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

The SARS-CoV-2 main protease (M-pro) is thedrug targetof Pfizer's oral drug nirmatrelvir. The emergence of SARS-CoV-2variants with mutations in M-pro raised the alarm of potentialdrug resistance. To identify potential clinically relevant drug-resistantmutants, we systematically characterized 102 naturally occurring M-pro mutants located at 12 residues at the nirmatrelvir-bindingsite, 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, showedcomparable 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 determinedforsix 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 showedthat M-pro mutants with reduced enzymatic activity had attenuatedviral replication. Overall, our study identified several drug-resistanthotspots in M-pro that warrant close monitoring for possibleclinical evidence of nirmatrelvir resistance, some of which have alreadyemerged in independent viral passage assays conducted by others. Systematic analysis of SARS-CoV-2 mainprotease sequencesin the GISAID database revealed naturally occurring mutations thatare resistant to nirmatrelvir without compromised fitness of replication.