Systematic analyses of the resistance potential of drugs targeting SARS-CoV-2 main protease

Abstract

Drugs that target the main protease (Mpro) 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 Mpro, we performed comprehensive surveys of amino acid changes that can cause resistance in a yeast screen to nirmatrelvir (contained in the drug Paxlovid), and ensitrelvir (Xocova) that is currently in phase III trials. The most impactful resistance mutation (E166V) recently reported in multiple viral passaging studies with nirmatrelvir showed the strongest drug resistance score for nirmatrelvir, while P168R had the strongest resistance score for ensitrelvir. Using a systematic approach to assess potential drug resistance, we identified 142 resistance mutations for nirmatrelvir and 177 for ensitrelvir. Among these mutations, 99 caused apparent resistance to both inhibitors, suggesting a strong likelihood for the evolution of cross-resistance. Many mutations that exhibited inhibitor-specific resistance were consistent with distinct ways that each inhibitor protrudes beyond the substrate envelope. In addition, mutations 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 secondary 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.

Contributions of hyperactive mutations in Mprofrom SARS-CoV-2 to drug resistance

“…To explore the potential mechanistic roles of hyperactive mutations in the response to drug selection (Figure 1), we examined their occurrence with and without mutations identified as reducing drug binding 6 in cell culture selected lineages 10 . In the drug selection experiments, most mutations that were reported had arisen close to fixation (110 mutations at a frequency >75%, 7 at 50-75%, 15 at 25-50%, and 11 at 5-25%), indicating a prevalence of selective sweeps of individual variants and relatively low levels of clonal interference.…”

“…Hyperactive mutations are more frequently observed than drug binding mutations in viruses selected for nirmatrelvir resistance and in sequenced isolates. (A) Graph of the fraction of substitutions identified in viruses selected for nirmatrelvir resistance 14 that exhibited M pro hyperactivity (this work), or reduced nirmatrelvir binding 10 . (B) Graph illustrating the relative fraction of all possible mutations that were identified as either hyperactive or having reduced nirmatrelvir binding.…”

“…Cultures were grown with shaking at 180 rpm for 45 min, at which time samples of ∼10 7 cells were collected. The cells were immediately washed three times with ice cold tris- buffered saline containing 0.1% Tween and 0.1% bovine serum albumin (TBST-BSA), diluted to 10 6 cells/mL and immediately sorted for YFP and CFP expression on a FACS Aria II cell sorter. Cells expressing the cut FRET pair (low FRET) were collected in one population and the uncut FRET pair (high FRET) in a second population.…”

“…We downloaded the latest variable call format file ( public-latest.all.masked.vcf.gz ) from http://hgdownload.soe.ucsc.edu/goldenPath/wuhCor1/UShER_SARS-CoV-2/ on Sept 7, 2023. We wrote a custom perl script to tabulate sequence observations of mutations identified as hyperactive in this work, or with reduced binding to nirmaltrevir or ensitrelvir based on a previously published yeast screen 6 . The frequency of these classes of mutations was calculated by dividing by the total number of sequenced isolates in the time window analyzed.…”

“…In the evolution of drug resistance, enzyme targets frequently accumulate mutations that impact the binding of drug and/or the turnover of substrate 9 . The impact of mutations on inhibitor binding are relatively straightforward to interpret and we have reported a comprehensive assessment of how all possible point mutations in M pro from SARS-CoV-2 impact binding to drugs 10 . In addition to drug binding, the effects of mutations on enzyme activity are also critical to understanding drug resistance.…”