Comprehensive analysis of epistasis in drug resistance potential of Mpro from SARS-CoV-2

PROJECT SUMMARY This project aims to systematically investigate a critical, but poorly understood aspect of drug resistance evolution: the interdependence of mutations that disrupt drug binding (usually also decreasing enzyme activity) and compensatory mutations that increase enzyme activity. Combinations of these types of mutations are typically observed in pathogens that evolve clinically relevant resistance. The mechanisms that underlie these mutations have not been extensively investigated. Here, we plan to comprehensively analyze all combinations of mutations in Mpro from SARS-CoV-2 that disrupt binding to nirmatrelvir with those that increase enzyme activity. Nirmatrelvir is the active component in Paxlovid that is currently an effective treatment for COVID19. We developed a yeast screen for Mpro activity that is both safe because it does not create or use virus and biologically relevant because it uses a cut-site that is used by the virus. Mutations we identified with this screen have been observed in SARS-CoV-2 viruses selected for resistance, further indicating the screens biological relevance. In the first aim of this work, we will quantify how all combinations of drug-binding and increased activity mutations impact Mpro activity and drug disruption in our yeast screen. The resulting data will be analyzed to elucidate patterns and their structural underpinnings. As compensatory mutations can be specific, in the second aim, we will perform an unbiased analysis of all possible point mutations in the background of two mutations that strongly disrupt nirmatrelvir binding. Together these aims will provide a new view of how mutational interdependencies impact the evolution of drug resistance in a clinically important pathogen.