High throughput screening of orthosteric inhibitors of flavivirus protease

翻译后摘要：许多黄病毒，如登革热病毒，寨卡病毒，西尼罗河病毒，黄热病病毒引起 重大人类疾病。然而，没有临床批准的抗病毒疗法可用于治疗 黄病毒感染因此，开发用于预防和治疗的疫苗和抗病毒剂， 黄病毒感染的治疗显然是公共卫生的优先事项。在感染宿主细胞的过程中， 病毒多蛋白由病毒基因组合成。这种多聚蛋白被切割成它的组分 通过宿主细胞蛋白酶和基因编码的双组分病毒蛋白酶的组合， NS 2B和NS 3。这种病毒蛋白酶的功能对于病毒组装和复制是不可或缺的。的 该提案的目标是开发高通量筛选策略，以识别和表征 正构抑制关键蛋白酶功能的化合物。在具体目标1和2中， 将开发和执行新的高通量筛选试验，能够筛选大的化学品， 文库来鉴定正构抑制剂。在具体目标3中，我们将评估所产生的 候选化合物具有阻断蛋白酶活性的能力。铅化合物对蛋白酶 将检测细胞毒性、细胞培养物中黄病毒滴度的降低和耐药变体。我们将 最终在活体动物中测试最好的铅化合物毒性和功效， 化合物.这项研究将产生关于黄病毒的蛋白质-酶相互作用的信息， 重要位点，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.