human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride (10.0 μg/ml) or selective human melanocortin MC3 receptor agonist PG-990 (10.0 μg/ml) for 6h. Cell viability was determined by MTT assay, Caspase -3 and -7 activity determined by Caspase-Glo 3/7 apoptosis assay. Glycosaminoglycan (GAG) content determined by alcian blue staining and anti-inflammatory heme-oxygenase-1 (HO-1) protein expression was determined by western blot. Data are expressed as Mean ±S.E.M ofn=4 samples repeated in triplicate. #p≤0.05vscontrol or *p≤0.05vsstimulus.Results:Cell viability analysis showed SNAP stimulation caused a maximal cell death of 23% (#p≤0.05), Dexamethasone, BMS-470539 dihydrochloride and PG-990 inhibited cell death by 2%, 98% and 129% respectively (*p≤0.05). SNAP stimulation caused a significant increase in Caspase -3 and -7 activity, which was inhibited by Dexamethasone, BMS-470539 dihydrochloride and PG-990 by 8%, 5% and 19% respectively (*p≤0.05). GAG content was significantly reduced by SNAP by 29% (#p≤0.05), which was inhibited by Dexamethasone, BMS-470539 dihydrochloride and PG-990 by 1%, 3% and 14% respectively (*p≤0.05). SNAP also caused a significant decrease in HO-1 protein expression, which was increased by Dexamethasone, BMS-470539 dihydrochloride and PG-990 by a 1.0-fold, 1.1-fold and 2.1-fold increase respectively (*p≤0.05).Conclusion:The selective human melanocortin MC3 receptor agonist PG-990 exhibited enhanced chondroprotection and modulation of inflammatory and tissue destructive mediators following SNAP activation compared to Dexamethasone and the selective human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride. This suggests that melanocortin peptides display enhanced chondroprotective and anti-inflammatory effects at the MC3 receptor sub-type in this cell line.

Novel anti-inflammatory and chondroprotective effects of the human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride and human melanocortin MC3 receptor agonist PG-990 on lipopolysaccharide activated chondrocytes

Human melanocortin MC1 and MC3 receptors expressed on C-20/A4 chondrocytes exhibit chondroprotective and anti-inflammatory effects when activated by melanocortin peptides. Nearly 9 million people in the UK suffer from osteoarthritis, and bacterial infections play a role in its development. Here, we evaluate the effect of a panel of melanocortin peptides with different selectivity for human melanocortin MC1 (α-MSH, BMS-470539 dihydrochloride) and MC3 ([DTrp8]-γ-MSH, PG-990) receptors and C-terminal peptide α-MSH11-13(KPV), on inhibiting LPS-induced chondrocyte death, pro-inflammatory mediators and induction of anti-inflammatory proteins. C-20/A4 chondrocytes were treated with a panel of melanocortin peptides prophylactically and therapeutically in presence of LPS (0.1 μg/ml). The chondroprotective properties of these peptides determined by cell viability assay, RT-PCR, ELISA for detection of changes in inflammatory markers (IL-6, IL-8 and MMP-1, -3 and -13) and western blotting for expression of the anti-inflammatory protein heme-oxygenase-1. C-20/A4 expressed human melanocortin MC1 and MC3 receptors and melanocortin peptides elevated cAMP. LPS stimulation caused a reduction in C-20/A4 viability, attenuated by the human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride, and MC3 receptor agonists PG-990 and [DTrp8]-γ-MSH. Prophylactic and therapeutic regimes of [DTrp8]-γ-MSH significantly inhibited LPS-induced modulation of cartilage-damaging IL-6, IL-8, MMPs −1,-3 and −13 mediators both prophylactically and therapeutically, whilst human melanocortin MC1 and MC3 receptor agonists promoted an increase in HO-1 production. In the presence of LPS, activation of human melanocortin MC1 and MC3 receptors provided potent chondroprotection, upregulation of anti-inflammatory proteins and downregulation of inflammatory and proteolytic mediators involved in cartilage degradation, suggesting a new avenue for osteoarthritis treatment.

Discovery of Novel Potent and Selective Agonists at the Melanocortin-3 Receptor.

The melanocortin receptors 3 and 4 control energy homeostasis, food-intake behavior, and correlated pathophysiological conditions. The melanocortin-4 receptor (MC4R) has been broadly investigated. In contrast, the knowledge related to physiological roles of the melanocortin-3 receptor (MC3R) is lacking because of the limited number of known MC3R selective ligands. Here, we report the design, synthesis, biological activity, conformational analysis, and docking with receptors of two potent and selective agonists at the human MC3 receptor.

