Discovery of novel SARS-CoV-2 3CLpro inhibitors from natural products by FRET-based assay

Abstract As a highly conserved protease, 3-chymotrypsin-like protease (3CLpro) plays a key role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, and drugs targeting 3CLpro have shown promising therapeutic effects. Promising applications have been achieved with direct-acting antivirals targeting 3CLpro. Natural products are an important source of medicinal compounds. In this study, we expressed and purified fusion proteins and obtained soluble 3CLpro, and the enzymatic activity was evaluated using fluorescence resonance energy transfer (FRET) assays at optimized concentrations of protease and substrate. This assay was further applied to validate the 3CLpro inhibitory activity of 30 compounds selected from 583 compounds via virtual screening. Epitheaflagallin 3-O-gallate (ETFGg) was identified with binding free energy of − 66.90 kcal/mol and an IC50 value of 8.73 ± 2.30 µM and was selected for the ligand-protein interaction study. Dynamics simulation results suggested that ETFGg interacted with HIE163, THR190 and GLN192 of 3CLpro, and was stable in the binding pocket of 3CLpro during the simulation. Together, this work identified ETFGg as a 3CLpro inhibitor with prominent binding capability, which could serve as a potential lead compound for drug development against COVID-19.