Project Summary/Abstract:Many flaviviruses such as Dengue virus Zika virus West Nile virus and Yellow Fever viruscause significant human diseases. However no clinically approved antiviral therapy is availablefor treatment of flavivirus infections. Therefore the development of vaccines and antiviral agentsfor prevention and treatment of flavivirus infections is a clear public health priority. The long-range goal of this proposal is to address this need by developing a new system to identifyscreen and validate a class of potential flavivirus inhibitors and successfully identify andanalyze candidate compounds that show promise in disrupting viral survival.During flaviviral infection of the host cell a single viral polyprotein is synthesized from the viralgenome. This polyprotein is cleaved into its component proteins by a combination of host cellproteases and a two-component viral protease encoded in genes NS2B and NS3. The functionof this viral protease is indispensable for virus assembly and replication. The objectives of thisproject are to develop innovative high throughput screening strategies to identify andcharacterize compounds that orthosterically inhibit the function of the critical protease NS2B-NS3.To achieve project objectives in Specific Aim 1 we will develop and perform novel high-throughput screening primary secondary and tertiary assays capable of screening largechemical libraries to identify orthosteric inhibitors. In Specific Aim 2 after optimizing theseassays we will perform large-scale HTS to screen a compound library of >400000 compoundsfor potential protease inhibitor candidates employing five levels of screening to fully eliminatefalse positives. In Specific Aim 3 we will evaluate the efficacy of the resulting candidatecompounds for their capacity to block protease activity and determined their modes of actions.Lead compounds against the protease will be tested for cellular toxicity reduction of flavivirustiter in cell culture resistant variants and limited structure-activity relationship. We will ultimatelytest the toxicity pharmacokinetics and efficacy in live animals for the most potent compounds.We anticipate the outcome of this study will be the generation of crucial new information onflavivirus inhibitor-enzyme interaction at structurally significant sites. This creates the potentialfor identifying novel classes of flaviviral inhibitors suitable for addressing the global publichealth need for new antiviral agents to fight flaviviruses.