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

The main protease (M ^Pro ) of SARS-CoV-2 is crucial for viral replication and is the target of nirmatrelvir (the active ingredient of Paxlovid) and ensitrelvir. The identification of new agents with differentiated pharmacokinetic and drug resistance profiles will increase therapeutic options for COVID-19 patients and bolster pandemic preparedness generally. Starting with a lead-like dihydrouracil chemotype from a large-library docking campaign, we improved M ^Pro inhibition >1,000-fold by engaging additional sub-sites in the M ^Pro active site, most notably by employing a latent propargyl electrophile to engage the catalytic Cys145. Advanced leads from this series, including AVI-4516 and AVI-4773 show pan-coronavirus antiviral activity in cells, very low clearance in mice, and for AVI-4773 a rapid reduction in viral titers more than a million-fold after just three doses, more rapidly and effectively than the approved drugs, nirmatrelvir and ensitrelvir. Both AVI-4516 and AVI-477 3 are well distributed in mouse tissues, including brain, where concentrations ten or fifteen-thousand times the EC ₉₀ , respectively, are observed eight hours after an oral dose. As exemplar of the series, AVI-4516 shows minimal inhibition of major CYP isoforms and human cysteine and serine proteases, likely due to its latent–electrophilic warhead. AVI-4516 also exhibits synergy in cellular infection models in combination with the RdRp inhibitor molnupiravir, while related analogs strongly inhibit nirmatrelvir-resistant M ^Pro mutant virus in cells. The in vivo and antiviral properties of this new chemotype are differentiated from existing clinical and pre-clinical M ^Pro inhibitors, and will advance new therapeutic development against emerging SARS-CoV-2 variants and other coronaviruses.