P.7.a.003 Affinity of alpha-MSH-reactive immunoglobulins in eating disorders and their effects on melanocortin 4 receptor signaling

Melanocortin-4 receptor (MC4R) plays a major role in regulating important biological functions including energy homeostasis. MC4 receptors are widely expressed in the brain and modulated by the endogenous peptide agonist a-melanocyte-stimulating hormone (MSH) and the protein antagonist agouti-related protein (AgRP). Both a-MSH and AgRP are nonselective, and because of the blood–brain barrier, a synthetic peptide or protein ligand of this receptor is not suitable for this centrally active target. Efforts to develop potent and selective agonists and antagonists of the human MC4 receptor (hMC4R) have led to the discovery of several classes of di-peptide and non-peptide compounds. Some of these compounds have been demonstrated to possess good pharmacokinetic properties in animals, including good oral bioavailability, because of their relatively low molecule weights. However, information on brain penetration is still very limited. These selective ligands have provided powerful tools for further delineating the role of MC4 receptor in feeding behavior, erectogenic activity, pain, and anxiety/depression.

High IGF-IR Activity in Triple-Negative Breast Cancer Cell Lines and Tumorgrafts Correlates with Sensitivity to Anti–IGF-IR Therapy

We previously reported an insulin-like growth factor (IGF) gene expression signature, based on genes induced or repressed by IGF-I, which correlated with poor prognosis in breast cancer. We tested whether the IGF signature was affected by anti-IGF-I receptor (IGF-IR) inhibitors and whether the IGF signature correlated with response to a dual anti-IGF-IR/insulin receptor (InsR) inhibitor, BMS-754807. An IGF gene expression signature was examined in human breast tumors and cell lines and changes were noted following treatment of cell lines or xenografts with anti-IGF-IR antibodies or tyrosine kinase inhibitors. Sensitivity of cells to BMS-754807 was correlated with levels of the IGF signature. Human primary tumorgrafts were analyzed for the IGF signature and IGF-IR levels and activity, and MC1 tumorgrafts were treated with BMS-754807 and chemotherapy. The IGF gene expression signature was reversed in three different models (cancer cell lines or xenografts) treated with three different anti-IGF-IR therapies. The IGF signature was present in triple-negative breast cancers (TNBC) and TNBC cell lines, which were especially sensitive to BMS-754807, and sensitivity was significantly correlated to the expression of the IGF gene signature. The TNBC primary human tumorgraft MC1 showed high levels of both expression and activity of IGF-IR and IGF gene signature score. Treatment of MC1 with BMS-754807 showed growth inhibition and, in combination with docetaxel, tumor regression occurred until no tumor was palpable. Regression was associated with reduced proliferation, increased apoptosis, and mitotic catastrophe. These studies provide a clear biological rationale to test anti-IGF-IR/InsR therapy in combination with chemotherapy in patients with TNBC.

Parallel Synthesis of a Lipopeptide Library by Hydrazone-Based Chemical Ligation

alpha-Melanocyte-stimulating hormone (alpha-MSH) is an endogeneous linear tridecapeptide with potential application for the modulation of skin tanning. To evaluate the interest of introducing a lipid moiety onto this peptide, we developed an efficient chemoselective parallel method to prepare a large series of analogues of alpha-melanocortin with high purity, varying the nature or the relative position of the lipid moiety. Two sets of building blocks containing lipidic alpha-oxo-aldehydes or alpha-hydrazinoacetyl peptides were combined to obtain a 102-membered library of amphiphilic alpha-MSH analogues. This library was pharmacologically tested at 1 x 10(-7) M for the ability to induce AMPc production in M4Be melanoma cell line after stimulation of the human melanocortin MC1 receptor. Among theses lipopeptides, 84 compounds exhibited an AMPc induction higher than Melitane, a patented alpha-MSH agonist. These results provide strong evidence of the interest of introduction of a lipid tail for the pharmacomodulation of bioactive peptides.

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.

Structure–activity relationship of a series of cyclohexylpiperidines bearing an amide side chain as antagonists of the human melanocortin-4 receptor

A series of cyclohexylpiperazines was synthesized as potent and selective antagonists of the human MC4 receptor. Compound 14t displayed binding affinity ( K i) of 4.2 and 1100 nM at MC4R and MC3R, respectively.

