Abstract SARS-CoV-2 has demonstrated extraordinary ability to evade antibody immunity by antigenic drift. Small molecule drugs may provide effective therapy while being part of a solution to circumvent SARS-CoV-2 immune escape. In this study we report an α-ketoamide based peptidomimetic inhibitor of SARS-CoV-2 main protease (M pro ), RAY1216. Enzyme inhibition kinetic analysis established that RAY1216 is a slow-tight inhibitor with a K i of 8.6 nM; RAY1216 has a drug-target residence time of 104 min compared to 9 min of PF-07321332 (nirmatrelvir), the antiviral component in Paxlovid, suggesting that RAY1216 is approximately 12 times slower to dissociate from the protease-inhibitor complex compared to PF-07321332. Crystal structure of SARS-CoV-2 M pro :RAY1216 complex demonstrates that RAY1216 is covalently attached to the catalytic Cys145 through the α-ketoamide warhead; more extensive interactions are identified between bound RAY1216 and M pro active site compared to PF-07321332, consistent with a more stable acyl-enzyme inhibition complex for RAY1216. In cell culture and human ACE2 transgenic mouse models, RAY1216 demonstrates comparable antiviral activities towards different SARS-CoV-2 virus variants compared to PF-07321332. Improvement in pharmacokinetics has been observed for RAY1216 over PF-07321332 in various animal models, which may allow RAY1216 to be used without ritonavir. RAY1216 is currently undergoing phase III clinical trials ( https://clinicaltrials.gov/ct2/show/NCT05620160 ) to test real-world therapeutic efficacy against COVID-19.

Inhibition mechanism and antiviral activity of an α-ketoamide based SARS-CoV-2 main protease inhibitor