The broad, long-term objective of this proposal is to lay the foundation for a collaborative structure aided drug design effort aimed at developing lead compounds for flaviviral drug development. Flaviviral infections, including those caused by West Nile virus and Yellow Fever virus, are of enormous worldwide and domestic importance. West Nile virus and Yellow Fever virus are both of significant concern as potential bioterrorism agents and are listed by NIAID as Category C and Category B agents respectively. There are no currently effective medications for the treatment of these flavivirus infections. The main aim of this initial proposal is to clone and express sufficient quantities of NS3A, NS2B and NS3B from West Nile virus and Yellow Fever virus for crystallization trials. These proteins represent the viral protease, its activating peptide and the viral helicase. Following expression and purification, crystallization of these constructs will be attempted. To date, several collaborators have joined the project. Genomic DNA has been used to successfully construct clones of several of these proteins. Expression trials are underway with positive preliminary results. More work is needed, however, in the optimization of expression and in purification before crystallization trials can take place. It is planned that in a continuation of this work these structures will be solved and used as templates for a comprehesive structure aided drug design effort. In this plan, promising lead compounds will be synthesized and tested against their target molecules in kinetic assays and then against virus grown in cell culture. Inhibitors developed in this way could serve as lead compounds for the development of effective therapy that would target West Nile and Yellow Fever virus. In parallel, structure-function analyses are planned for the viral protease, the viral helicase, the intact bifunctional protein and the protease with activating peptide bound. It is anticipated that these experiments will aid in understanding flaviviral pathogenesis.