Isolation and characterization of antagonist and agonist peptides to the human melanocortin 1 receptor

We identified a large number of peptide mimotopes of the adrenocorticotropic hormone (ACTH) and the α-melanocyte stimulating hormone (α-MSH) to analyze better the structure–function relationships of these hormones with the human MC1 receptor (hMC1R). We have investigated the use of phage-display technology to isolate specific peptides of this receptor by using three monoclonal anti-ACTH antibodies (mAbs). A library of 10 8 phage-peptides displaying randomized decapeptides was constructed and used to select phage-peptides that bind to mAbs. Forty-five phage-peptides have been isolated and from their amino acid sequences, we have identified two consensus sequences, EXFRWGKPA and WGXPVGKP, corresponding to the regions 5–13 and 9–16 of ACTH, respectively. A biological assay on cells expressing the hMC1-R was developed to determine the capacity of phage-peptides to stimulate the receptor. Only two phage-peptides showed detectable activity. Thirty-one peptides were synthesized to analyze their biological effect. We identified two weak agonists, EC 50 = 16 and 11 μM, two strong agonists, EC 50 = 25 and 14 nM and a partial antagonist, IC 50 = 36 μM. This work confirmed the modulator agonist role of the regions 11–12 of α-MSH and ACTH, and the importance of the methionine residue at position 4 for the stimulation of the hMC1-R. We also identified analogues of the regions 8–17 of ACTH that exhibited a weak activator effect, and of one analogue of the N-terminal regions 1–9 of ACTH and α-MSH having a partial antagonist effect. These results may be useful in the development of potential agonists or antagonists of the hMC1R.

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>500nM) at the MC3 receptor. Compound 14c was identified to be a potent MC4R antagonist (Ki=3.2nM) 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.

Pharmacological Characterization of Melanocortin Receptors in Fish Suggests an Important Role for ACTH

The melanocortin (MC) receptor subtypes have distinctive characteristic binding profiles. We found that the trout and Fugu MC4 receptors have similar affinity for alpha-MSH and beta-MSH and a much higher affinity for ACTH than does the human MC4 receptor. The Fugu MC1 and the trout and Fugu MC5 receptors also have higher affinity for ACTH-derived peptides than alpha-, beta-, or gamma-MSH. It is tempting to speculate that ACTH-derived peptides may have played an important role as "original" ligands at the MC receptors, while the specificity of the different subtypes for the alpha-, beta-, and gamma-MSH peptides may have appeared at later stages during vertebrate evolution.

4-{(2R)-[3-Aminopropionylamido]-3-(2,4-dichlorophenyl)propionyl}-1-{2-[(2-thienyl)ethylaminomethyl]phenyl}piperazine as a Potent and Selective Melanocortin-4 Receptor AntagonistDesign, Synthesis, and Characterization

Recent studies have demonstrated that melanocortin-4 receptor (MC4R) antagonists can prevent weight loss in tumor-bearing mice, which indicates clinical usage for the treatment of cachexia. In our efforts to develop potent and selective antagonists of the human MC4R, we designed piperazinebenzylamines bearing a 2,4-dichlorophenylalanine, by utilizing information derived from structure--activity relationships of MC4R agonists and mutagenesis results of the MC4R and peptide ligands. On the basis of known MC4R agonists such 6, we successfully synthesized potent MC4R antagonists exemplified by 10, which possesses a K(i) value of 1.8 nM in binding affinity. 10 does not stimulate cAMP release in HEK 293 cells expressing the human MC4 receptor at 10 microM concentration. It was demonstrated by Schild analysis that 10 was a competitive functional antagonist with a pA(2) value of 7.9 in the inhibition of alpha-MSH-stimulated cAMP accumulation. 10 also penetrated into the brain when dosed intravenously in rats.

