Coronavirus Disease 2019 (COVID-19) represents an ongoing pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and a current death toll of about 5 million. There are only limited antiviral treatment options for patients who contracted the disease. In March 2020, we initiated collaborative small molecule drug development including the laboratories of Dr. Tuschl (high-throughput screening (HTS) assay development), Dr. Glickman (HTS operations), Dr. Patel (structural biology), Dr. Rice (virology), and the Tri- Institutional Therapeutics Discovery Institute (medicinal chemistry) focusing on the two SARS-CoV-2 proteases, NSP3/PLpro and NSP5/3CLpro, required for viral polyprotein processing, the cap N7-G methyltransferase (MTase) NSP14, required for efficient viral protein synthesis and escape from host cell innate immune response, and the RNA helicase NSP13 important for viral replication. We developed robust, HTS fluorescent-substrate-based assays for monitoring PLpro, 3CLpro, and NSP13 activities as well as a luminescence-based assay for monitoring NSP14 N7-G-MTase activity. By HTS of the diverse library of 430,000 compounds available at Rockefeller University (RU), we identified hits in all assays, validated hits, and pursued the most promising non-covalent hits in medicinal chemistry programs. Our lead compounds for NSP14 and PLpro show IC50 values of 2 nM and 100 nM, respectively, and cell-based inhibition of SARS-CoV-2 replication with EC50 values of 80 nM and 7 µM, without cellular toxicity. Here, we propose further lead optimization to improve inhibitor potency and drug-like properties, as well as apply our HTS expertise to target proteases, MTases, and helicases of other RNA viruses with pandemic potential, including MERS, flavi- and alphaviruses. Our specific aims are: (1) Development of SARS-CoV-2 NSP14 and PLpro clinical candidates. Guided by structural insights from inhibitor-PLpro and NSP14 complexes, we will further optimize inhibitor potency and drug-like characteristics with industry-quality medicinal chemistry support from TDI and this program, and the goal to develop 3-5 advanced leads that are subjected to in vitro and in vivo DMPK/ADME-Tox studies utilizing all scientific cores designated for hit-to-lead and lead optimization (2) Develop antiviral lead series against MTases, proteases and helicases of select members of the Coronaviridae, Flaviviridae and Togaviridae families. We will adapt and develop HTS assays using recombinant proteins and identify covalent and non-covalent inhibitors in the RU drug collection and accompany the medicinal chemistry lead development to provide 3-5 leads per target for comprehensive DMPK/ADME-Tox analysis. (3) Monitor the selectivity of novel small molecule antivirals in biochemical assays (using increasingly distant viral and human recombinant proteins) and cell-based viral propagation, as well as resistance mutation identification. We will express recombinant protein...