High throughput screening of orthosteric inhibitors of flavivirus protease

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都能引起 重大的人类疾病。然而，目前还没有临床批准的抗病毒疗法可用于治疗 黄病毒感染。因此，预防和预防疾病的疫苗和抗病毒药物的发展 治疗黄病毒感染是一个明确的公共卫生优先事项。在宿主细胞感染期间，单个 病毒多蛋白是由病毒基因组合成的。这种多聚蛋白被裂解成其组成部分。 由宿主细胞蛋白酶和双组分病毒蛋白酶组合而成的蛋白质，编码在基因中 NS2B和NS3。这种病毒蛋白水解酶的功能对于病毒组装和复制是不可或缺的。这个 该提案的目标是开发高通量筛查策略，以识别和表征 以邻位构型方式抑制关键的蛋白酶功能的化合物。在具体目标1和2中，我们 将开发和执行能够筛选大型化学品的新型高通量筛选分析 用于鉴定正构体抑制剂的文库。在具体目标3中，我们将评估由此产生的 候选化合物的能力，以阻断蛋白酶活性。抗蛋白酶的先导化合物 将进行细胞毒性、细胞培养中黄病毒滴度的降低和抗药性变异的测试。我们会 最终测试最好的铅化合物在活体动物中的毒性和效力，以获得最有效的 化合物。这项研究将在结构上产生关于黄病毒抑制剂-酶相互作用的信息 重要的位点，并可能导致新的黄病毒抑制剂类别。

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. During 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 goal of this proposal is to develop high throughput screening strategies to identify and characterize compounds that orthosterically inhibit the function of the critical protease. In Specific Aims 1 and 2 we will develop and perform novel high-throughput screening assays capable of screening large chemical libraries to identify orthosteric inhibitors. In Specific Aim 3 we will evaluate the efficacy of the resulting candidate compounds for their capacity to block protease activity. Lead compounds against the protease will be tested for cellular toxicity, reduction of flavivirus titer in cell culture, and resistant variants. We will ultimately test the best lead compound toxicity and efficacy in live animals for the most potent compounds. This study will generate information on flavivirus inhibitor-enzyme interaction at structurally significant sites and potentially lead to novel classes of flaviviral inhibitors.