High throughput screening of orthosteric inhibitors of flavivirus protease

项目概要/摘要： 许多黄病毒，如登革热病毒、寨卡病毒、西尼罗河病毒和黄热病病毒 导致严重的人类疾病。然而，没有临床批准的抗病毒治疗是可用的 用于治疗黄病毒感染。因此，疫苗和抗病毒剂的开发 预防和治疗黄病毒感染显然是公共卫生的优先事项。很长的- 本提案的范围目标是通过开发一个新系统来满足这一需求， 筛选并验证了一类潜在的黄病毒抑制剂，并成功鉴定和 分析有望破坏病毒存活的候选化合物。 在宿主细胞的黄病毒感染期间，从病毒多聚蛋白合成单个病毒多聚蛋白。 基因组这种多聚蛋白被宿主细胞的组合切割成其组成蛋白。 蛋白酶和双组分病毒蛋白酶，其在基因NS 2B和NS 3中编码。功能 这种病毒蛋白酶是病毒组装和复制所不可缺少的。这一目标 项目是开发创新的高通量筛选策略，以确定和 表征正构抑制关键蛋白酶NS 2B-的功能的化合物， NS3。 为了实现项目目标，在具体目标1中，我们将开发和执行新的高- 通量筛选一级、二级和三级试验，能够筛选大的 化学文库来鉴定正构抑制剂。在具体目标2中，在优化这些之后， 我们将进行大规模的HTS，以筛选超过40万种化合物的化合物库。 对于潜在的蛋白酶抑制剂候选物，采用五个级别的筛选，以完全消除 假阳性在具体目标3中，我们将评估最终候选人的有效性 化合物阻断蛋白酶活性的能力，并确定其作用模式。 将测试针对蛋白酶的先导化合物的细胞毒性、黄病毒减少 细胞培养物中的滴度、抗性变体和有限的结构-活性关系。最终肯定我们会 在活体动物中测试最有效化合物的毒性、药代动力学和功效。 我们预计这项研究的结果将是产生关键的新信息， 在结构上重要的位点上的黄病毒介导物-酶相互作用。这就创造了 用于鉴定新的黄病毒抑制剂类别，适用于全球公众 健康需要新的抗病毒药物来对抗黄病毒。

Project Summary/Abstract: Many flaviviruses such as Dengue virus, Zika virus, West Nile virus, and Yellow Fever virus cause significant human diseases. However, no clinically approved antiviral therapy is available for treatment of flavivirus infections. Therefore, the development of vaccines and antiviral agents for prevention and treatment of flavivirus infections is a clear public health priority. The long- range goal of this proposal is to address this need by developing a new system to identify, screen and validate a class of potential flavivirus inhibitors, and successfully identify and analyze candidate compounds that show promise in disrupting viral survival. During flaviviral infection of the host cell, a single viral polyprotein is synthesized from the viral genome. This polyprotein is cleaved into its component proteins by a combination of host cell proteases and a two-component viral protease, encoded in genes NS2B and NS3. The function of this viral protease is indispensable for virus assembly and replication. The objectives of this project are to develop innovative high throughput screening strategies to identify and characterize compounds that orthosterically inhibit the function of the critical protease NS2B- NS3. To achieve project objectives, in Specific Aim 1, we will develop and perform novel high- throughput screening primary, secondary, and tertiary assays capable of screening large chemical libraries to identify orthosteric inhibitors. In Specific Aim 2, after optimizing these assays, we will perform large-scale HTS to screen a compound library of >400,000 compounds for potential protease inhibitor candidates, employing five levels of screening to fully eliminate false positives. In Specific Aim 3, we will evaluate the efficacy of the resulting candidate compounds for their capacity to block protease activity, and determined their modes of actions. Lead compounds against the protease will be tested for cellular toxicity, reduction of flavivirus titer in cell culture, resistant variants, and limited structure-activity relationship. We will ultimately test the toxicity, pharmacokinetics, and efficacy in live animals for the most potent compounds. We anticipate the outcome of this study will be the generation of crucial new information on flavivirus inhibitor-enzyme interaction at structurally significant sites. This creates the potential for identifying novel classes of flaviviral inhibitors, suitable for addressing the global public health need for new antiviral agents to fight flaviviruses.