Structural basis for the broad-spectrum inhibition of the coronavirus main protease by Zevotrelvir.

Beta-coronaviruses have caused three major coronavirus outbreaks, underscoring the urgent need for broad-spectrum antivirals. The coronavirus main protease (Mpro) is an ideal drug target due to its high conservation and absence of human homologs. Zevotrelvir (EDP-235) is an oral Mpro inhibitor with broad-spectrum activity against betacoronaviruses, but its molecular mechanism remains unclear. Here, we systematically evaluated Zevotrelvir against SARS-CoV-2 Mpro and its mutants (G15S, M49I, K90R, S144A), as well as SARS-CoV and MERS-CoV Mpro, using enzyme activity assays, crystallography, and molecular dynamics simulations. Zevotrelvir potently inhibited SARS-CoV-2 Mpro (IC50 = 23 nM) and maintained micromolar activity against mutants and other coronaviruses. Structural analysis revealed that Zevotrelvir forms a conserved covalent bond with the catalytic C145 (C148 in MERS-CoV) via its nitrile warhead, while its spirolactam and trifluoroindole groups occupy the S1', S1, S2, and S4 subsites through an E166-centered hydrogen bond network and hydrophobic interactions. Notably, conformational asymmetry was observed within the Mpro dimer, with the trifluoroindole moiety rotating ~80° in protomer B. Molecular dynamics simulations confirmed complex stability and identified the key hydrogen bonds. This study elucidates the molecular basis of Zevotrelvir's broad-spectrum inhibition, informing its clinical development and next-generation drug design.