Discovery of <i>bis</i> -thiourea derivatives as potent tyrosinase inhibitors: combined experimental and computational study

Tyrosinase, a key enzyme in melanin synthesis, serves as a primary target for developing depigmenting agents. The search for novel tyrosinase inhibitors is needed due to the adverse effects of current inhibitors. This study evaluated 16 <i>bis</i>-thiourea derivatives using <i>in vitro</i> and <i>in silico</i> methods, identifying compound <b>4</b>, with chlorine substituents, as the most potent inhibitor. Compound <b>4</b> outperformed kojic acid in inhibiting mushroom tyrosinase activity and interacted with catalytic copper ions and active site residues, as revealed by molecular docking and copper-chelating assay. Molecular dynamics simulation and MM/PBSA-based free energy calculations confirmed the greater stability and binding affinity of the compound <b>4</b>-tyrosinase complex in an aqueous environment compared to kojic acid-tyrosinase complex. Melanin assay revealed that compound <b>4</b> significantly suppressed melanin production in B16F10 melanoma cells, showing stronger anti-melanogenic activity than kojic acid. Drug-likeness predictions confirmed its compliance with Lipinski's rule of five, supporting <i>bis</i>-thiourea derivatives as promising tyrosinase inhibitors.