Flaviviruses are the causative agents of a number of diseases of public health importance including West Nile encephalitis, yellow fever, dengue fever and Japanese encephalitis. However, current treatment options for these diseases are limited and there is a real need for new antiviral agents with specific activity against these viruses. The long-term objectives of this project are to meet this need by identifying and characterizing new small molecule compounds with broad spectrum activity against medically important f1aviviruses. The project that we propose has three specific aims. Specific Aim 1: High Throughput Screening Identification of Novel Lead Compounds. Using a West Nile virus (WNV) virus-like particle (VLP) assay that we have developed and, which is suitable for use under low bio-containment conditions we will conduct high-throughput screening of a large library of compounds at the National Screening Laboratory for the Regional Centers of Excellence. The data generated during screening will be analyzed and the most promising antiviral compounds identified. The activity of the selected compounds against WNV will be confirmed using live virus assays and, their broad spectrum anti-flavivirus activity determined in live virus assays with dengue virus and yellow fever virus. Specific Aim 2: Medicinal Chemistry Modification and Iterative in vitro Antiviral Testing. Based on their activity against all three flaviviruses, toxicity profiles and potential for chemical modification, three compounds will be identified as leads and will undergo a series of medicinal chemical modifications in the Parallel Synthesis and Medicinal Chemistry facility at UTMB. Each round of chemical modifications will involve the synthesis of a small number of compounds which will then undergo rapid in vitro toxicity and antiviral activity testing in live virus assays. This process will result in several rounds of controlled iterative synthesis and evaluation that we expect will lead to the identification of more potent antiviral compounds. Specific Aim 3: In Vivo Evaluation of Activity in a Small Animal Model of WNV Encephalitis In this aim the two most promising compounds identified during the chemical modification studies will be evaluated for activity in vivo using a mouse model of WNV encephalitis. Thus by the end of the project period we expect to have identified a number of new compounds with broad spectrum activity against medically important flaviviruses and to have undertaken preliminary studies to evaluate their potential therapeutic utility in a well defined animal model with virologic and pathologic endpoints that are relevant to those seen in the clinical situation.