Skin pigmentation is a complex process including melanogenesis within melanocytes and melanin transfer to the keratinocytes. system. We identified candidate pigmentation inhibitors from 4 0 screened compounds including zoxazolamine 3 and alpha-mangostin which were also shown to modulate expression of MITF and several key pigmentation factors and are worthy of further evaluation for potential translation to clinical use. co-culture systems were used to validate melanin transfer and to observe the effects on the pigmentation (26-29). Moreover tyrosinase TRP-1 and TRP-2 which are key eumelanogenesis enzymes are induced under co-culture conditions containing keratinocytes and Afatinib dimaleate melanocytes (30). For the mechanism of retinoic Afatinib dimaleate Afatinib dimaleate acid’s inhibition of melanogenesis cellular retinoic acid binding protein-1 of melanocytes is thought to be influenced by keratinocytes (31). As one of the regulatory factors of pigmentation activation or inhibition Afatinib dimaleate of protease-activated receptor 2 in keratinocytes also has been suggested to modulate a key keratinocyte-melanocyte interaction required for pigment transfer (32). Keratinocytes participate in additional crosstalk with melanocytes (33-35). UV induced DNA damage in keratinocytes results in p53-mediated upregulation of Melanocyte Stimulating Hormone which stimulates melanocytic cyclic AMP and pigment synthesis (8). A number of hit-compounds exhibited little or no effect on expression of melanogenesis related genes suggesting that their primary targets might be post-transcriptional suppression of pigmentation machinery rather than affecting expression of genes involved in melanogenesis. We used immortalized murine cell lines for the co-culture instead of human primary keratinocytes and melanocytes due to greater ease and reproducibility of in vitro culturing as compared to donor variability NS1 of human primary cultures (33) (although human primary melanocytes were utilized for followup validation studies). Importantly the reported assay integrated measurement of cell viability and excluded from further analysis compounds whose toxicity reduced cellular adherence to the tissue culture well. These efforts were further expanded by the measurement of toxicities in primary human melanocytes and keratinocytes at higher doses for top hits from the primary assay. In previously published studies using a co-culture assay melanin content was measured by NaOH lysis and absorbance (11) or spectrophotometric analysis (27 28 These methods may be less suitable for high-throughput scale-up due to the fact that smaller well sizes will reflect lower amounts of melanin which may fall below detection sensitivity. We Afatinib dimaleate therefore utilized this assay for reconfirmation of our primary hits. In this assay we Afatinib dimaleate observed a higher sensitivity of the co-culture assay. Among the hits from the screen reported here Resveratrol and Miconazole have been previously demonstrated to inhibit pigmentation and tyrosinase activity in B16 cells (14 36 The identification of previously known agents as pigmentation inhibitors provided validation of the current screening approach. Alpha-mangostin is reported to have a role in inhibition of lipoprotein oxidation (37) and methoxycathechol inhibits carcinogenesis in the rat (38) however their roles in suppression of pigmentation have not been previously elucidated. Carapin was isolated from the Brazilian Hard-Nut tree and has been used in Brazilian traditional medicine for anti-inflammatory anti-allergic purposes although no evidence regarding regulation of pigmentation was found in the literature (39). Of course it is always important to consider the possibility of unpredicted effects such as the described role of forskolin in producing pigment-independent epidermal thickening (40). Taken together we executed a high-throughput high-content image-based screen with 4 0 individual compounds and found several previously described agents (carnosic acidity resveratrol and miconazole) in addition to book modulators of melanin deposition. The clinical relevance and utility of the agents is going to be investigated additional. Supplementary Materials Supp FigS1Body S1. (a) Dose-dependent inhibition of melanin creation by top strikes. Ten compounds displaying.