The high throughput YESS 2.0 platform was used to screen a large library of SARS-CoV-2 Mpro variants in the presence of nirmatrelvir. Of the 100 individual most prevalent mutations identified in the screen and reported here, the most common were E166V, L27V, N142S, A173V, and Y154N, along with their various combinations. In vitro analysis revealed that resistance to nirmatrelvir for these individual mutations, as well as all of the combinations we analyzed, was accompanied by decreased catalytic activity with the native substrate. Importantly, the mutations we identified have not appeared as significantly enriched in SARS-CoV-2 Mpro sequences isolated from COVID-19 patients following the introduction of nirmatrelvir. We also analyzed three of the most common SARS-CoV-2 Mpro mutations that have been seen in patients recently, and only a measured increase in nirmatrelvir resistance was seen when the more recently appearing A285V is added to both P132H and K90R. Taken together, our results predict that resistance to nirmatrelvir will be slower to develop than expected based on experience with other viral protease inhibitors, perhaps due in part to the close structural correspondence between nirmatrelvir and SARS-CoV-2 Mpro's preferred substrates.
© 2024 The Authors. Published by American Chemical Society.