Project Abstract The world is currently under siege by the spread of a novel positive-strand RNA virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). All positive-strand RNA viruses require their virus-encoded RNA- dependent RNA polymerase (RdRp) to synthesize RNA to serve as either mRNA for production of viral proteins or as genomic RNA for progeny virions. Viral polymerases are well-established targets for antiviral therapy with clear potential for broad-spectrum activity. The RdRp from hepatitis C virus (HCV), another positive-strand RNA virus, is a key target of the antiviral cocktails developed to cure HCV infection. The most efficacious inhibitors of viral polymerases with broad-spectrum activity are nucleoside or nucleotide analogs. This class of compounds binds to the nucleotide substrate-binding pocket of the viral polymerase and is incorporated into nascent RNA, leading to termination, mutagenesis, or recombination, all of which can be lethal to the virus. Given the conserved structural and sequence requirements of the nucleotide substrate-binding pocket of viral RdRps, in general, it is not at all surprising that a nucleotide analog developed to treat one virus would exhibit antiviral activity against another. Potency in vitro and/or in vivo can differ. The problem in vivo often relates to failure of compound activation and/or accumulation in the relevant tissue/organ. A coronavirus-specific complication for use of nucleotide analogs is the presence of a 3’→5’ exoribonuclease activity capable of excising not only mismatched basepairs but also some antiviral nucleotides. The primary motivation of this project, in alignment with the Stanford AViDD Center (SyneRx), is development of safe, effective nucleoside/nucleotide candidates for use in the treatment and/or prevention of SARS CoV-2 infection and perhaps other viral pathogens of the respiratory system as well. A unique asset of our program is a panel of nucleotide analogs with activity against the SARS- CoV-2 RdRp discovered by screening a library owned by Riboscience LLC. To increase the potency of these candidates, we will antagonize excision by inhibiting the exoribonuclease. To achieve these goals we will pursue the following specific aims: (1) Identify nucleotide analogs for development. (2) Develop strategies to antagonize the exoribonuclease. (3) Synthesize and characterize ProTides and second-generation nucleotide analogs for use as anti-coronavirus therapeutics. (4) Biological analysis of polymerase and exoribonuclease inhibitors. Together, these studies have the potential to discover safe, efficacious nucleotide analogs for use in the treatment of SARS-CoV-2 infection and perhaps other viral infections of the respiratory system.