1 Thiopurine Drugs Repositioned as Tyrosinase 2 Inhibitors 3

In this study, we repositioned thiopurine drugs used for the treatment of acute 12 leukaemia as new tyrosinase inhibitors. Tyrosinase catalyses two distinct and successive 13 oxidations in melanin biosynthesis: the conversions of tyrosine to dihydroxyphenylalanine (DOPA) 14 and DOPA to dopaquinone. Continuous efforts are underway to discover small molecule 15 inhibitors of tyrosinase for therapeutic, cosmetic, and agricultural purposes. Structure-based 16 virtual screening has predicted inhibitor candidates for mushroom tyrosinase from drugs 17 approved by the US Food and Drug Administration (FDA). Enzyme assays have confirmed the 18 thiopurine leukaemia drug, thioguanine, as a tyrosinase inhibitor. Two other thiopurine drugs, 19 mercaptopurine and azathioprine, were also evaluated for their tyrosinase inhibitory activity; 20 mercaptopurine caused stronger inhibition than thioguanine did, whereas azathioprine was a 21 poor inhibitor. The inhibitory constants of thioguanine and mercaptopurine were calculated as 22 52 and 16 μM, respectively, and the value of mercaptopurine was comparable to that of the 23 well-known inhibitor kojic acid (13 μM). The cell lysate and melanin content assay in B16F10 24 melanoma cells confirmed that the compounds inhibited mammalian tyrosinase. In particular, 25 50 μM thioguanine reduced the melanin content by 57% without cytotoxicity. Furthermore, 26 the thiopurine drugs shared little chemical similarity with the known tyrosinase inhibitors. 27