Development of Novel Small Molecule Flavivirus Inhibitors

DESCRIPTION (provided by applicant): We have identified TYT-1 as a novel sultam thiourea inhibitor of West Nile virus (WNV) replication. The compound has a 50% effective concentration (EC50) of 0.7 micromolar and a 50% cytotoxic concentration (CC50) of gt70 micromolar, yielding a therapeutic (selectivity) index of gt100. We have demonstrated that TYT-1 protects animals from the lethality of WNV infection, that the compound reduces viral RNA levels over a hundred-fold in infected cells, and that TYT-1 analogues inhibit replication of related flaviviruses such as the Japanese encephalitis virus (JEV) and the category A biodefense pathogen Dengue virus (DENV). The development of TYT-1-based antivirals will provide a new approach to the treatment of flavivirus infections, and will complement vaccine approaches. Because very few sultam thioureas have been described, our work also will establish a foundation for their efficient preparation, and for their use in biomedical applications. To achieve these goals, we propose phase I studies to perfect sultam thiourea synthesis methodology, to assess analogue inhibition efficacy, to characterize mechanisms of inhibition, and to evaluate compound toxicity and metabolism in animals. Our specific aims are as follows: 1.Synthesis and evaluation of sultam thiourea analogues: Methods for the synthesis of TYT-1 and analogues will be optimized, and compound cellular toxicities and antiviral effects against flavivirus strains will be quantitated. These studies will perfect pathways for analogue preparation, and determine chemical and viral requirements for inhibition. 2. Characterization of the mechanism of compound inhibition: The mechanism of TYT-1 inhibition of WNV will be characterized. Examination of compound effects on wild type infections and replicon expression will help delineate inhibitor activities, and assays on viral protein activities will help define inhibition mechanisms. Analysis of putative drug resistant mutants will help assess whether resistance is acquired at a fitness cost to the virus, and will identify viral genes targeted by inhibitors. Our results will establish a mode of antiviral action, and ways to optimize inhibitor activities. 3. Examination of TYT-1 toxicity and metabolism: To examine the safety of sultam thioureas, toxicology studies on TYT-1 will be performed in mice. To gain an understanding of how these unique compounds are metabolized, pharmacokinetic studies will be performed in vitro and in vivo. These studies will serve as an essential basis for development of sultam thioureas as antivirals, and for other possible therapeutic purposes investigations are designed to develop new antivirals against the human pathogens West Nile virus, Japanese encephalitis virus, and Dengue virus. PUBLIC HEALTH RELEVANCE: We have identified a novel class of flavivirus inhibitor, representatives of which inhibit West Nile virus (WNV) and Japanese encephalitis virus (JEV) replication in cell culture, and protect animals from WNV lethality. We propose to perfect compound synthesis methodology, to assess analogue inhibition efficacy against flavivirus strains, to characterize mechanisms of inhibition, and to evaluate compound toxicity and metabolism in animals. Our results will lead to the development of new antivirals, and an understanding of how they work.