Tyrosinase Oxidative\nCross-Linking in the Cell-Like\nCrowded Microenvironment for Visible Inhibitor Screening

Owing to many diseases and disorders being caused by\ndysfunctional\nor over/underexpressed enzymes, screening for inhibitors of pharmacologically\nrelevant enzymes has emerged as a promising tool for drug discovery,\nclinical diagnostics, enzyme engineering, and other medical fields.\nHowever, despite the recent advances, most inhibitor screenings are\nstill usually conducted in dilute media, at concentrations far from\nthe media in which the enzymes are actually found, which may cause\ndrugs to fail when translated to <i>in vivo</i>. Herein,\nwe build a gel-like intracellular biological environment <i>in\nvitro</i> using a tyrosinase oxidative cross-linking hydrogel\nsystem that is closer to the real catalytic environment of enzymes.\nWe report a straightforward and effective inhibitor evaluation strategy\nthat can quickly compare the inhibitory strengths of inhibitors based\non the principle that adding inhibitors causes color changes and mechanical\nchanges in the system. Enabled by molecular docking, we further demonstrate\nthe different performances of the inhibitors at different concentrations.\nBy construction of the cell-like crowded environment <i>in vitro</i>, this system shows an appealing application prospect for new drug\ndevelopment.