The significant burden that results from dengue virus infection combined with the absence of effective vaccines or drugs makes the development of novel therapeutics a high priority. We have previously identified disubstituted pyrimidines that inhibit dengue virus entry by binding to the pre-fusion E dimer on the virion surface, perhaps in the beta-octoglucoside binding pocket previously identified by Harrison and colleagues. Here, we propose to develop assays to identify other ligands of this site that can inhibit dengue virus entry. The subsequent high-throughput screening (HTS) effort and downstream activities are needed to interrogate the chemical diversity that can bind in this pocket, to generate additional lead compounds, and to elucidate the structural and mechanistic correlates of potent anti-dengue activity. In Aim 1, we will use these compounds to develop a high-throughput fluorescence polarization assay to identify ligands of the dengue virus envelope protein that inhibit dengue virus infection. In Aim 2, we propose to develop an assay to monitor bulk fusion of viral particles in vitro. In Aim 3, we will validate our assay pipeline by performing a pilot screen. We will use assays developed in Aims 1 and 2 as well as other assays previously developed in our laboratory to characterize the mechanisms of action of compounds with activity against dengue virus. At the conclusion of this proposal, we anticipate having robust assays that are ready for high-throughput screening. The goal of these downstream HTS efforts is the development of molecular probes to study the structural and biochemical mechanisms of dengue entry, to validate the beta-octoglucoside pocket as a molecular target for inhibition of dengue entry, and to define the SAR governing the anti-dengue activity of ligands of the beta-octoglucoside pocket. In the longer term, this work will also contribute to our future goals of developing small molecule inhibitors of DV entry as preclinical candidates for anti-dengue drug discovery efforts.