Studies on the SARS-CoV-2 Main Protease Dimerization Mechanism - ABSTRACT The main protease enzyme (Mpro) of SARS-CoV-2 cleaves the viral polyprotein into functional units responsible for virus replication and pathogenesis. Interestingly, Mpro functions only as a homodimer. However, the structure- function relationship and conformational plasticity of the Mpro dimerization mechanism remains poorly understood. To address these gaps in knowledge, I have developed quantitative luciferase-based reporter assays for Mpro dimerization in living cells and in vitro. I propose first to use these assays to study the Mpro dimerization mechanism through the construction and analysis of a panel structure-guided and evolution- informed mutants, as well as through targeted deep-mutational scanning of dimerization interface residues. Second, I will use my luciferase-based reporter assays to identify candidate dimerization inhibitors. Third, I additionally propose to use my luciferase-based reporter assays to perform studies on the (likely allosteric) mechanism of inhibitor-facilitated dimerization. Altogether, the proposed studies will yield new assays for studying coronavirus protease biology, unique insights into the mechanisms of Mpro dimerization and its allosteric modulation, and novel compounds that can be used as chemical probes of these molecular mechanisms.
