α-Melanotropin: The Minimal Active Sequence in the Frog Skin Bioassay

Victor J. Hruby, Brian C. Wilkes, Mac E. Hadley, Fahad Al-obeidi, Tomi K. Sawyer, Douglas J. Staples, Ann E. Devaux, Orin Dym, Ana Maria De L Castrucci, Mary F. Hintz, John P. Riehm, K. Ranga Rao

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Abstract

The minimal sequence required for biological activity of a-MSH (a-melanotropin, a-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-a-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 10–4M. We were unable to demonstrate biological activity for the tetrapeptide, Ac-Phe-Arg-Trp-Gly-NH2(Ac-a-MSH7_10-NH2), and for several carboxy terminal analogues including Ac-Lys-Pro-Val-NH2(Ac-α-MSH11_13-NH2). We prepared a series of fragment analogues of a-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-α-MSH6_9-NH2could be enhanced about a factor of 16 by the addition of glycine to the C-terminus, yielding Ac-α-MSH6_10-NH2(Ac-His-Phe-Arg-Trp-Gly-NH2). Addition of glutamic acid to the N-terminus provided the peptide, Ac-a-MSH5_10-NH2, which was only slightly more potent than Ac-a-MSH6_10-NH2, indicating that position 5 contributes little to the biological potency of a-MSH in this assay. Addition of methionine to the N-terminus of Ac-α-MSH5-10-NH2resulted in the heptapeptide, Ac-α-MSH4_10-NH2, which was only about 4-fold more potent than Ac-α-MSH5_10NH2. Addition of lysine and proline to the C-terminal of the Ac-α-MSH4_10-NH2sequence yielded the peptide, Ac-a-MSH4_12-NH2with a 360-fold increase in potency relative to Ac-a-MSH4_10-NH2. This peptide was only about 6-fold less potent than a-MSH. A series of Nle-4-substituted analogues also were prepared. Ac-[Nle4]-α-MSH4- 10-NH2was about 4 times more potent than Ac-a-MSH4_10-NH2. Ac-[Nle4]-α-MSH4_11-NH2also was about 4 times more potent than Ac-a-MSH4_10-NH2, demonstrating that lysine-11 contributes somewhat to the biological activity of a-MSH on the frog skin melanocyte receptor. However, addition of proline-12 to this fragment, yielding Ac-[Nle4]-α-MSH4_l2-NH2, resulted in about a 90-fold increase in relative potency of the melanotropin. Addition of the final C-terminal amino acid, valine-13, provided the decapeptide, Ac-[Nle4]-a-MSH4_13-NH2, which showed only a small further increase in potency. This analogue was, however, only about 2-3-fold less active than a-MSH. Addition of the N-terminal tripeptide Ac-Ser-Tyr-Ser to yield the tridecapeptide [Nle4]-α-MSH resulted in an analogue that was 3 times more potent than a-MSH. The importance of the amino acids in the primary structure of a-MSH in contributing to the biological activity of a-MSH in the frog skin bioassay can be summarized as follows: (1) the central tetrapeptide sequence, Ac-His-Phe-Arg-Trp-NH2, represents the minimum chain length for observable biological activity; (2) the active sequence of a-MSH is contiguous in that no two structurally noncontiguous fragment sequences were found to have biological activity; (3) Met-4, Gly-10, and Pro-12 are important potentiating amino acids and contribute significantly to the biopotency of a-MSH; and (4) Ser-1 and -3, Tyr-2, Glu-5, Lys-11, and Val-13 apparently contribute only minimally to the biological potency of a-MSH at the frog melanocyte skin receptor.

Original languageEnglish (US)
Pages (from-to)2126-2130
Number of pages5
JournalJournal of Medicinal Chemistry
Volume30
Issue number11
DOIs
StatePublished - Nov 1 1987

ASJC Scopus subject areas

  • Molecular Medicine
  • Drug Discovery

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    Hruby, V. J., Wilkes, B. C., Hadley, M. E., Al-obeidi, F., Sawyer, T. K., Staples, D. J., Devaux, A. E., Dym, O., De L Castrucci, A. M., Hintz, M. F., Riehm, J. P., & Rao, K. R. (1987). α-Melanotropin: The Minimal Active Sequence in the Frog Skin Bioassay. Journal of Medicinal Chemistry, 30(11), 2126-2130. https://doi.org/10.1021/jm00394a033