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

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-MSH_5-10-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-MSH_{4- 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.