Development of Novel Small Molecule Flavivirus Inhibitors

描述（由申请人提供）：我们已经鉴定TYT-1为西尼罗河病毒（WNV）复制的新型磺内酰胺硫脲抑制剂。该化合物具有0.7微摩尔的50%有效浓度（EC 50）和>70微摩尔的50%细胞毒性浓度（CC 50），产生>100的治疗（选择性）指数。我们已经证明，TYT-1保护动物免受WNV感染的致死性，该化合物使感染细胞中的病毒RNA水平降低超过一百倍，并且TYT-1类似物抑制相关黄病毒如日本脑炎病毒（JEV）和A类生物防御病原体登革热病毒（DENV）的复制。基于TYT-1的抗病毒药物的开发将为黄病毒感染的治疗提供新的方法，并将补充疫苗方法。由于很少有磺内酰胺硫脲已被描述，我们的工作也将建立一个基础，其有效的制备，并为他们在生物医学应用。为了实现这些目标，我们提出了I期研究，以完善磺内酰胺硫脲合成方法，以评估类似物的抑制效果，表征抑制机制，并评估化合物的毒性和代谢在动物中。具体研究内容如下：1.磺内酰胺硫脲类似物的合成与评价：优化TYT-1及其类似物的合成方法，并对化合物的细胞毒性和抗黄病毒作用进行定量研究。这些研究将完善类似物制备的途径，并确定抑制的化学和病毒要求。2.化合物抑制机制的表征：将表征TYT-1抑制WNV的机制。化合物对野生型感染和复制子表达的影响的检查将有助于描述抑制剂活性，并且对病毒蛋白活性的测定将有助于定义抑制机制。对假定的耐药突变体的分析将有助于评估耐药性是否是以病毒的适应性为代价获得的，并将鉴定抑制剂靶向的病毒基因。我们的研究结果将建立一种抗病毒作用的模式，以及优化抑制剂活性的方法。3. TYT-1毒性和代谢的检查：为了检查磺内酰胺硫脲的安全性，将在小鼠中进行TYT-1的毒理学研究。为了了解这些独特化合物的代谢方式，将在体外和体内进行药代动力学研究。这些研究将作为开发磺内酰胺硫脲作为抗病毒药物的重要基础，并为其他可能的治疗目的进行研究，以开发针对人类病原体西尼罗河病毒、日本脑炎病毒和登革热病毒的新的抗病毒药物。公共卫生相关性：我们已经确定了一类新的黄病毒抑制剂，其代表抑制西尼罗河病毒（WNV）和日本脑炎病毒（JEV）在细胞培养中的复制，并保护动物免受WNV致死。我们建议完善化合物的合成方法，以评估类似物对黄病毒株的抑制效果，表征抑制机制，并评估化合物在动物中的毒性和代谢。我们的研究结果将导致新的抗病毒药物的开发，并了解它们是如何工作的。

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 >70 micromolar, yielding a therapeutic (selectivity) index of >100. 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.