ABSTRACT Alphaviruses (Togaviridae genus) several medically important viruses, including chikungunya virus (CHIKV), Venezuelan equine encephalitis virus (VEEV), and Eastern equine encephalitis virus (EEEV). These re- emerging viruses are categorized as Biodefense Category B and C priority pathogens due to their pandemic risk or potential as bioterror threats. Despite the threat posed by these viruses, there are no approved antivirals for treating any alphavirus infection. Therefore, our highly experienced and interactive team will leverage our world class expertise in alphavirus biology, structural biology, high throughput screening, medicinal chemistry, and drug development to generate small molecule inhibitors targeting conserved viral enzymes with the goal of producing new, broad spectrum, direct acting anti-alphaviral drugs. The central premise of this effort is that small molecule-based inhibitors of essential viral proteins across multiple members of the Alphavirus family can be developed employing a platform that (a) integrates target identification and validation (Aim 1), (b) confirms and optimizes cellular antiviral activity and SAR against the given target (Aim 2a), (c) establishes and improves in vitro ADME and in vivo pharmacokinetics (Aim 2b), and progresses leads into in vivo efficacy and toxicity animal models (Aim 3). Collaboration with the Discovery Core B will functionally validate structurally conserved compound binding pockets in alphaviruses enzymes (RNA dependent RNA polymerase (RDRP; nsP4), the RNA helicase (nsP2), and the nsP2 protease). These analyses will inform the selection of viral targets for hit discovery using a combination of physical and in silico screening in coordination with Core B. Hit compounds that show robust target engagement will be optimized in collaboration with the Medicinal Chemistry Core (Core C) for target inhibition, stability, selectivity, solubility, permeability, pharmacokinetics, and antiviral activity in vitro against multiple alphaviruses (CHIKV, EEEV, and VEEV). As our goal is to identify broadly active compounds, lead compounds will also be tested agAainst additional alphaviruses (e.g. RRV, MAYV, ONNV, WEEV, and SINV) and other viruses in our program, including coronaviruses (Proj. 1 and 2), flaviviruses (Proj. 4), and filoviruses (Proj. 5). We will also collaborate with Enzymology Core D to identify their antiviral mechanism of action. Lead compounds with optimal in vivo biodistribution and stability will be tested for in vivo antiviral efficacy using our established models of alphavirus-induced arthritis (CHIKV) or encephalitis (VEEV or EEEV). Candidates with potent in vivo antiviral efficacy will be further optimized for formulation, toxicity, and advanced PK/PD.