ABSTRACTa-Melanocyte-stimulating hormone (a-MSH) reversibly darkens frog skins by stimulating melanosome movement (dispersion) within melanophores. Heat-alkali treatment of a-MSH results in prolonged biological activity of the hormone. Quantitative gas chromatographic analysis of the hydrolyzed heat-alkali-treated peptide revealed partial racemization particularly at the 4 (methionine) and 7(phenylalanine) a-Melanotropin (a-MSH, a-melanocyte-stimulating hormone) is a tridecapeptide (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-ArgTrp-Gly-Lys-Pro-Val-NH2) that is synthesized and secreted by the pars intermedia of the vertebrate pituitary (1). The amino acid residues that are important in the expression of melanotropic activity have been elucidated through systematic structure-function investigations of a-MSH and a-MSH fragments on amphibian melanophores (2, 3) and, to a lesser extent, on mammalian melanoma cells (4-6). Very little information is available, however, regarding the stereochemical and conformational correlates of biological activity in either of these two biological systems.Earlier reports have shown that heat-alkali treatment of crude or purified preparations of naturally occurring a-MSH produces a partially racemized product with altered activity on amphibian melanophores both in vivo and in vitro. Such changes in biological effects have been discussed in terms of "potentiation," "prolongation," and "retardation" (7-12). Although the precise biochemical mechanism by which these unusual biological properties were produced is unknown, it appeared possible that synthetic stereostructural tailoring of a-MSH might produce an analogue that would also possess these properties. Utilizing a high-resolution gas chromatographic method to localize and quantitate specific sites of racemization within the primary sequences of peptides, we obtained additional evidence which suggested that stereochemical substitution at position 7 (replacement of L-phenylalanine by D-phenylalanine) of a-MSH or [Nle4]-a-MSH would provide an analogue with the desired biological properties. Previous investigations have shown that [Nle4]-a-MSH is more potent than a-MSH on both amphibian melanophores (2, 6) and on stimulating melanoma adenylate cyclase (6, 13), and it is also resistant to inactivation by chloramine-T (14, 15), an oxidant used in peptide iodination. Because heat-alkali treatment of this analogue also resulted in "potentiation," "prolongation," and "retardation," it was clear that alteration of the methionine residue was not a requirement for the expression of these properties. Thus, it was decided to retain the benefits of the norleucine substitution in position 4 in the synthesis of the "definitive" peptide.We report here the synthesis of [Nle4, D-Phe7]-a-MSH and present data demonstrating its unique biological properties. These include prolonged biological activity, enhanced potency relative to a-MSH in a number of biological systems, and resistance to degradation by serum enzymes. The biological properties of this analogue provide...