Pharmacological Characterization of Loss of Function Mutations of the Human Melanocortin 1 Receptor That Are Associated with Red Hair

Variation in skin color is the major host risk factor for melanoma and other forms of skin cancer. Individuals with red hair show an increased ratio of phaeomelanin to eumelanin in both hair and skin. This ratio is regulated by the melanocortin (MC) 1 receptor. There are several common point mutations in the human MC1 receptor that are overrepresented in North European red-heads, and in individuals with pale skin. In order to determine the functional significance of these mutations, we expressed the Asp84Glu, Val92Met, Arg163Gln, and Asp294His variants of the human MC1 receptors in eukaryotic cells and determined their ability to bind alpha-melanocyte stimulating hormone (MSH) peptides and increase intracellular cAMP. The mutants Asp84Glu and Asp294His showed a much lower response to alpha-MSH in cAMP and a slightly impaired ability to bind alpha-MSH, and the Val92Met mutant bound alpha-MSH with 100-fold lower affinity as compared with the wild-type. The Arg163Gln variant, widely found in some Asian populations, reached normal level of cAMP response but had just slightly lower potency for alpha-MSH in binding and second messenger studies. The results provide important pharmacological characterization of common MC1 receptor variants in various world populations.

End-capping of the modified melanocortin tetrapeptide ( p-Cl)Phe- D-Phe-Arg-Trp-NH 2 as a route to hMC4R agonists

Of the 42 R′-X-( p-Cl)Phe- D-Phe-Arg-Trp-NH 2 (X = CO, SO 2, PO, PS) tested at the human (h)MC1, hMC3, and hMC4 receptors (R), the most potent MC4R agonists (EC 50 of 8–20 nM) were obtained by end-capping with R′ = CH 2 CHCH 2 ( 9), NCCH 2 ( 16), NH 2COCH 2 ( 17), HCONHCH 2 ( 18), CH 3NH ( 19), CH 2 CHCH 2NH ( 21), 2-Th ( 23), PhCH 2 ( 30) and X = CO. These compounds possess 35–60-fold hMC4 versus hMC1Rs selectivity with urea LK-71 ( 19) being the most potent at hMC4R and MC4/1R selective (EC 50 = 8.5 nM, MC4/1R = 100). LK-75 ( 16) combines high potency at hMC4R and MC4/3R selectivity (EC 50 = 10.5 nM, MC4/3R = 290). SAR is discussed.

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.

Evaluation of the human melanoma targeting properties of radiolabeled alpha-melanocyte stimulating hormone peptide analogues.

The purpose of this study was to evaluate the human MC1 receptor-mediated melanoma targeting properties of two metal cyclized alpha-MSH peptide analogues, (188)Re-(Arg(11))CCMSH and (188)Re-CCMSH. Initially, the presence and density of the MC1 receptor were determined on a bank of human melanoma cell lines. All eight human melanoma cell lines tested in this study displayed the MC1 receptor at a density of 900 to 5700 receptors per cell. Receptor affinity and biodistribution properties of (188)Re-(Arg(11))CCMSH and (188)Re-CCMSH were evaluated in a cultured TXM13 human melanoma-xenografted Scid mouse model. Biodistribution results demonstrated that 3.06 +/- 0.68 %ID/g of (188)Re-(Arg(11))CCMSH accumulated in the tumors 1 h postinjection and greater than 65% of the activity at 1 h postinjection remained in the tumors at 4 h after dose administration. Whole body clearance of (188)Re-(Arg(11))CCMSH was very rapid, with approximately 82% of injected dose cleared through urinary system at 4 h postinjection. There was very little activity in blood and major organs such as liver, lung, and muscle except for the kidney. (188)Re-CCMSH exhibited similar tumor uptake and retention in TXM13 human melanoma-xenografted Scid mice as (188)Re-(Arg(11))CCMSH. However, the kidney uptake value of (188)Re-CCMSH was two times higher than that of (188)Re-(Arg(11))CCMSH. The results of this study indicate that the MC1 receptor is present on the surface of a large number of human melanoma cells, which makes the MC1 receptor a good imaging or therapeutic target. Moreover, the biodistribution properties of (188)Re-(Arg(11))CCMSH and (188)Re-CCMSH highlight their potential as therapeutic agents for human melanoma.

Association of feather colour with constitutively active melanocortin 1 receptors in chicken.

