TY - JOUR T1 - A yeast-based system to study SARS-CoV-2 M<sup>pro</sup> structure and to identify nirmatrelvir resistant mutations JF - bioRxiv DO - 10.1101/2022.08.06.503039 SP - 2022.08.06.503039 AU - Jin Ou AU - Eric M. Lewandowski AU - Yanmei Hu AU - Austin A. Lipinski AU - Ryan T. Morgan AU - Lian M.C. Jacobs AU - Xiujun Zhang AU - Melissa J. Bikowitz AU - Paul Langlais AU - Haozhou Tan AU - Jun Wang AU - Yu Chen AU - John S. Choy Y1 - 2022/01/01 UR - http://biorxiv.org/content/early/2022/08/08/2022.08.06.503039.abstract N2 - The SARS-CoV-2 main protease (Mpro) is a major therapeutic target. The Mpro inhibitor, nirmatrelvir, is the antiviral component of Paxlovid, an orally available treatment for COVID-19. As Mpro inhibitor use increases, drug resistant mutations will likely emerge. We have established a non-pathogenic system, in which yeast growth serves as a proxy for Mpro activity, enabling rapid identification of mutants with altered enzymatic activity and drug sensitivity. The E166 residue is known to be a potential hot spot for drug resistance and yeast assays showed that an E166R substitution conferred strong nirmatrelvir resistance while an E166N mutation compromised activity. On the other hand, N142A and P132H mutations caused little to no change in drug response and activity. Standard enzymatic assays confirmed the yeast results. In turn, we solved the structures of Mpro E166R, and Mpro E166N, providing insights into how arginine may drive drug resistance while asparagine leads to reduced activity. The work presented here will help characterize novel resistant variants of Mpro that may arise as Mpro antivirals become more widely used.Competing Interest StatementThe authors have declared no competing interest. ER -