The cloning of the melanocyte-stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) receptors (MC1-R and MC2-R, respectively) recently has led to the identification of three additional melanocortin receptors, MC3-R, MC4-R, and MC5-R. The MC2 receptor primarily recognizes only ACTH peptides, but the other four receptors all recognize alpha-melanocyte-stimulating hormone (alpha-MSH) and potent alpha-MSH agonists such as [Nle4,D-Phe7]alpha-MSH-NH2 and Ac-Nle4-c[Asp5,D-Phe7,Lys10]alpha-MSH-(4-10)-NH2 as well as ACTH. The absence of any known physiological role for these new receptors, expressed both in the brain (MC3-R and MC4-R) and throughout a number of peripheral tissues (MC5-R), has necessitated as search for potent and receptor selective agonists and antagonists. We report here that analogues of the superpotent cyclic agonist analogue Ac-Nle4-c[Asp5,D-Phe7, Lys10]alpha-MSH-(4-10)-NH2, in which a bulky aromatic amino acid is substituted in the 7-position, can produce potent and selective antagonists for melanocortin receptors. Thus, the D-p-iodophenylalanine7-containing analogue Ac-Nle4-c[Asp5,D-Phe(pI)7,Lys10]alpha-MSH-(4-10)-NH2 is a potent antagonist (pA2 = 10.3) in the classical frog skin (Rana pipiens) assay (MC1-R), as is the D-2'-naphthylalanine7 (D-Nal(2)7)-containing analogue Ac-Nle4-c[Asp5,D-Nal(2)7,Lys10]alpha-MSH-(4-10)-NH2 (pA2 > 10.3). Interestingly, the D-p-chloro- and D-p-fluorophenylalanine7-containing analogues lacked antagonist activities at all melanotropin receptors, and both exhibited full agonist potency in the frog skin assay. The activity of these analogues also was examined at four mammalian melanocortin receptors. Interestingly, Ac-Nle4-c[Asp5,(D-Nal(2)7,Lys10] alpha-MSH-(4-10)-NH2 was found to be a potent antagonist of the MC4-R (pA2 = 9.3) with minimal agonist activity, a less potent antagonist of the MC3-R (pA2 = 8.3) with minimal agonist activity, and a full agonist of the MC1 and MC5 receptors. Surprisingly, Nle4-c[Asp5,D-Phe(pI)7,Lys10]alpha-MSH was found to be a potent agonist at the cloned human MC1-R (EC50 = 0.055 nM) and mouse MC1-R (EC50 = 0.19 nM) but had potent antagonist activities at the human MC4-R (pA2 = 9.7) and human MC3-R (pA2 = 8.3) with significant partial agonist activities (EC50 = 0.57 and 0.68 nM, respectively) as well. Thus, highly potent and receptor selective antagonist analogues can arise from substitution of the D-Phe7 residue with a bulky aromatic amino acid. These analogues can be used to help determine the functional roles of these receptors.

Utilizing results from previous structure-activity relationships and theoretical studies of alpha-melanotropin (alpha-MSH, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2) and its related superpotent analogues, Ac-[Nle4,D-Phe7]-alpha-MSH and Ac-[Cys4,Cys10]-alpha-MSH, we have designed a new class of alpha-MSH4-13 and alpha-MSH4-10 cyclic lactam fragment analogues of alpha-melanotropin. The cyclic peptides have the following general structures: Ac-[Nle4,Xxx5,D-Phe7,Yyy10,Gly11]-alpha-MSH4-13- NH2 and Ac-[Nle4,Xxx5,D-Phe7,Yyy10]-alpha-MSH4-10-NH2, where Xxx = Glu or Asp and Yyy = Lys, Orn, Dab, or Dpr. Formation of the lactam bridge between the side-chain groups Xxx and Yyy was performed either in solution or on a solid-phase support. Seven cyclic peptides were prepared and bioassayed for their melanotropic potency by using standard frog (Rana pipiens) and lizard (Anolis carolinensis) skin bioassays. Relative to alpha-MSH (relative potency = 1), the potencies of the cyclic peptides in the lizard skin bioassay were as follows: alpha-MSH (1); Ac-[Nle4,Glu5,D-Phe7,Lys10,Gly11]-alpha-MSH4-13- NH2 (6); Ac-[Nle4,Asp5,D-Phe7,Lys10,Gly11]-alpha-MSH4-13- NH2 (100); Ac-[Nle4,Glu5,D-Phe7,Lys10]-alpha-MSH4-10-NH2 (9); Ac-[Nle4,Asp5,D-Phe7,Lys10]-alpha-MSH4-10-NH2 (90); Ac-[Nle4,Asp5,D-Phe7,Orn10]-alpha-MSH4-10-NH2 (20); Ac-[Nle4,Asp5,D-Phe7,Dab10]-alpha-MSH4-10-NH2 (5); Ac-[Nle4,Asp5,D-Phe7,Dpr10]-alpha-MSH4-10-NH2 (5). Similar results were obtained in the frog skin bioassay, but the analogues were much less potent. Cyclic melanotropins with 23-membered rings exhibited 100-fold higher melanotropic potency than alpha-MSH with selectivity for the lizard melanocyte receptors over the frog melanocyte receptors. Increasing or decreasing the ring size of these cyclic melanotropins from 23 diminishes the biological potency of the resulting cyclic peptide. The 23- and 24-membered ring analogues showed prolonged (residual) biological activities in both biological assays, but the smaller ring systems (20, 21, 22) did not. These results provide new insights into the structural and conformational requirements of alpha-MSH and its analogues at two different types of pigment cell (melanocyte) receptors.

