Naturally Occurring Mutations of SARS-CoV-2 Main Protease Confer Drug Resistance to Nirmatrelvir

[Image: see text] The SARS-CoV-2 main protease (M(pro)) is the drug target of Pfizer’s oral drug nirmatrelvir. The emergence of SARS-CoV-2 variants with mutations in M(pro) raised the alarm of potential drug resistance. To identify potential clinically relevant drug-resistant mutants, we systematically characterized 102 naturally occurring M(pro) mutants located at 12 residues at the nirmatrelvir-binding site, among which 22 mutations in 5 residues, including S144M/F/A/G/Y, M165T, E166 V/G/A, 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) while being resistant to nirmatrelvir (K(i) > 10-fold increase). X-ray crystal structures were determined for six representative mutants with and/or without GC-376/nirmatrelvir. Using recombinant SARS-CoV-2 viruses generated from reverse genetics, we confirmed the drug resistance in the antiviral assay and showed that M(pro) mutants with reduced enzymatic activity had attenuated viral replication. Overall, our study identified several drug-resistant hotspots in M(pro) that warrant close monitoring for possible clinical evidence of nirmatrelvir resistance, some of which have already emerged in independent viral passage assays conducted by others.