Ensemble-Based Virtual Screening Led to the Discovery of New Classes of Potent Tyrosinase Inhibitors.

Abstract

In this study, we report new classes of potent tyrosinase inhibitors identified by enhanced structure-based virtual screening prediction; the enzyme and melanin content assays were also confirmed. Tyrosinase, a type-3 copper protein, participates in two distinct reactions, hydroxylation of tyrosine to DOPA and conversion of DOPA to dopaquinone, in melanin biosynthesis. Although numerous inhibitors of this reaction have been reported, there is a lag in the discovery of the new functional moieties. In order to improve the performance of virtual screening, we first produced an ensemble of 10,000 structures using molecular dynamics simulation. Quantum mechanical calculation was used to determine the partial charges of catalytic copper ions based on the met and deoxy states. Second, we selected a structure showing an optimal receiver operating characteristic (ROC) curve with known direct binders and their physicochemically matched decoys. The structure revealed more than 10-fold higher enrichment at 1% of the ROC curve than those observed in X-ray structures. Third, high-throughput virtual screening with DOCK 3.6 was performed using a library consisting of approximately 400,000 small molecules derived from the ZINC database. Fourth, we obtained the top 60 molecules and tested their inhibition of mushroom tyrosinase. The extended assays included 21 analogs of the 21 initial hits to test their inhibition properties. Here, the moieties of tetrazole and triazole were identified as new binding cores interacting with the dicopper catalytic center. All 42 inhibitors showed inhibitory constant, Ki, values ranging from 11.1 nM and 33.4 μM, with a tetrazole compound exhibiting the strongest activity. Among the 42 molecules, five displayed more than 30% reduction in melanin production when treated in B16F10 melanoma cells; cell viability was >90% at 20 μM. Particularly, a thiosemicarbazone-containing compound reduced melanin content by 55%.