A novel hybrid melanocortin pharmacophore was designed based on the topographical similarities between the pharmacophores of Agouti related protein (AGRP) an endogenous melanocortin antagonist, and α-melanocyte-stimulating hormone (α-MSH), an endogenous melanocortin agonist. When employed in two different 23-membered macrocyclic lactam peptide templates, the designed hybrid AGRP/MSH pharmacophore yielded non-competitive ligands with nanomolar range binding affinities. The topography-based pharmacophore hybridization strategy will prove useful in development of unique non-competitive melanocortin receptor modulators.

The major pharmacophore for the melanocortin 1, 3, 4 and 5 receptors is the sequence -His-Phe-Arg-Trp-. There is a need for potent, biologically stable, receptor selective ligands, both agonists and antagonists, for these receptors. In this report we briefly examine the structural and biophysical approaches we have taken to develop selective agonist and antagonist ligands that can cross (or not) the blood brain barrier. Remaining questions and unmet needs are also discussed.

A novel hybrid melanocortin pharmacophore was designed based on the pharmacophores of the agouti-signaling protein (ASIP), an endogenous melanocortin antagonist, and α-melanocyte-stimulating hormone (α-MSH), an endogenous melanocortin agonist. The designed hybrid ASIP/MSH pharmacophore was explored in monomeric cyclic, and cyclodimeric templates. The monomeric cyclic disulfide series yielded peptides with hMC3R-selective non-competitive binding affinities. The direct on-resin peptide lactam cyclodimerization yielded nanomolar range (25-120 nM) hMC1R-selective full and partial agonists in the cyclodimeric lactam series which demonstrates an improvement over the previous attempts at hybridization of MSH and agouti protein sequences. The secondary structure-oriented pharmacophore hybridization strategy will prove useful in development of unique allosteric and orthosteric melanocortin receptor modulators. This report also illustrates the utility of peptide cyclodimerization for the development of novel GPCR peptide ligands.

A new series of melanotropin analogues with His or Arg residues in the core pharmacophores of MTII, SHU9119, and Ac-NDP-gamma-MSH-NH2 replaced by Pro or trans-/cis-4-guanidinyl-Pro derivatives were designed and synthesized to introduce selectivity toward the human melanocortin 4 receptor (hMC4R). Analogues 1,. 2, 3, 6, 7, 8 were found to be hMC4R selective. Second messenger studies have demonstrated that analogues 1 and 2 are insurmountable inhibitors of MTII agonist activity at the hMC4R. Molecular modeling studies suggest that the hMC4R selectivity is due to a beta-turn shift induced by the Pro ring that makes the global minimum structures of these analogues resemble the NMR solution. structure of the hASIP melanocortin receptor binding motif. Substitution of His in MTII also provided functional selectivity for the hMC3R or the hMC4R. These findings are important for a better understanding of the selectivity mechanism at the hMC3R/hMC4R and the development of therapeutic ligands selectively targeting the hMC4R.

A variety of dicarboxylic acid linkers introduced between the alpha-amino group of Pro(6) and the -amino group of Lys(10) of the cyclic lactam alpha-melanocyte-stimulating hormone (alpha-MSH)-derived Pro(6)-D-Phe(7)/D-Nal(2')(7)-Arg(8)-Trp(9)-Lys(10)-NH2 pentapeptide template lead to nanomolar range and selective hMC3R agonists and antagonists. Replacement of the Pro(6) residue and the dicarboxylic acid linker with 2,3-pyrazine-dicarboxylic acid furnished a highly selective nanomolar range hMC3R partial agonist (analogue 12, c[CO-2,3-pyrazine-CO-D-Phe-Arg-Trp-Lys]-NH2, EC50 = 27 nM, 70% max cAMP) and an hMC3R antagonist (analogue 13, c[CO-2,3-pyrazine-CO-D-Nal(2')-Arg-Trp-Lys]-NH2, IC50 = 23 nM). Modeling experiments suggest that 2,3-pyrazinedicarboxylic acid stabilizes a beta-turn-like structure with the D-Phe/D-Nal(2') residues, which explains the high potency of the corresponding peptides. Placement of a Nle residue in position 6 produced a hMC3R/hMC5R antagonist (analogue 15, c[CO-(CH 2)2-CO-Nle-D-Nal(2')-Arg-Trp-Lys]-NH2, IC50 = 12 and 17 nM, respectively), similarly to the previously described cyclic gamma-melanocyte-stimulating hormone (gamma-MSH)-derived hMC3R/hMC5R antagonists. These newly developed melanotropins will serve as critical biochemical tools for elucidating the full spectrum of functions performed by the physiologically important melanocortin-3 receptor.

