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.

Grant Number: 5R21AI191052-02
NIH Institute/Center: NIH
Principal Investigator: DANIEL BOLON