SARS-CoV-2 Mpro inhibitor ensitrelvir: asymmetrical cross-resistance with nirmatrelvir and emerging resistance hotspots

SARS-CoV-2 main protease (Mpro) inhibitors are the first-line COVID-19 treatment. Nirmatrelvir is used worldwide, while ensitrelvir, licensed in Japan and Singapore, has received FDA fast-track designation. To facilitate population monitoring for viral resistance and guide next-generation inhibitor design, we investigated SARS-CoV-2 resistance and cross-resistance to ensitrelvir and nirmatrelvir. SARS-CoV-2 escape variants with high fitness and high ensitrelvir resistance were selected under clinically relevant concentrations in infectious cell culture assays. Using infectious cell culture, replicon, and Mpro assays, reverse genetics revealed synergistic combinations of resistance-associated substitutions (RAS), specifically M49L + S144A and M49L + S144A + T169I, that conferred high resistance with a low fitness cost. Molecular dynamics simulations confirmed that M49L + S144A or M49L + S144A + T169I weakened ensitrelvir-Mpro binding. M49L + S144A and M49L + S144A + T169I exhibited a lower fitness cost and conferred higher resistance than the previously identified ensitrelvir RAS M49L + E166A. Cross-resistance between these ensitrelvir RAS and previously described nirmatrelvir RAS L50F + E166V was asymmetrical, with nirmatrelvir RAS showing greater resistance to ensitrelvir than vice versa. Amino acid changes at Mpro-position 166, an emerging resistance hotspot with natural variation, had differential impacts on viral fitness and Mpro inhibitor resistance in infectious cell culture assays. The most frequently naturally occurring substitution, E166Q, did not confer significant resistance to either ensitrelvir or nirmatrelvir. However, the second most frequent substitution, E166H, conferred high resistance to nirmatrelvir, but not to ensitrelvir. This comparative resistance analysis can inform COVID-19 treatment strategies and contribute to pandemic preparedness.