We have identified compound 1 as a novel ligand for opioid and melanocortin (MC) receptors, which is derived from the overlapping of a well known structure for the delta opioid receptor, 2,6-dimethyltyrosine (Dmt)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), and a small molecule for the MC receptor, Tic-DPhe(p-Cl)-piperidin-4-yl-N-phenyl-propionamide. Ligand 1 showed that there is an overlapping pharmacophore between opioid and MC receptors through the Tic residue. The ligand displayed high biological activities at the delta opioid receptor (Ki = 0.38 nM in binding assay, EC(50) = 0.48 nM in GTP-gamma-S binding assay, IC(50) = 74 nM in MVD) as an agonist instead of an antagonist and showed selective binding affinity (IC(50) = 2.3 muM) at the MC-3 receptor rather than at the MC-5 receptor. A study of the structure-activity relationships demonstrated that the residues in positions 2, 3, and the C-terminus act as a pharmacophore for the MC receptors, and the residues in positions 1 and 2 act as a pharmacophore for the opioid receptors. Thus, this structural construct can be used to prepare chimeric structures with adjacent or overlapping pharmacophores for opioid and MC receptors.

Differentiation of the physiological role of the melanocortin receptor 5 MC5R from that of other melanocortin receptors will require development of high affinity and selective antagonists. To date, a few synthetic antagonist ligands active at hMC5 receptor are available, but most do not have appreciable selectivity. With the aim to gain more potent and selective antagonists for the MC5R ligands, we have designed, synthesized, and pharmacologically characterized a series of alkylthioaryl-bridged macrocyclic peptide analogues derived from MT-II and SHU9119. These 20-membered macrocycles were synthesized by a tandem combination using solid phase peptide synthesis and microwave-assisted reactions. Biological assays for binding affinities and adenylate cyclase activities for the hMC1R, hMC3R, hMC4R, and hMC5R showed that three analogues, compounds, 9, 4, and 7, are selective antagonists at the hMC5 receptor. In particular, compound 9(PG-20N) is a selective and competitive hMC5R antagonist, with IC 50 of 130 +/- 11 nM, and a pA 2 value of 8.3, and represents an important tool for further biological investigations of the hMC5R. Compounds 4 and 7 (PG14N, PG17N) show potent and selective allosteric inhibition at hMC5R with IC 50 values of 38 +/- 3 nM and 58 +/- 6 nM, respectively. Compound 9 will be used to further investigate and more clearly understand the physiological roles played by the MC5 receptor in humans and other animals.

Recently we have demonstrated that replacing His(6) by constrained amino acids(2) in the well-known antagonist SHU-9119 resulted in potent and selective antagonist ligands especially at the hMC3R and hMC5 receptors. With the aim to further explore position 6 in the sequence of SHU-9119 and MT-II, we have designed, synthesized, and pharmacologically characterized a series of peptide analogues of MT-II and SHU-9119 at the human melanocortin receptors subtypes MC3R, MC4R and MC5R. All these peptides were modified at position 6 with constrained amino acids which are commercially available. In this study, we have identified new selective ligands for the hMC4R, and an antagonist for the hMC3/hMC4 receptors. Additionally, we have discovered an interesting new selective antagonist at the hMC3R, Ac-Nle-c[Asp-beta Ala-DNal(2')-Arg-Trp-Lys]-NH2 (2, PG-106) which represents an important tool in further biological investigations of the hMC3R. PG-106 will be useful in further efforts to differentiate the substructural features responsible for selectivity at the hMC3R, hMC4R, and hMC5R. (c) 2007 Elsevier Inc. All rights reserved.

