Identification of novel antimelanogenic hexapeptides via positional scanning of a synthetic peptide combinatorial library (2016)

Abnormal accumulation or distribution of melanin causes melasma, freckles and senile lentigines, and controlling unwanted melanin accumulation has received considerable interest in the field of cosmetics.1, 2 Peptides are potentially useful as active ingredients for cosmetics because of their bioactivity relevant to skin care and biosafety.3, 4 A method called “positional scanning synthetic peptide combinatorial library” is useful for the screening of various bioactive peptides.5, 6 The aim of this study was to identify antimelanogenic peptides by “positional scanning of a synthetic peptide combinatorial library.” The synthetic hexapeptide combinatorial library consisted of six positional sublibraries, namely OXXXXX, XOXXXX, XXOXXX, XXXOXX, XXXXOX, and XXXXXO, where O positions are any of the 19 amino acids excluding cysteine, and the X positions are equimolar mixtures of the 19 amino acids. The activities of different mixtures of peptides against melanin synthesis were examined in murine melanoma B16-F10 cells stimulated by α-melanocyte-stimulating hormone (α-MSH). The activity differences between the 19 peptide mixtures in a single positional sublibrary were used to predict the ideal amino acid for each position in the active hexapeptide. Preliminary positional scanning (Fig. S1) predicted the sequences of active hexapeptides as (I/F)-(N/S)-H-(H/N)-L-G-NH2. In the next step, we examined the activities of individual peptides (Fig. S2) and identified hexapeptide B6 as the most active. Hexapeptide B6 did not affect the viability of B16-F10 cells and human epidermal melanocytes (HEM) at concentrations up to 300 μmol/L (Fig. S3). The binding of α-MSH to melanocortin 1 receptors activates cyclic AMP-dependent signalling pathways that trigger the expression of melanogenic enzymes such as tyrosinase (TYR), TYR-related protein 1 (TYRP1), and dopachrome tautomerase (DCT).7-9 As shown in Figure 1, 30-300 μmol/L hexapeptide B6 attenuated the increases in melanin levels and TYR activities in B16-F10 cells stimulated with α-MSH. Western blot analysis showed that hexapeptide B6 decreased the levels of TYR, TYRP1, and DCT. Hexapeptide B6 also attenuated the increases in melanin level and TYR activity of cells stimulated with forskolin, an adenylate cyclase activator (Fig. S4). In HEM, hexapeptide B6 attenuated the increases in melanin level, TYR activity, and TYR expression in response to L-tyrosine (Fig. S5) as effectively as arbutin, a well-known melanogenesis inhibitor. In addition, hexapeptide B6 attenuated the increases in the melanin level and TYR activity of HEM stimulated with α-MSH (Fig. S6). In this study, novel antimelanogenic hexapeptides including hexapeptide B6 (FSHHLG-NH2) were identified through “positional scanning of a synthetic peptide combinatorial library.” Hexapeptide B6 attenuated melanin synthesis in B16-F10 cells and HEM stimulated with α-MSH, forskolin, or L-tyrosine. This study was supported by the INNOPOLIS Foundation (Daegu) funded by the Ministry of Science, ICT & Future Planning, Republic of Korea (No 2015-01-7005), and by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2014R1A1A2053768). JKS and SWL performed research and analysed data. JC and YMK contributed essential reagents and tools. JKS and YCB designed the research study and wrote the manuscript. The authors have no conflict of interest to declare. Data S1 Materials and methods Figure S1 Positional scanning of synthetic peptide combinatorial library for cellular melanin synthesis Figure S2 Effects of numerous hexapeptides on cell viability and cellular melanin synthesis Figure S3 Effects of hexapeptide B6 on cell viability Figure S4 Effects of hexapeptide B6 on basal and forskolin-induced melanogenesis in B6-F10 cells Figure S5 Effects of hexapeptide B6 on the melanin contents, cellular tyrosinase (TYR) activity, and TYR level in human epidermal melanocytes (HEM) stimulated with L-tyrosine Figure S6 Effects of hexapeptide B6 on basal and α-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis in human epidermal melanocytes (HEM) Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.