Pan-3C Protease Inhibitor Rupintrivir Binds SARS-CoV-2 Main Protease in a Unique Binding Mode

Lockbaum, Gordon J.; Henes, Mina; Lee, Jeong Min; Timm, Jennifer; Nalivaika, Ellen A.; Thompson, Paul R.; Kurt Yilmaz, Nese; Schiffer, Celia A.

[Image: see text] Rupintrivir targets the 3C cysteine proteases of the picornaviridae family, which includes rhinoviruses and enteroviruses that cause a range of human diseases. Despite being a pan-3C protease inhibitor, rupintrivir activity is extremely weak against the homologous 3C-like protease of SARS-CoV-2. In this study, the crystal structures of rupintrivir were determined bound to enterovirus 68 (EV68) 3C protease and the 3C-like main protease (M(pro)) from SARS-CoV-2. While the EV68 3C protease–rupintrivir structure was similar to previously determined complexes with other picornavirus 3C proteases, rupintrivir bound in a unique conformation to the active site of SARS-CoV-2 M(pro) splitting the catalytic cysteine and histidine residues. This bifurcation of the catalytic dyad may provide a novel approach for inhibiting cysteine proteases.