SUMMARY The SARS-CoV-2 RNA-dependent RNA polymerase (RDRP), non-structural protein 12 (NSP12), which in complex with viral proteins nsp7 and nsp8 carries out essential RNA synthesis reactions, is an attractive and well-validated target for antivirals. To date, nucleoside analogs that inhibit RDRP activity show promise as COVID-19 treatments. Among these is remdesivir (RDV), a prodrug of the adenosine analog GS-441524 and the first FDA-approved antiviral for the treatment of COVID-19. Recent phase 3 clinical data for molnupiravir, a prodrug of β-D-N4-hydroxycytidine (NHC), has encouraged Merck to seek Emergency Authorization Use. Whereas RDV suffers from lack of oral bioavailability, limiting its outpatient use, molnupiravir is orally administered but requires frequent high doses. Further, none of the most advanced nucleoside inhibitors were developed specifically to treat coronavirus infections. Here, we propose three parallel approaches to provide novel drugs optimized to target the SARS-CoV-2 RDRP and treat coronavirus infections. In the first approach, we build on promising preliminary data in non-human primates as we aim to develop a prodrug with improved oral exposure vs. RDV so as to enable oral administration for use in the outpatient setting, preferably a once a day dosing regimen. Our second approach seeks to identify and optimize novel nucleoside analogs against SARS-CoV-2 RDRP. Key to this approach will be the screening of a nucleoside library that includes 167 novel analogs for activity against SARS-CoV-2 in human bronchial epithelial cells differentiated in an air-liquid interface. Third, we will use a cell-based assay of SARS-CoV-2 RDRP to screen a novel library of structurally diverse “fully functionalized fragments” and a second library of cysteine- and lysine-reactive compounds to identify allosteric inhibitors. Promising RDRP inhibitors will be tested for pan-coronavirus potential by testing against SARS-CoV, MERS-CoV and seasonal coronaviruses. Because RDRPs share conserved structures, broad-spectrum activity is possible and would be desirable. Therefore, we will evaluate top RDRP inhibitors from this Project and from Projects 5 and 6, which will focus on arboviruses and hemorrhagic fever viruses, in established biochemical RDRP assays for a panel of emerging viruses. These assays will provide mechanistic insight into the basis for broad-spectrum activity. In parallel, deep-sequencing approaches will be used to define MOA in infected cells and animals. For these studies, compound progression will be driven by iterative optimization by the Medicinal Chemistry Core (Core B), biological profiling against coronavirus replication assays in the HTS Core (Core A), ADME, PK and toxicology profiling from the Pharmacology Core (Core C), animal efficacy studies in collaboration with the Organoid and Animal Model Core (Core D), and structure-based drug discovery with the Structural and Modeling Core (Core E) to deliver a pan-active coronav...