Introduction Despite the central importance of peptides as drugs such as insulin, oxytocin, calcitonin, ace inhibitors, etc., there has been a prejudice against developing peptide drugs. Instead large efforts are made to develop non-peptide small molecules (“peptide mimetics”) to replace peptides. But are they true mimetics (same SAR, same bioactivity, same toxicity profile, etc.)? This issue generally has not been addressed directly. This is especially the case when the targets for the ligands are GPCRs. When the ligands are antagonists (for receptors, enzymes, etc.) this issue is not as critical as when the ligand is an agonist for a GPCR (or growth factors, or cytokines, etc.). The issue is highly relevant because biological function will depend on activation of a particular signaling pathway, and to obtain the same bioactivity will require the same SARs. To examine this conundrum we have begun to carefully evaluate whether peptide and non-peptide mimetics utilize the same signaling pathways. We find that often they do not, which raises questions regarding drug design. Here we examine the signaling of two agonist (a peptide and a nonpeptide) ligands for the human melanocortin 4 receptor (hMC4R) utilizing molecular biological, pharmacological, biophysical, and biochemical methods and demonstrate that though both are agonists they utilize quite different signaling pathways. The implications for drug design are briefly discussed.

The effects of the linker arm rigidity and size on melanocortin receptor selectivity were explored in a series of compounds using cyclic lactam α‐melanocyte‐stimulating hormone template. A variety of dicarboxylic acid linkers introduced between the α‐amino group of His6 and the ɛ‐amino group of Lys10 lead to high‐affinity, selective human melanocortin receptor‐1 and ‐5 (hMC1R and hMC5R) antagonists. The incorporation of hydrophilic functions into the linker arm was found to be unfavorable for both binding potency and receptor selectivity. Analogs 8 and 9 containing highly conformationally constrained hydrophobic linkers (m‐ and p‐phthalic acids) were found to be selective nanomolar range hMC1R antagonists (IC50 = 7 and 4 nm, respectively), whereas the employment of a small conformationally constrained linker (maleic acid) resulted in a high‐affinity (IC50 = 19 nm) and selective hMC5R antagonist (analog 12). These newly developed melanotropins will serve as critical biochemical tools for elucidating the full spectrum of functions performed by the physiologically important melanocortin‐1 and ‐5 receptors.

A series of cyclic lactam analogues of gamma-MSH (H-Tyr1-Val2-Met3-Gly4-His5-Phe6-Arg7-Trp8-Asp9-Arg10-Phe11-Gly12-OH) with a bulky hydrophobic residue in the direct proximity to the pharmacophore (Xaa-D-Phe/D-Nal(2')-Arg-Trp) were designed and synthesized by solid-phase methods. A variety of amino acids with a broad range of hydrophobic/hydrophilic properties was introduced in position 5 to further explore their complementary role in receptor selectivity. Biological evaluation of these peptides revealed several analogues with potent hMC3R agonist and hMC3R/hMC5R antagonist activities, and good receptor selectivity. Analogue 4, c[Nle-Arg-D-Phe-Arg-Trp-Glu]-NH2, was found to be a very potent and selective hMC3R agonist (EC50=1.2 nM, 112% act). In addition, analogue 13, c[Nle-Val-D-Nal(2')-Arg-Trp-Glu]-NH2, was identified as an hMC3R/hMC5R antagonist with the best selectivity against the hMC4R in this series (pA2(hMC3R)=8.4; pA2(hMC5R)=8.7). These results indicate the significance of steric factors in melanocortin receptor selectivity and suggest that introduction of bulky residues in the direct proximity to the melanocortin pharmacophore is an effective approach to design of novel hMC3R and hMC5R selective ligands.

Melanocortin hormones and neurotransmitters regulate a vast array of physiologic processes by interacting with five G-protein-coupled melanocortin receptor types. In the present study, we have systematically studied the regulation of individual human melanocortin receptor wild subtypes using a synthetic rhodamine-labeled human melanotropin agonist and antagonist, arrestins fused to green fluorescent protein in conjunction with two-photon fluorescence laser scanning microscopy and confocal microscopy. Stimulation of the melanocortin receptors by its cognate agonist triggered rapid arrestin recruitment and receptor internalization for all four human melanocortin receptors examined. Antagonists-bound melanocortin receptors, on the other hand, did not recruit beta-arrestins, and remained in the cell membrane even after long-term (30 min) treatment. Agonist-mediated internalization of all melanocortin receptor subtypes was sensitive to inhibitors of clathrin-dependent endocytosis, but not to caveolae inhibitors. In summary, agonist-mediated internalization of all subtypes of melanocortin receptors are dependent upon beta-arrestin-mediated clathrin-coated pits, whereas, beta-arrestin-2 conjugated green fluorescence protein (beta-arrestin-2-GFP) recruitment is not dependent on protein kinase A activation. Real time two-photon fluorescence laser scanning microscopy is a most powerful tool to study the dynamic processes in living cells and tissues, without inflicting significant and often lethal damage to the specimen.