PROJECT SUMMARY Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) cause severe diseases in humans (COVID- 19) that presents a major threat for global public health. Since it was first reported in 12/2019, COVID-19 has become a pandemic that continues to spread, with >246 million confirmed cases and >5 million deaths as of 11/02/2021. In addition to the human tragedy, the magnitude of the pandemic-driven implosion of global economies is enormous. Although vaccines are now available, their efficacy appears to be reduced with spreading viral strains. Remdesivir is the only approved antiviral targeting SARS-CoV-2, but it has little effect on COVID-19 mortality. Therefore, it is critical to identify and develop additional antivirals to combat viral infection. The following strong preliminary data enable targeting of SARS-CoV-2: 1) Construction of an extensive collection of SARS-CoV-2 replicon systems that enable cutting-edge, rapid, and economical high-throughput screening. 2) Preparation of cell lines that stably express SARS-CoV-2 replicon. 3) Preliminary screening of chemical libraries led to the discovery of a novel SARS-CoV-2 antiviral, which has already been improved with one round of optimization through medicinal chemistry efforts. 4) Multiplex visualization of single-genomic or subgenomic (+) or (-) SARS-CoV-2 RNA and simultaneously viral and/or host proteins in individual infected cells. 5) Cutting-edge rapid high-throughput infectious virus BSL3 assays that enable kinetic, mechanistic, drug resistance studies. 6) In-house cloning expression and purification of 10 SARS-CoV-2 non-structural proteins (nsps). 7) Biochemical and biophysical assays to measure the enzymatic activities of several SARS-CoV and SARS-CoV-2 nsp proteins and to measure compound binding to nsps. We hypothesize that our recently developed replicon systems can be used for the discovery of anti-SARS- CoV-2 hits, which upon hit-to-lead optimization can become COVID-19 drug candidates. To address this hypothesis, we propose the following specific aims: 1. Use of SARS-CoV-2 replicon plasmid systems and SARS-CoV-2 replicon-expressing cell lines to screen chemical libraries for antiviral hits. 2. Inhibition and resistance studies with prioritized inhibitors. 3. Hit-to-lead optimization These studies will lead to antivirals with strong potency and pharmacokinetic profiles, setting the stage for development of SARS-CoV-2 antivirals and combination therapies.

PROJECT NARRATIVE We have developed a non-infectious, self-replicative SARS-CoV-2 replicon system that is a safe and effective tool for antiviral screening and amenable to genetic engineering for studying drug resistance. We have identified a small molecule inhibitor of SARS-CoV-2 antiviral activity, and early medicinal chemistry efforts have optimized this lead. This project aims to further identify and characterize compounds toward the development of novel inhibitors against SARS-CoV-2 with potent, nanomolar antiviral activity.