Multidisciplinary studies on overcoming SARS-CoV-2 main protease drug resistance and on fitness costs - ABSTRACT The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused a global pandemic since first emerging in 2019. Since, Pfizer, Inc. (USA) developed Paxlovid™, an FDA-approved antiviral containing the protease inhibitor nirmatrelvir (NIR) that has seen moderate success in treating SARS-CoV-2 infections. The main protease (Mpro) of SARS-CoV-2 cleaves the viral polyproteins to release proteins essential for replication. NIR mimics the Mpro consensus sequence, thus covalently blocking Mpro activity and viral replication. However, resistance mutations to NIR have started to develop as its use increases globally. Understanding how such substitutions confer resistance and balance the impacts on fitness will enable the strategic design of the next generation of Mpro inhibitors. My preliminary data and published experiments have identified drug resistance mutations (DRMs) in Mpro that confer significant resistance to NIR including E166V. Further, I have demonstrated that the E166V substitution remains susceptible to GC376, a feline coronavirus protease inhibitor, and PF-00835231, developed during the SARS-CoV-1 epidemic. E166V also severely decreases viral fitness and requires compensatory mutations such as L50F to rescue fitness. Despite this fitness cost, E166V and L50F/E166V were both observed in patients treated with Paxlovid™ during the clinical trial, and recent case studies also identified the L50V/E166V combination. It is unclear how E166V decreases fitness and why mutations at Leu50 restore fitness. Structural work from other labs and my preliminary data indicate Mpro forms an active homodimer with the N-terminus from the opposite protomer interacting with Glu166. Loss of these interactions due to the E166V mutation are likely to disrupt Mpro dimerization and, consequently, activity. Of note, dimerization has not been studied in the context of drug resistance, and the role of Leu50 mutations in restoring fitness is poorly understood. Building upon my previous work, I will analyze E166V and L50V/E166V using virological, biophysical, and structural techniques. The goal of my project is to characterize mechanisms of NIR resistance, determine how DRMs impact Mpro activity and fitness, and investigate strategies for overcoming NIR resistance. I hypothesize that DRMs alter the intermolecular interactions in the active site resulting in decreased binding of inhibitors and Mpro dimer formation. Aim 1 will characterize the effect of the selected substitutions on Mpro resistance to NIR, GC376, PF-00835231, and a novel inhibitor shown to inhibit E166V Mpro (NIP-22c) using a virus-like particle (VLP) assay (Aim 1.1) and elucidate the mechanism of and strategies for overcoming NIR resistance using biochemical (Aim 1.2) and crystallographic studies (Aim 1.3). Aim 2 will investigate the effect of E166V and L50V on viral replication efficiency in cells (Aim 2.1) and on Mpro dimerization in vitro using biochemical methods (Aim 2.2). This project will provide valuable insights into mechanisms of drug resistance, impacts on viral fitness, and strategies for overcoming NIR-resistant SARS-CoV-2 Mpro informing the design of next-generation antivirals targeting Mpro and future development of pan-coronavirus antivirals. Completing this project will train me in techniques and skills essential for my future career as an independent scientist.
