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Skin color is determined by the amount of melanin. Tyrosinase is a key enzyme for melanin synthesis (melanogenesis). Its transcription is started by MITF and E-box binding. MITF (Microphthalmia-associated transcription factor) is the basic helix-loop-helix-zip transcription factor that regulates gene expression by E-box binding. To screen depigmenting agents by a HTS (high-throughput screening) system, an EMSA (Electrophoretic Mobility Shift Assay) was performed. Based on the computer-simulated structure, 10 compounds isolated from natural plants and microbial sources were selected and were investigated as MITF-DNA binding inhibitors. First, we performed a cell test to check if the compounds have depigmenting activity. From the results of the melanin assay and MTT assay, 3 compounds exhibited depigmenting activity in a dose dependent manner without toxicity. Among these 3 compounds, we choose compound #7 for further experiments. To check its inhibitory activity against MITF-DNA binding, an EMSA (Electrophoretic Mobility Shift Assay) was performed. As a result, intensities of MITF-DNA binding bands were dose-dependently reduced by compound #7. In addition, to investigate the MITF and tyrosinase protein level, western blot was performed by using B16F10 melanoma cell extract. As a result, tyrosinase protein was down-regulated by compound #7 in a dose dependent manner but the MITF protein not was reduced. Thus, compound #7 which
was isolated from a natural plant can be used as an inhibitor of MITF-DNA binding for depigmenting agents.