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

Abstract

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

Resistance mechanisms of SARS-CoV-2 3CLpro to the non-covalent inhibitor WU-04

“…Our finding aligns with the observation that the L50F mutation compensated for the replicative fitness loss caused by the E166V mutation 10 , 11 . A yeast screen also indicated that the L50F mutation can increase the catalytic activity of 3CLpro, however, there was a lack of enzymatic assay data 19 . A recent study showed that the double mutant L50F/E166V had increased catalytic activity compared to the E166V single mutant, indicating that the L50F mutation increased the 3CLpro catalytic activity 20 .…”

“…With the increasing clinical use of 3CLpro inhibitors, the emergence of drug resistance has become a growing concern. Although no SARS-CoV-2 variants resistant to 3CLpro inhibitors have been reported in patients to date, several mutations in 3CLpro conferring resistance to nirmatrelvir have been identified through in vitro studies 8 – 19 . According to the crystal structure of the 3CLpro/nirmatrelvir complex (PDB code: 7RFS) 4 , most of these mutation sites are located at three segments within 5 Å of nirmatrelvir, including residues 140–144, 163–168, and 186–192.…”

“…All clinically approved inhibitors of SARS-CoV-2 3CLpro are designed to target the substrate-binding pocket of 3CLpro, which raises concern about cross-resistance. Many of the nirmatrelvir-resistant mutations also confer resistance to ensitrelvir 16 – 19 . However, mutations that confer resistance to one drug but not the other have also been reported.…”

Resistance mechanisms of SARS-CoV-2 3CLpro to the non-covalent inhibitor WU-04

“…Our finding aligns with the observation that the L50F mutation compensated for the replicative fitness loss caused by the E166V mutation ( 8, 9 ). A yeast screen also indicated that the L50F mutation can increase the catalytic activity of 3CLpro, however, there was a lack of enzymatic assay data ( 17 ). A recent study showed that the double mutant L50F/E166V had increased catalytic activity compared to the E166V single mutant, indicating that the L50F mutation increased the 3CLpro catalytic activity ( 18 ).…”

A comprehensive study of SARS-CoV-2 main protease (Mpro) inhibitor-resistant mutants selected in a VSV-based system

“…To date, several studies have addressed the issue of nirmatrelvir-resistant/escaping variants using different approaches: highlighting mutational hotspots (14,15), in silico investigation of specific mutations (16), studying resistance phenotypes (17)(18)(19)(20), addressing which mutations are the most prone to decrease inhibitor susceptibility (21)(22)(23) or more comprehensive studies describing either mutation resistance, fitness costs, or both (24)(25)(26)(27). Notably, a few groups have performed gain-offunction selection experiments using the Wuhan-1 strain of SARS-CoV-2.…”