High throughput screening of orthosteric inhibitors of flavivirus protease

项目摘要/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 会导致重大的人类疾病。然而，目前还没有临床批准的抗病毒疗法。 用于治疗黄病毒感染。因此，疫苗和抗病毒药物的发展 预防和治疗黄病毒感染是一个明确的公共卫生优先事项。长的- 这项提议的范围目标是通过开发一种新的系统来识别、 筛选和验证一类潜在的黄病毒抑制剂，并成功识别和 分析在破坏病毒生存方面有希望的候选化合物。 在黄病毒感染宿主细胞期间，病毒合成一种单一的病毒多蛋白。 基因组。这种多聚蛋白被宿主细胞组合裂解成其组成蛋白 NS2B和NS3基因中编码的一种双组分病毒蛋白水解酶。该功能 这种病毒蛋白水解酶对于病毒组装和复制是必不可少的。这样做的目的是 该项目旨在开发创新的高通量筛选策略，以识别和 表征以邻位构象方式抑制关键蛋白酶NS2B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性能 吞吐量筛查一次、二次和三次化验能够筛查大 用于鉴定邻位构型抑制剂的化学文库。在具体目标2中，在优化这些之后 在化验方面，我们将进行大规模的HTS以筛选包含400,000个化合物的化合物库 对于潜在的酶抑制剂候选者，采用五个级别的筛选来完全消除 假阳性。在具体目标3中，我们将评估由此产生的候选人的效果 测定化合物阻断蛋白酶活性的能力，并确定它们的作用模式。 针对蛋白酶的先导化合物将进行细胞毒性测试，减少黄病毒 细胞培养中的滴度、耐药变异体和有限的构效关系。我们最终会 测试最有效的化合物在活体动物中的毒性、药代动力学和疗效。 我们预计这项研究的结果将是在以下方面产生关键的新信息 黄病毒抑制物-结构上有意义的部位的酶相互作用。这创造了潜在的 用于识别适合全球公众使用的新型黄病毒抑制剂 卫生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.