Homology modelling and virtual screening of P-protein in a quest for novel antimelanogenic agent and In vitro assessments Vivek K. Morya, Nguyen H. Dung, Birendra K. Singh, Hyang-Bok Lee and Eun-ki Kim Department of Biological Engineering, Inha University, Incheon, Korea Correspondence: Eun-Ki Kim, Department of Biological Engineering, Inha University, Incheon 402-751, Korea, Tel.: +82-32-860-7514(O)/ 32-872- 2978(L), Fax: +82-872-4046, e-mails: moryavivek@gmail.com and ekkim@inha.ac.kr Abstract: An adequate knowledge on molecular mechanism of melanogenesis provides an opportunity to find the novel molecular targets for the discovery and development of new cosmetics. Among various genes, the OCA2 is being essential for proper melanin synthesis, and mutation or deletion of this gene leads to oculocutaneous albinism type 2. Thus, for this study, the product of this gene, that is P-protein, was targeted in quest for novel inhibitors as antimelanogenic agents. Based on pattern search of amino acid sequence and homology analysis, the protein structure was modelled. The role of this protein has been predicted as a tyrosine transporter of melanosomes. Thus, the molecular library was generated on the basis of tyrosine transporter inhibitor. Based on the dock score, 20 molecules have been considered as putative inhibitors for P-protein. Among these compounds, five molecules (compound #1, #4, #8, #13 and #17) were found to be quite effective as antimelanogenic without having any toxicity. Further investigations to establish the mechanism of action, the indirect methods such as tyrosinase assay, analysis for eumelanin and pheomelanins and investigation of mRNA levels were being carried out. The results from the studies offered a new lead in antimelanogenic therapy and may be very useful for further optimization work in developing them as novel depigmenting agents. Key words: antimelanogenic – melan-a melanocytes – OCA-2 – P-protein – virtual screening Accepted for publication 12 September 2014 Introduction The eternity and beauty are the two unfilled desires since the exis- tence of human civilization. In the cosmetics market, many func- tional products such as those developed for skin moisturizing, skin whitening, wrinkle care, anti-ageing, pimple care and improv- ing atopic skin have been launched. Specifically, the category of whitening products makes up 22.4% of the local functional cos- metics market (1). To reduce the melanin content, many approaches were attempted for example, by regulation of the tran- scription and activity of tyrosinase, tyrosinase-related protein-1 (TRP-1), tyrosinase-related protein-2 (TRP-2) and peroxidase; the uptake and distribution of melanosomes in recipient keratinocytes; melanin and melanosome degradation and turnover of pigmented keratinocytes (2). However, all these approaches come with signifi- cant side effects including oedema, carcinogenesis and allergy. About 200 alleles spread over 90 loci are known to regulate cutaneous pigmentation, and the products of these loci which are acting as structural proteins, enzymes, transporters, transcriptional regulators, receptors and growth factors have a wide array of func- tions and cellular targets (3). Among these proteins, total of 68 proteins have been identified as melanosomal proteins (4). Only nine proteins were identified as melanosome-specific proteins. P-protein, the product of OCA2 gene is one of them. The P-protein is consisting of 833 amino acids arranged in 12 membrane spanning domains (5). From the predicted protein structure and the phenotype of melanocytes with OCA2 gene mutations, it has been hypothesized that the OCA2 gene encodes a transport or pore protein vital for melanosome function (5). It has been reported that the OCA2 gene transcript is expressed in the black dorsal skin, but not the yellow ventral skin in mice (6), corroborating earlier notions that the OCA2 gene mutations affect eumelanins, but not pheomelanin biosynthesis. Thus, the P-pro- tein may play a role in eumelanin synthesis. Recessive mutations in the gene cause oculocutaneous albinism type 2, the most com- mon form of albinism among people of African heritage (7). OCA2 is often referred to as tyrosinase positive albinism, as hypo- pigmented hair bulbs plucked from these patients will become pigmented when incubated with the tyrosinase substrates L-tyro- sine or L-DOPA (8). The non-essential aromatic amino acid L-tyrosine serves as precursor to melanin biosynthesis (9). L-tyro- sine and L-DOPA (or phosphorylated isomers of L-DOPA) are key molecules in regulation of the melanin synthesis (10–15). The availability of the L-tyrosine for melanogenesis depends on either In vivo synthesis of L-tyrosine from phenylalanine or import of extracellular L-tyrosine to melanosomes (3,9). It was reported that L-DOPA can stimulate melanoma cell proliferation (16), and L-tyrosine can stimulate the differentiation as well as melanogene- sis in human melanoma cells (17). It was reported that L-tyrosine supplementation into hamster melanoma lines stimulates melanin synthesis as well as tyrosinase activity in a dose- and time-depen- dent manner; however, the expression of mRNA remains unaf- fected (9,13,18). Similar observations were also reported in B16 melanoma cells (19). Indeed, OCA2 patients can exhibit light to moderate pigmentation. Like all other OCA subtypes, OCA2 patients present phenotypes that are restricted to tissues involving dysfunction due to loss of pigment. Study from immortalized melan-p1 skin melanocytes derived from the p cp /p 25H mouse showed a very few mature, pigmented melanosomes, but instead 838 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 838–842 DOI: 10.1111/exd.12549 www.wileyonlinelibrary.com/journal/EXD Original Article