Naturally occurring mutations of SARS-CoV-2 main protease confer drug resistance to nirmatrelvir

The SARS-CoV-2 main protease (M(pro)) is the drug target of Pfizer’s oral drug Paxlovid. The emergence of SARS-CoV-2 variants with mutations in M(pro) raised the alarm of potential drug resistance. In this study, we identified 100 naturally occurring M(pro) mutations located at the nirmatrelvir binding site, among which 20 mutants, including S144M/F/A/G/Y, M165T, E166G, H172Q/F, and Q192T/S/L/A/I/P/H/V/W/C/F, showed comparable enzymatic activity to the wild-type (k(cat)/K(m) <10-fold change) and resistance to nirmatrelvir (K(i) >10-fold increase). X-ray crystal structures were determined for seven representative mutants with and/or without GC-376/nirmatrelvir. Viral growth assay showed that M(pro) mutants with reduced enzymatic activity led to attenuated viral replication. Overall, our study identified several drug resistant hot spots that warrant close monitoring for possible clinical evidence of Paxlovid resistance.