Identification of small peptides and glycinamide that inhibit melanin synthesis using a positional scanning synthetic peptide combinatorial library

Background: Antimelanogenic peptides are potentially useful to treat hyperpigmentation, but many peptides have limited application because of high cost and/or low activity. Objectives: To identify small and potent peptide inhibitors of cellular melanin synthesis that are useful for cosmetic and medical applications. Methods: A positional scanning synthetic tetrapeptide combinatorial library was used for screening of potentially active peptides. Antimelanogenic activities of the peptide pools and individual peptides were evaluated in B16-F10 melanoma cells and human epidermal melanocytes treated with alpha-melanocyte-stimulating hormone (α-MSH). Results: Predicted active tetrapeptide sequences were R-(F/L)-(C/W)-(G/R)-NH₂. Of the individual tetrapeptides tested, D3 (RFWG-NH₂) and D5 (RLWG-NH₂) exhibited high antimelanogenic activities. Tetrapeptide D9 (FRWG-NH₂) with a sequence identical to that of a portion of α-MSH also showed antimelanogenic activity. Of the tripeptides tested, E5 (FWG-NH₂), E6 (LWG-NH₂) and E7 (RWG-NH₂) were relatively more active. Dipeptide F1 (WG-NH₂) and monopeptide G1 (G-NH₂, glycinamide) retained activity, but G2 (Ac-G-NH₂) and G3 (glycine) did not. The antimelanogenic activities of peptides D3, E5, F1 and G1 were verified in α-MSH-stimulated human epidermal melanocytes. Commercially available G-NH₂·HCl suppressed the phosphorylation levels of cAMP-responsive element binding protein, protein levels of microphthalmia-associated transcription factor and tyrosinase, l-tyrosine hydroxylase activity of tyrosinase, and the melanin levels in stimulated cells. Conclusions: Small peptides, including glycinamide and tryptophanyl glycinamide, are potent antimelanogenic agents with potential value for the treatment of skin hyperpigmentation.