Seven alleles of the chicken melanocortin (MC) 1 receptor were cloned into expression vectors, expressed in mammalian cells and pharmacologically characterized. Four of the clones e(+R), e(+B&D), e(wh)/e(y), E(Rfayoumi) gave receptors to which melanocortin stimulating hormone (alpha-MSH) and NDP-MSH bound with similar IC50 values and responded to alpha-MSH by increasing intracellular cAMP levels in a dose-dependent manner. Three of the cMC1 receptors; e(b), E and E(R), did not show any specific binding to the radioligand, but were found to be constitutively active in the cAMP assay. The E and E(R) alleles are associated with black feather colour in chicken while the eb allele gives rise to brownish pigmentation. The three constitutively active receptors share a mutation of Glu to Lys in position 92. This mutation was previously found in darkly pigmented sombre mice, but constitutively active MC receptors have not previously been shown in any nonmammalian species. We also inserted the Glu to Lys mutation in the human MC1 and MC4 receptors. In contrast with the chicken clones, the hMC1-E94K receptor bound to the ligand, but was still constitutively active independently of ligand concentration. The hMC4-E100K receptor did not bind to the MSH ligand and was not constitutively active. The results indicate that the structural requirements that allow the receptor to adapt an active conformation without binding to a ligand, as a consequence of this E/K mutation, are not conserved within the MC receptors. The results are discussed in relationship to feather colour in chicken, molecular receptor structures and evolution. We suggest that properties for the 'E92K switch' mechanism may have evolved in an ancestor common to chicken and mammals and were maintained over long time periods through evolutionary pressure, probably on closely linked structural features.

2,3-Diaryl-5-anilino[1,2,4]thiadiazoles as melanocortin MC4 receptor agonists and their effects on feeding behavior in rats

The melanocortin-4 receptor (MC4) modulates physiological functions such as feeding behavior, nerve regeneration, and drug addiction. Using a high throughput screen based on 125I-NDP-MSH binding to the human MC4 receptor, we discovered 2,3-diaryl-5-anilino[1,2,4]thiadiazoles 3 as potent and selective MC4 receptor agonists. Through SAR development on the three attached aryl rings, we improved the binding affinity from 174 nM to 4.4 nM IC 50. When delivered intraperitoneally, compounds 3a, 3b, and 3c induced significant inhibition of food intake in a fasting-induced feeding model in rats. When delivered orally, these compounds lost activity, mainly due to rapid metabolism to inactive imidoylthiourea reduction products.

D-Amino acid scan of gamma-melanocyte-stimulating hormone: importance of Trp(8) on human MC3 receptor selectivity.

In our search for potent and receptor-selective agonists and antagonists, we report here the results of D-amino acid substitution at each position of the short peptide gamma-melanocyte-stimulating hormone (gamma-MSH). The native gamma-MSH shows weak binding at all three receptors (i.e., the human MC3, MC4, and MC5) and a selectivity of 1-2 orders of magnitude at the MC3R over the MC4R and MC5R. Sequential replacement of each residue in the gamma-MSH sequence with the corresponding D-isomer results in analogues which mostly have weaker binding affinity than the native peptide, except for two analogues. For the DTrp(8) analogue, there is an increase in binding affinity by about 1 order of magnitude (IC(50) = 6 nM) at the MC3R compared with that of the natural molecule and an increase in selectivity for the MC3R by 2 orders of magnitude compared with the activity at the MC4R and MC5R. The DPhe(6) analogue is about 10-fold more potent (IC(50) = 8.8 nM) at the MC3R compared with the native peptide but lacks subtype selectivity. Measurement of the intracellular cAMP accumulation in human MC3R, MC4R, and MC5R revealed that the native peptide shows potent activity at the MC3R (EC(50) = 5.9 nM) and is about 50-100-fold selective at this receptor compared with the MC4R and MC5R. The DArg(10) (EC(50) = 35 nM) and DPhe(11) (EC(50) = 11 nM) analogues are selective for the MC3R by 1 and 2 orders of magnitude compared with the MC4R and MC5R, respectively. The DTrp(8) compound (EC(50) = 0.33 nM) shows about 300- and 250-fold increase in selectivity at the MC3R compared with the MC4R and MC5R, respectively. Finally, the DTyr(1) peptide is selective for the MC3R (EC(50) = 12 nM) by 40-200-fold compared with the MC4R and MC5R. In general, the trend is that D-amino acid substitutions of the aromatic residues 1, 6, 8, and 11 and the basic residue Arg(10), but not Arg(7), result in an increase in MC3R selectivity over the MC4R and MC5R and only agonist activity is observed. Thus, the key residues of gamma-MSH identified in this study include the aromatic residues 1, 6, 8, and 11 and the basic residue Arg(10) (but not Arg(7)), as important for MC3 selectivity over the MC4 and MC5 subtypes. Further, the study reveals the extreme importance of DTrp at position 8 in imparting potency and selectivity since this is the most selective analogue for the human MC3R reported thus far.