Project Summary – Core F The coronavirus disease (COVID-19) pandemic has led to devastating impacts on public health and the global economy. Without effective means of controlling the spread of severe acute respiratory virus 2 (SARS- CoV-2), the etiological agent of COVID-19, the number of new infections exploded in early 2020. In less than two years, over 230 million individuals have been infected, leading to more than 4.7 million deaths. The lack of effective antiviral countermeasures contributed greatly to the inability of public health organizations to halt the initial spread of SARS-CoV-2. While efficacious vaccines are currently available, key factors, such as vaccine hesitancy and the rise of SARS-CoV-2 variants capable of escaping natural and vaccine induced immunity, have limited our ability decrease the spread of new SARS-CoV-2 cases and end the current pandemic. The COVID- 19 pandemic has underscored the urgent need for effective antiviral countermeasures to limit the spread and severity of current and future viral threats. Orally-available direct-acting antivirals (DAA) would provide a valuable weapon in the war to halt COVID-19. The objective of the Antiviral Countermeasure Development Center (AC/DC) will be to identify and develop DAA therapeutics to mitigate current and future viral threats. To fulfill this objective and identify new DAA chemotypes, the AC/DC High-Throughput Screening (HTS) Core (Core F) will perform high-throughput screening campaigns against the various pathogens of concern investigated within AC/DC. Building on our expertise in drug discovery and previous success in identifying and developing orally available antivirals, such molnupiravir/EIDD-2801, EIDD-2749, GS-621763, GHP-88309, ERDRP-0519, and AVG-233, Core F will utilize a state-of-the-art HTS facility under BSL3 conditions to identify new antiviral hit candidates against various RNA viruses of pandemic potential under investigation within AC/DC. Core F will work individual AC/DC research projects to miniaturize, optimize, and validate assays for use in HTS campaigns. We will implement fully automated HTS protocols to screen our extensive library of small molecules to identify new hit scaffolds for further development within AC/DC (specific aim 1). In pilot studies with Research Projects 1 and 3, we have developed and optimized fully automated HTS assays using Cedar henipavirus (CedV) and SARS-CoV-2 luciferase reporter viruses. We then implemented these HTS protocols and completed successful screening campaigns against CedV and SARS-CoV-2, identifying several promising hit scaffolds for future mechanistic characterization and synthetic optimization. In addition to screening, Core F will generate bioactivity profiles of new analogs of hit candidates created by Core B to promote cross-core cooperation to significantly increase the speed of hit-to-lead development (specific aim 2). Core F will also acquire and perform HTS on new compound libraries, providing ...