Contributions of hyperactive mutations in M^profrom SARS-CoV-2 to drug resistance

Abstract

The appearance and spread of mutations that cause drug resistance in rapidly evolving diseases, including infections by SARS-CoV-2 virus, are major concerns for human health. Many drugs target enzymes, and resistant mutations impact inhibitor binding and/or enzyme activity. The most widely used inhibitors currently used to treat SARS-CoV-2 infections, including nirmatrelvir, target the main protease (Mpro) preventing it from processing viral polyproteins into active subunits. Previous work has systematically analyzed resistance mutations in Mpro that reduce binding to inhibitors, and here we investigate mutations that affect enzyme function. Hyperactive mutations that increase Mpro activity can contribute to drug resistance both directly by requiring elevated inhibitor concentrations to reduce function to critical levels and indirectly by increasing tolerance to mutations that reduce both substrate turnover and inhibitor binding. We comprehensively assessed how all possible individual mutations in Mpro affect enzyme function using a mutational scanning approach with a FRET-based yeast readout. We identified hundreds of mutations that significantly increased Mpro activity. Hyperactive mutations occurred both proximal and distal to the active site, consistent with protein stability and/or dynamics impacting activity. Hyperactive mutations were observed three times more than mutations that reduced apparent binding to nirmatrelvir in laboratory grown viruses selected for drug resistance and were also about three times more prevalent than nirmatrelvir binding mutations in sequenced isolates from circulating SARS-CoV-2. Our findings indicate that hyperactive mutations are likely to contribute to the natural evolution of drug resistance in Mpro and provide a comprehensive list for future surveillance efforts.