Structure-Activity Relationships of γ-MSH Analogues at the Human Melanocortin MC3, MC4, and MC5 Receptors. Discovery of Highly Selective hMC3R, hMC4R, and hMC5R Analogues Preeti Balse-Srinivasan, † Paolo Grieco, ‡ Minying Cai, Dev Trivedi, and Victor J. Hruby* Department of Chemistry, University of Arizona, Tucson, Arizona 85721 Received March 13, 2003 It has been shown by extensive studies that melanotropin bioactivities are critically dependent on the core or central tetrapeptide sequence, His-Phe-Arg-Trp, and in R-MSH it has been demonstrated further that a reverse-turn type conformation exists at this pharmacophore. To probe the receptor active conformation of the pharmacophore His-Phe-Arg-Trp in γ-MSH, two different series of γ-MSH analogues have been designed and synthesized and their biological activities determined at hMC3R, hMC4R, and hMC5R. The 1st series consists of a cyclic scan using different disulfides or lactam bridges. It was found that cyclization of the native γ-MSH around the highly conserved sequence can lead to shifts in affinity and selectivity for hMC4R instead of the hMC3R as seen in the native peptide. Furthermore, a 23-membered ring is desirable for potency (e.g., analogues 6 and 10) whereas a 26-membered ring (analogue 1, H-Tyr- Val-c[Cys-Gly-His-Phe-Arg-Trp-Cys]-Arg-Phe-Gly-NH 2 with Gly 4 ) is more important for selectiv- ity. The 2nd series is made of D-2-naphthylalanine (D-Nal(2′)) scan of the γ-MSH sequence at position 6 and 8 and the replacement of His 5 with Pro (analogue 13). Analogue 12, H-Tyr-Val- Nle-Gly-His-Phe-Arg-D-Nal(2′)-Asp-Arg-Phe-Gly-NH 2 , is a potent and selective antagonist at the hMC4R, and analogue 15, H-Tyr-Val-Nle-Gly-Aib-Phe-Arg-D-Nal(2′)-Asp-Arg-Phe-Gly-NH 2 , is a highly selective and potent agonist of the hMC5R. A most promising analogue is 13, H-Tyr- Val-Nle-Gly-Pro-D-Nal(2′)-Arg-Trp-Asp-Arg-Phe-Gly-NH 2 , which is a very potent agonist of the hMC4R, and this analogue can be further evaluated for feeding behavior and the regulation of fat stores. Introduction γ-Melanocyte-stimulating hormone (γ-melanotropin, γ-MSH, Table 1, Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp- Arg-Phe-Gly-OH) belongs to the family of melanotropin peptides, that also includes R-MSH, -MSH, and adreno- corticotropin (ACTH), and is derived by the posttrans- lational cleavage of the precursor pro-opiomelanocortin (POMC). Physiological effects of the melanotropins have been extensively studied for many years in vertebrates and mammals including humans. 1-3 These peptides have been implicated in numerous biological functions including regulation of skin pigmentation, regulation of steroid production, stimulation of nerve regeneration, modulation of immune responses, feeding behavior, erectile function, learning processes, and cell growth. 1,4,5 At present, five subtypes of human receptors that bind melanotropins have been characterized, hMC1R- hMC5R. 6-10 The receptors are G-protein-coupled recep- tors and for the most part their activation leads to an elevation in cAMP. Each of the receptors possesses unique affinities for the melanotropins. The MC1 recep- tor, binding mainly to R-MSH, is found in skin and melanoma cells and plays a role in skin pigmentation. The MC2 receptor, found in the adrenal cortex, binds only to ACTH and is involved in steroidogenesis. The MC3 and MC5 receptors are found both in the brain and periphery, but their functions are not very clear, though they appear to be involved in energy homeostasis and glandular secretions, respectively. 11 Both R- and γ-MSH show about equal affinity for the MC3R, while the MC5R binds mainly to R-MSH. The MC4 receptor, found mainly in the central nervous system, has a higher affinity for R-MSH and -MSH than for γ-MSH. While most of the research on the melanotropins has concentrated on R-MSH, the most potent of the three melanotropins, little is known of γ-MSH in terms of its structure-activity relationships (SAR) to the various melanocortin receptors. So our initial SAR study of γ-MSH involved an L-alanine scan 12 and a D-amino acid scan, 13 to understand the importance of each of the residues in its sequence and their topographical re- quirements for biological activity, as determined from activities at the cloned human MCRs. 12,13 The next step would be to further probe the conformation or topo- graphical distribution of the core pharmacophoric resi- dues in the sequence. As shown before, all the melano- tropins are characterized by the core or central tetra- peptide sequence, His-Phe-Arg-Trp, flanked by N- and C-terminal residues. This core sequence is the minimal fragment essential for biological activity 14,15 and is believed to form the pharmacophore. 14-17 Furthermore, the “bioactive conformation” of R-MSH was first shown to possibly exist as a reverse-turn-type conformation at this His-Phe-Arg-Trp sequence by the design of the * To whom correspondence should be addressed: Phone: (520) 621- 6332. Fax: (520) 621-8407. Email: hruby@u.arizona.edu. † Current address: Bexel Pharmaceuticals, Inc., 32990 Alvarado Niles Rd., Suite 910, Union City, CA 94587. ‡ Current address: Department of Pharmaceutical Chemistry and Toxicology, University of Napoli “Federico II”, Napoli 80131, Italy. 4965 J. Med. Chem. 2003, 46, 4965-4973 10.1021/jm030119t CCC: $25.00 © 2003 American Chemical Society Published on Web 10/04/2003