Rapid color changes of amphibians are mediated by three types of dermal chromatophores, xanthophores, iridophores, and melanophores, which comprise a morphologically and physiologically distinct structure, the dermal chromatophore unit. Xanthophores, the outermost element, are located immediately below the basal lamella. Iridophores, containing light-reflecting organelles, are found just beneath the xanthophores. Under each iridophore is found a melanophore from which processes extend upward around the iridophore. Finger-like structures project from these processes and occupy fixed spaces between the xanthophores and iridophores. When a frog darkens, melanosomes move upward from the body of the melanophore to fill the fingers which then obscure the overlying iridophore. Rapid blanching is accomplished by the evacuation of melanosomes from these fingers. Pale coloration ranging from tan to green is provided by the overlying xanthophores and iridophores. Details of chromatophore structure are presented, and the nature of the intimate contact between the chromatophore types is discussed.

The minimal sequence required for biological activity of alpha-MSH (alpha-melanotropin, alpha-melanocyte stimulating hormone) was determined in the frog (Rana pipiens) skin bioassay. The sequence required to elicit measurable biological activity was the central tetrapeptide sequence, Ac-His-Phe-Arg-Trp-NH2 (Ac-alpha-MSH6-9-NH2), which was about 6 orders of magnitude less potent than the native tridecapeptide. Smaller fragments of this sequence (Ac-His-Phe-NH2, Ac-Phe-Arg-NH2, Ac-His-Phe-Arg-NH2) were devoid of melanotropic activity at concentrations as high as 10(-4) M. We were unable to demonstrate biological activity for the tetrapeptide, Ac-Phe-Arg-Trp-Gly-NH2 (Ac-alpha-MSH7-10-NH2), and for several carboxy terminal analogues including Ac-Lys-Pro-Val-NH2 (Ac-alpha-MSH11-13-NH2). We prepared a series of fragment analogues of alpha-MSH in an attempt to determine the contribution of each individual amino acid to the biological activity of the native hormone. The minimal potency of Ac-alpha-MSH6-9-NH2 could be enhanced about a factor of 16 by the addition of glycine to the C-terminus, yielding Ac-alpha-MSH6-10-NH2 (Ac-His-Phe-Arg-Trp-Gly-NH2). Addition of glutamic acid to the N-terminus provided the peptide, Ac-alpha-MSH5-10-NH2, which was only slightly more potent than Ac-alpha-MSH6-10-NH2, indicating that position 5 contributes little to the biological potency of alpha-MSH in this assay. Addition of methionine to the N-terminus of Ac-alpha-MSH5-10-NH2 resulted in the heptapeptide, Ac-alpha-MSH4-10-NH2, which was only about 4-fold more potent than Ac-alpha-MSH5-10-NH2. Addition of lysine and proline to the C-terminal of the Ac-alpha-MSH4-10-NH2 sequence yielded the peptide, Ac-alpha-MSH4-12-NH2 with a 360-fold increase in potency relative to Ac-alpha-MSH4-10-NH2. This peptide was only about 6-fold less potent than alpha-MSH. A series of Nle-4-substituted analogues also were prepared. Ac-[Nle4]-alpha-MSH4-10-NH2 was about 4 times more potent than Ac-alpha-MSH4-10-NH2. Ac-[Nle4]-alpha-MSH4-11-NH2 also was about 4 times more potent than Ac-alpha-MSH4-10-NH2, demonstrating that lysine-11 contributes somewhat to the biological activity of alpha-MSH on the frog skin melanocyte receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

