Discovery of Novel SARS-CoV-2 3CLpro Inhibitors from Natural Products using FRET-based Assay

The coronavirus 3C-like protease (3CLpro) is essential for SARS-CoV-2 replication, making it a key target for antiviral drug development. Natural products represent a valuable source of bioactive compounds. This study aimed to identify novel 3CLpro inhibitors from natural compounds through a combination of virtual screening and experimental validation.

Recombinant 3CLpro was expressed, purified, and evaluated for enzymatic activity using Fluorescence Resonance Energy Transfer (FRET) assays under optimized conditions. Out of 583 virtually screened compounds, 30 were selected for experimental validation. Epitheaflagallin 3-O-gallate (ETFGg) was further analyzed for binding interactions using Molecular Dynamics (MD) simulations.

ETFGg exhibited strong binding affinity (-66.90 kcal/mol) and inhibitory activity (IC50 = 8.73 ± 2.30 μM) against 3CLpro. MD simulations revealed stable interactions with key residues (HIE163, THR190, GLN192) in the 3CLpro active site.

Our findings demonstrate that ETFGg is a potent and stable inhibitor of SARS-CoV-2 3CLpro, with a binding profile distinct from known inhibitors such as ebselen. The substrate inhibition kinetics observed suggest a novel allosteric mechanism, which may provide a new strategy for targeting 3CLpro. This study supports the value of natural product libraries combined with computational and FRET-based screening for discovering antiviral leads.

ETFGg was identified as a promising 3CLpro inhibitor with high binding stability, highlighting its potential as a lead compound for the development of anti-- COVID-19 drugs.