Development of Novel Small Molecule Flavivirus Inhibitors

描述(申请人提供)：我们已经确定TYT-1是一种新型的西尼罗河病毒(WNV)复制的磺胺硫脲抑制剂。该化合物的50%有效浓度(EC50)为0.7微摩尔，50%细胞毒性浓度(CC50)为&gt；70微摩尔，治疗(选择性)指数为&gt；100。我们已经证明，TYT-1可以保护动物免受西尼罗河病毒的致命感染，该化合物可以将感染细胞中的病毒RNA水平降低100倍以上，并且TYT-1类似物可以抑制相关黄病毒的复制，如日本脑炎病毒(JEV)和A类生物防御病原体登革病毒(DENV)。基于TYT-1的抗病毒药物的开发将为治疗黄病毒感染提供一种新的方法，并将补充疫苗方法。由于所描述的硫脲类化合物很少，我们的工作也将为它们的有效制备和在生物医学应用中的应用奠定基础。为了实现这些目标，我们建议进行第一阶段的研究，以完善舒坦硫脲的合成方法，评估类似物的抑制效果，表征抑制机制，并评估化合物在动物体内的毒性和代谢。1.磺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，定量测定化合物对黄病毒株的细胞毒性和抗病毒作用。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.复合抑制机制的表征：将对TYT-1抑制西尼罗河病毒的机制进行表征。对野生型感染和复制子表达的复合效应的检测将有助于描述抑制物的活性，而对病毒蛋白活性的分析将有助于确定抑制机制。对假定的耐药突变的分析将有助于评估是否以适应病毒的成本获得抗药性，并将识别抑制剂靶向的病毒基因。我们的结果将建立一种抗病毒作用模式，以及优化抑制剂活性的方法。3.TYT-1毒性和代谢的检测：为了考察舒坦硫脲的安全性，对TYT-1进行了小鼠的毒理学研究。为了了解这些独特的化合物是如何代谢的，我们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舒坦硫脲作为抗病毒药物的重要基础，出于其他可能的治疗目的，研究旨在开发针对人类病原体西尼罗河病毒、乙型脑炎病毒和登革病毒的新抗病毒药物。公共卫生相关性：我们已经确定了一类新的黄病毒抑制剂，其代表物在细胞培养中抑制西尼罗河病毒(WNV)和乙型脑炎病毒(JEV)的复制，并保护动物免受西尼罗河病毒的致死。我们建议完善化合物合成方法学，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体内的毒性和代谢。我们的结果将导致新的抗病毒药物的开发，并了解它们的工作原理。

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.