Baicalein from Scutellaria baicalensis Allosterically Inhibits TMPRSS2 and Suppresses Coronavirus Entry

Abstract

Transmembrane protease serine 2 (TMPRSS2) is an important host serine protease involved in the activation of viral envelope glycoproteins. To identify natural bioactive constituents that modulate TMPRSS2, we screened extracts from 28 natural product extracts using an affinity-guided ligand fishing approach. Four flavonoids from Scutellaria baicalensis were identified as TMPRSS2-binding compounds, with baicalein exhibiting the strongest inhibitory activity. Enzyme kinetic analysis revealed that baicalein inhibits TMPRSS2 through a non-competitive mechanism. Surface plasmon resonance and multiple spectroscopic analyses confirmed that baicalein directly binds to TMPRSS2 and induces conformational changes in the enzyme. Molecular docking combined with site-directed mutagenesis suggested a putative allosteric site involving residues Q276, N277, H307 and G323. Baicalein also inhibited the entry of multiple coronavirus pseudoviruses in TMPRSS2-expressing cells. These findings demonstrate that baicalein inhibits TMPRSS2 through a putative allosteric site, highlighting plant-derived flavonoids as potential inhibitors of host protease-mediated viral entry.