Abstract
Tyrosinase catalyzes two distinct sequential reactions in melanin biosynthesis: The hydroxylation of tyrosine to dihydroxyphenylalanine (DOPA) and the oxidation of DOPA to dopaquinone. Developing functional modulators of tyrosinase is important for therapeutic and cosmetic purposes. Given the abundance of thiourea moiety in known tyrosinase inhibitors, we studied other thiourea-containing drugs as potential tyrosinase inhibitors. The thiourea-containing drugs in clinical use were retrieved and tested for their ability to inhibit tyrosinase. We observed that methimazole, thiouracil, methylthiouracil, propylthiouracil, ambazone, and thioacetazone inhibited mushroom tyrosinase. Except for methimazole, there was limited information regarding the activity of other drugs against tyrosinase. Both thioacetazone and ambazone significantly inhibited tyrosinase, with IC50 of 14 and 15 μM, respectively. Ambazone decreased melanin content without causing cellular toxicity at 20 μM in B16F10 cells. The activity of ambazone was stronger than that of kojic acid both in enzyme and melanin content assays. Kinetics of enzyme inhibition assigned the thiourea-containg drugs as non-competitive inhibitors. The complex models by docking simulation suggested that the intermolecular hydrogen bond via the nitrogen of thiourea and the contacts via thione were equally important for interacting with tyrosinase. These data were consistent with the results of enzyme assays with the analogues of thiourea.
Highlights
Type-3 copper proteins, including tyrosinase, catechol oxidase, and hemocyanin, possess six evolutionarily-conserved histidines
We have reported that the analogues of ethionamide, a drug used for multidrug-resistant tuberculosis, share remote similarity with PTU and exhibit potent inhibition of tyrosinase [18]
The high content of the thiourea moiety in the inhibitors raised the question whether thiourea itself would inhibit the function of tyrosinase
Summary
Type-3 copper proteins, including tyrosinase, catechol oxidase, and hemocyanin, possess six evolutionarily-conserved histidines. Catalytic activities of tyrosinase are coupled with four possible oxidation states of copper ions: oxy-, met-, deoxy-, and deact-states [1]. A couple of complex structures, one between PTU and sweet potato catechol oxidase (PDB ID: 1BUG) [14] and the other between tropolone and mushroom tyrosinase (PDB ID: 2Y9X) [15], have been published. These structures revealed that the inhibitors bind to the proteins in met and not in oxy-states. The simulated binding modes between protein and small molecules are discussed
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