2023 AIChE Annual Meeting

Yeast-Based High-Throughput Screening to Isolate Inhibitor-Resistant Sars-CoV2 Protease Variants

RNA viruses have a rapid evolution rate (between anywhere from 10−4 to 10−6 substitutions per nucleotide site per cell infection), giving rise to drug-resistant mutants. This concerning evolutionary rate has been seen most recently in the years following the SARS-CoV-2 viral outbreak as researchers attempt to predict the next iteration of the virus. The key actor in the virus’ ability to rapidly proliferate is cleavage of various sites in the viral polyprotein by the cystine protease 3Clpro, making the protease a focal point of many research studies. SARS-CoV-2 is often treated with drugs that covalently bind to 3Clpro and block its viral replication mechanism. Current in vitro studies seek out variants of 3Clpro that may be of concern due to this rapid proclivity to evolve, possibly towards resistance against such covalent inhibitors. However, present screens lack the ability to detect drug-resistant variants with low enzymatic activity (1-10% of the wild-type enzyme’s activity). To address this issue, we built a high-throughput yeast-based surface display screen that can 1) identify low activity 3Clpro variants by tuning co-expression of 3Clpro and its biologically relevant substrate in the endoplasmic reticulum (ER) of the displaying yeast and 2) comprehensively profile resistance of these mutants to Nirmatrelvir, a known covalent inhibitor of 3Clpro. Adjusting our endoplasmic retention signal to retain one or both protease and substrate in the ER for differing periods of time gives each low resistant variant a dynamic potential to be detected in our screening system, with and without introduction of a drug inhibitor. In this way, our comprehensive screen provides a novel means to characterize previously undetected drug-resistant SARS-CoV-2 protease mutations.