Abstract: Flaviviruses are primarily insect-borne, associated with global morbidity and mortality, and found on every inhabited continent. Unfortunately, current therapeutic options for treating diseases associated with these viruses are limited. All flaviviruses encode methyltransferases (MTases)—flaviviral NS5 for both N-7 and 2'-O methylations of viral genomic RNA. The N-7 MTase function is essential for replication of the viral RNA genome, whereas 2'-O MTase function is required for the virus to evade the host innate immune response. These activities are conserved among the flaviviruses. For this project, our collaborative team will optimize the current lead compounds, perform high throughput screening (HTS) to identify additional lead candidates, chemically optimize the lead candidates, and define structure activity relationships. Optimizing current lead compounds using cutting- edge medicinal chemistry, the team will perform a large scale HTS campaign using innovative fluorescence chemical probes to identify additional small molecule inhibitors of flavivirus RNA capping MTases. We will perform an in-depth investigation of the model of action and antiviral efficacy using in vitro biochemistry, structural biology, virology, in vivo pharmacokinetics, and in vivo animal models, which will allow the development of novel, effective, broad-spectrum, and druglike therapeutic agents against both flaviviruses. Preliminary progress has been made in the identification of initial lead inhibitors of these MTases, demonstrating low nanomolar antiviral activity. We will advance these compounds to further develop potent antiviral compounds while conducting large- scale screening in parallel for additional structural scaffold discoveries. Complementary expertise among our investigators will synergize and expedite the progress of this research. Our collaborative objective is to provide first-in-class drug candidates for the treatment or prevention of these viral infections.