4-Norleucine, 7-D-phenylalanine-alpha-melanocyte-stimulating hormone: a highly potent alpha-melanotropin with ultralong biological activity.

Abstract

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...

Interactions of Human Melanocortin 4 Receptor with Nonpeptide and Peptide Agonists,

“…The changes in IC 50 and EC 50 values for NDP-MSH were also parallel in the majority of tested mutants, with the notable exception of H264A and D126A mutations, which led to significant reduction of NDP-MSH binding affinity such that no specific binding was detected by the filtration method.…”

“…The relative contributions of the replaced side chains in the ligand-receptor interactions are reflected in the degree of change in the binding affinity in the mutants. Our data indicate that the most important interaction likely involve charged residues (Glu 100 , Asp 122 , Asp 126 , and His 264 ), residues from TM6 (Phe 261 Leu 265 ,) and from TM7 (Leu 288 ), as their mutations decrease IC 50 and EC 50 of both agonists more than 10 fold. These residues are relatively conserved for MCRs 1, 3, 4, and 5 and therefore are not likely to be involved in the specific interactions with MC4R that provide >100 fold selectivity of these agonists over MC1R, MC3R, and MC5R (18,19).…”

“…Alanine-scanning mutagenesis was done in thirteen positions in the area of the putative ligand binding pocket (16,33,37,38,46), whose mutation has been shown to affect binding of linear peptide agonists to MC4R and MC1R (37,38,46). The mutated residues include Trp 258 , which is highly conserved in rhodopsin-like GPCRs and is presumed to be involved in activation mechanism (31) 50 values are similar for each of the three agonists at the wild type receptor. All thirteen mutations alter potencies but do not significantly affect the efficacies of the agonists.…”

A chelating derivative of α-melanocyte stimulating hormone as a potential imaging agent for malignant melanoma

“…The relatively high levels of bisMSH-DTPA-Il'In injected, however, would have ensured that the concentration in the blood remained significantly higher than that of naturally circulating MSH for several hours. The maximum levels of bisMSH-DTPA in the blood following injection would have been about 0.8p1gml-, and, assuming that bisMSH-DTPA was broken down at approximately the same rate as the native hormone, the time taken to reach a concentration equivalent to the naturally circulating hormone (30pgml-'; Eberle, 1988) would be between 7 h and 8 h. The use of analogues of MSH with greater resistance to proteolysis (Sawyer et al, 1980;Castrucci et al, 1984) may enable lower doses to be used and higher tumour/tissue ratios to be obtained.…”

Third Transmembrane Domain of the Adrenocorticotropic Receptor Is Critical for Ligand Selectivity and Potency

“…We then performed a site-directed mutagenesis study to determine whether these amino acid residues are crucial for ligand binding affinity and potency. Three MC2R mutations were made (D104N, I105V, and L109V), and effect of We also examined the effects of ligands ACTH(1-39), ACTH , and ACTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), as well as ␣-MSH, at chimeric MC4R/TM3 MC2R (Fig. 7).…”