A new highly potent, receptor-selective and prolonged-acting cyclic lactam analogue of -melanotropin (a-MSH) has been designed and synthesized. Molecular dynamics simulations and molecular mechanics calculations were used in conjunction with results from previous conformational structure-biological activity studies to design a new class of linear and cyclic -melanotropin ( -MSH) analogues. Examination of these properties of -MSH and [Nle4,D-Phe7] -MSH led to the design of the potent linear fragment analogue Ac-[Nle4,Asp5,D-Phe7,Lys10] a-MSH4_10-NH2, in which the Gly10 residue of a-MSH4-10 was replaced by Lys10 as the major novel change from previous investigations. This in turn led to the synthesis of the cyclic lactam analogue Ac-[Nle4,Asp5,D-Phe7,Lys10]a-MSH4_10-NH2, which was exceptionally potent in the lizard skin (90 times that of -MSH) and mammalian melanoma tyrosinase (100 times that of -MSH) assays and in addition exhibited prolonged

Melanotan-II is a potent initiator of erections in men with psychogenic erectile dysfunction and has manageable side effects at a dose of 0.025 mg./kg.

a-Melanocyte-stimulating hormone (ai-melanotropin; a-MSH) is a linear tridecapeptide (Ac-Ser-Tyr-Ser-MetGlu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2) that reversibly darkens amphibian skins by stimulating melanosome (pigment granule) dispersion within melanophores. By using a number of in vitro melanocyte assays, we have examined the conformational requirements for a-MSH activity. Synthesis of [half-Cys4,half-Cys 0.l-a MSH, a cyclic, conformationally restricted, "isosteric" analogue of a-MSH, provided a melanotropin with a potency > 10,000 times that of the native hormone in stimulating frog (Rana pipen) skin darkening. The cyclic analogue also showed substantially prolonged activity relative to the native hormone. Cys'l0-a-MSH was =30 times more potent than a-MSH in stimulating lizard (Anolis carolinensis) skin melanophores in vitro. By using a cell-free Cloudman S-91 mouse melanoma plasma membrane preparation, we found the cyclic analogue to be "'3 times as potent as the native hormone in stimulating adenylate cyclase activity. These results provide insight into the conformational requirements for biological activity of a-MSH, and the comparative conformational requirements of a-MSH at a number of pigment cell receptors.Peptide hormones and their analogues provide useful molecular probes for investigating the chemical-physical basis ofbiological information transfer at specific target tissues. a-Melanocytestimulating hormone (a-MSH; a-melanotropin) is a linear tridecapeptide, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-GlyLys-Pro-Val-NH2, that is synthesized and secreted by the pars intermedia of the vertebrate pituitary (1). The hormone may have important physiological roles in the control of vertebrate pigment cell melanogenesis (2), neural functioning related to learning and behavior (3, 4), and fetal development (4).a-Melanotropin structure-function relationships have been studied by analysis of the biological activities of a number of structurally or stereochemically modified a-MSH analogues and fragments in amphibian melanophores (5-8) and, more recently, on mammalian melanoma cells (9-11). The amino acid residues believed to be important in the expression of melanotropic activity of a-MSH have been systematically examined and, based on these studies, a-MSH apparently contains two message sequences, (Glu)-His-Phe-Arg-Trp and Gly-Lys-ProVal-NH2. Each ofthese sequences can independently stimulate melanosome dispersion in amphibian melanophores in vitro (7). It has been reported (12) that a-MSH, like other so-called sychnologically organized hormones [e.g., corticotropin (ACTH), f3-lipotropin, cholecystokinin] is a conformationally flexible molecule in aqueous solution. Perhaps because of the postulated inherent conformational flexibility of this peptide, the threedimensional topochemical requirements of a-MSH, which may be important for its biologically active conformation § at the melanotropin receptor, have not been experimentally examined.The various conformational requirements in a peptide hormone that ...