PROJECT SUMMARY This R43 proposal answers the call of the RFA PA-22-176 for assay development and chemical probe screening by addressing the unmet need for development of antiviral treatments for Ebola patients through an innovative high-content small molecule screen for antagonists of the Ebola VP40 matrix protein. There is a need for a fast-acting therapy that is independent of the immune system and targets essential viral proteins. Such a novel therapeutic is anticipated to enhance the survival probability for infected people in hot zones of an Ebola outbreak. We chose to target EBOV VP40 because it is absolutely required for EBOV particle assembly at the cell membrane, is capable of budding virus- like particles (VLPs) when expressed in isolation and VP40 protein-protein interaction domains have been structurally determined. Further guiding this application is our published pilot screen which established proof-of-concept by demonstrating the accessibility of Ebola VP40 protein-protein interactions to sangivamycin, a dual acting small molecule antagonist of both EBOV VP40 assembly of virions and the viral replication machinery. Given this success and due to anticipated complications with efficacy and MOA studies inherent to compounds with dual targets, our Specific Aims propose to screen a library of ~123,000 small molecule compounds to identify antagonists of VP40 accumulation at the cell membrane for VLP formation and release from cells through a fully automated, quantitative, and high-content assay. The assay has been vetted to quantify the effect of small molecules on the cellular distribution of a fluorescent VP40 expressed in 293T cells. Hits validated as dose-dependent by qHTS and displaying low cytotoxicity will be further prioritized based on their absolute requirement for VP40 in antiviral mechanism of action through counter screening with the VP40- independent minigenome assay. Lead compounds also will be prioritized by their favorable ADMET profiles. Our proposed critical path anticipates identifying 2-4 dose-dependent, VP40- selective antagonists with potent antiviral activity that display low cytotoxicity for future medicinal chemistry and preclinical development.