ABSTRACT The ongoing SARS-CoV-2 pandemic continues to be a significant threat to global health. First reported in November 2021, the Omicron variant (B.1.1.529) is more transmissible and can evade immunity better than previous SARS-CoV-2 variants, fueling an unprecedented surge in cases. To produce functional proteins from this polyprotein, SARS-CoV-2 relies on the cysteine proteases Nsp3/papain-like protease (PLpro) and Nsp5/Main Protease (M pro )/3C-like protease to cleave at three and more than 11 sites, respectively. 1 Therefore, M pro and PL pro inhibitors are considered to be some of the most promising SARS-CoV-2 antivirals. On December 22, 2021, the Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) for PAXLOVID, a ritonavir-boosted formulation of nirmatrelvir. Nirmatrelvir is a first-in-class orally bioavailable SARS-CoV-2 M pro inhibitor. 2 Thus, the scientific community must vigilantly monitor potential mechanisms of drug resistance, especially because SARS-CoV-2 is naïve to M pro inhibitors. Mutations have been well identified in variants to this point. 3 Notably, Omicron M pro (OM pro ) harbors a single mutation– P132H. In this study we characterize the enzymatic activity, drug inhibition, and structure of OM pro while evaluating the past and future implications of M pro mutations.

The P132H mutation in the main protease of Omicron SARS-CoV-2 decreases thermal stability without compromising catalysis or small-molecule drug inhibition