The HIV pandemic has grown to ~40 million individuals world-wide. Although Highly Active Anti- Retroviral Therapy (HAART) has been transformative for millions of people, less than half of those infected are on therapy. And while the current medications are typically well-tolerated and efficacious, because of both acute and chronic drug toxicities, drug-drug interactions, and virus resistance, there is always room for novel HIV therapeutics, especially for those considered first-in-class. Rev is an essential regulatory protein of the virus, which multimerizes on the Rev-response element or RRE to export intron-containing HIV mRNAs from the nucleus to the cytoplasm. Although there are some compounds in clinical trials that purportedly interfere with Rev function, there are no FDA-approved agents that specifically target Rev’s known mechanism of action. Notably, an anti-Rev compound should reduce the amount of both cytoplasmic and virion-associated unspliced or genomic RNA and also plasma viral loads in the absence of viral replication, an activity that none of the currently FDA-approved drugs possesses. We have developed a cell-based firefly luciferase (FFLUC) complementation system that quantifies Rev-Rev interaction but then transitioned over to a cell-free one that has an excellent Z’ (Z factor) of 0.64 in 384-well format. Here we first wish to further optimize the assay to then permit testing of compounds in 384-well format, in collaboration with Yale Center for Molecular Discovery (YCMD), which has multiple collections of compounds and requisite instrumentation to allow fully automated testing of ~125,000 small molecules. These compounds will initially be screened at a single concentration, and positives will be retested at a range of concentrations, in replicates. Secondary assays for specificity of inhibition of Rev-Rev interaction include non-inhibition of FFLUC activity and transcription, non-inhibition of unrelated protein-protein interaction, and inhibition of Rev function using a variety of assays, including cell-free multimerization, production of Gag, and both X4 and R5 HIV replication in vitro. Therapeutic index (TI) of any hits will be determined using a cell-based assay. Based upon these results, the physicochemical properties of any hits will be assessed and in silico hit expansion along with any necessary synthesis will be achieved by the commercial entity Life Chemicals. These select hit compounds will be retested in the above assays in order to identify even more potent and active drug-like compounds. At the end of two years it is hoped that a number of advanced hit and close-to-lead compounds will have been identified that have high TI (>100) and specifically prevent Rev-Rev interaction and thus interfere with known Rev function, which will allow future medicinal chemistry testing and iterative in vitro and in vivo studies to identify lead compounds of enhanced potency, activity, and specificity. Thus, a long-term goal of this...