Structure-based discovery of highly bioavailable, covalent, broad-spectrum coronavirus M inhibitors with potent in vivo efficacy.

The main protease (M) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a validated drug target. Starting with a lead-like dihydrouracil chemotype identified in a large-library docking campaign, we improved M inhibition >1000-fold by engaging additional M subsites and using a latent electrophile to engage Cys. Advanced leads from this series show pan-coronavirus antiviral activity, low clearance in mice, and for , a rapid reduction in viral titers >1,000,000 after just three doses. Both compounds are well distributed in mouse tissues, including brain, where concentrations >1000× the 90% effective concentration are observed 8 hours after oral dosing for . shows minimal inhibition of major cytochrome P450s and human proteases. also exhibits synergy with the RNA-dependent RNA polymerase inhibitor, molnupiravir, in cellular infection models. Related analogs strongly inhibit nirmatrelvir-resistant M mutant virus. The properties of this chemotype are differentiated from existing clinical and preclinical M inhibitors and will advance therapeutic development against emerging SARS-CoV-2 variants and other coronaviruses.