Systematic Analyses of the Resistance Potential of Drugs Targeting SARS-CoV-2 Main Protease

Drugs that target the main protease (M-pro)of SARS-CoV-2are effective therapeutics that have entered clinical use. Wide-scaleuse of these drugs will apply selection pressure for the evolutionof resistance mutations. To understand resistance potential in M-pro, we performed comprehensive surveys of amino acid changesthat can cause resistance to nirmatrelvir (Pfizer), and ensitrelvir(Xocova) in a yeast screen. We identified 142 resistance mutationsfor nirmatrelvir and 177 for ensitrelvir, many of which have not beenpreviously reported. Ninety-nine mutations caused apparent resistanceto both inhibitors, suggesting likelihood for the evolution of cross-resistance.The mutation with the strongest drug resistance score against nirmatrelvirin our study (E166V) was the most impactful resistance mutation recentlyreported in multiple viral passaging studies. Many mutations thatexhibited inhibitor-specific resistance were consistent with the distinctinteractions of each inhibitor in the substrate binding site. In addition,mutants with strong drug resistance scores tended to have reducedfunction. Our results indicate that strong pressure from nirmatrelviror ensitrelvir will select for multiple distinct-resistant lineagesthat will include both primary resistance mutations that weaken interactionswith drug while decreasing enzyme function and compensatory mutationsthat increase enzyme activity. The comprehensive identification ofresistance mutations enables the design of inhibitors with reducedpotential of developing resistance and aids in the surveillance ofdrug resistance in circulating viral populations.