Novel Nirmatrelvir-Resistance Mutations in SARS-CoV-2 Mpro Using a Yeast Screening Platform

SARS-CoV-2 is a positive-sense single-stranded RNA virus of the Coronaviridae family. Emerging coronaviruses posing pandemic risk and the swift appearance of new antigenic variants of circulating SARS-CoV-2 urge the need for new and diverse effective treatments. The most outstanding targets to inhibit viral replication are its cysteine-proteases, Mpro and PLpro, which are essential for virus life cycle. Here, we investigated the functional consequences of heterologously expressed SARS-CoV-2 proteases in the eukaryotic model Saccharomyces cerevisiae. Both overproduced Mpro and PLpro caused yeast growth inhibition. This phenotype was dependent on their protease activities, since it was abrogated by the expression of catalytically inactive mutants or counteracted by the treatment with known protease inhibitors. Mpro reproduced in yeast the proteolytic cleavage of the pore-forming protein Gasdermin D, previously described in human cell lines. We generated a yeast-based screening platform to detect mutations in Mpro conferring resistance to inhibitors. To this end, we evaluated the behaviour of the purported resistant mutant, Mpro^E166V, and found a resistant pattern to nirmatrelvir. Taking advantage of this model, we performed a random mutagenesis screen to isolate nirmatrelvir-resistant mutations in Mpro. Our screening identified nine clones. Five harbored previously described mutations, either identical (A260V) or involving alternative substitutions (G15N/F159I, G23E/D33N/R60K, C128R/R188K, S139I). Importantly, our study also revealed four novel residues that have not yet been associated with nirmatrelvir resistance (K88R, L220M, Y209F, L262M). In sum, we report a suitable model to screen compounds in search of new Mpro and PLpro inhibitors and to predict mutations associated to drug resistance.