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

The main protease (Mpro) of SARS-CoV-2 is essential for viral replication and is considered to be one of the most promising SARS-CoV-2 drug targets. While Mpro mutations have occurred in many variants, including Omicron (B.1.1.529), their structural and biochemical importance have, until this point, remained uncharacterized. Using X-ray crystallography, we show the Omicron Mpro mutant (P132H) induces a small rearrangement of residues distal from the active site. While enzymatic activity and small-molecule inhibition appears unchanged, the melting temperature (Tm) of Omicron Mpro is 2.6 ° C lower than Alpha and Delta Mpro. Yet, when incubated with inhibitors, these enzymes have nearly identical Tm values. The physiological importance of the P132H mutant is unclear; however, we show residue 132 is located at the interface of the dimerization and catalytic domains and is frequently mutated in other variants. Lower thermal stability may indicate increased flexibility that can potentially broaden substrate profile or alter inhibitor binding. Therefore, structural insights are key to anticipating future mutations that may promote drug-resistance and are especially important at present, given the recent approval of nirmatrelvir (PAXLOVID), an oral SARS-CoV-2 Mpro inhibitor. ### Competing Interest Statement The authors have declared no competing interest.