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

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 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 inhibitor-specific 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.