Structure-based Design of Zika Virus Inhibitors Targeting Envelope Glycoprotein (E)

项目摘要/摘要 由蚊子传播的寨卡病毒(ZIKV)，世卫组织已宣布为“全球紧急状态”，正在“蔓延” 在美洲“爆炸性地”，到2016年底可能有多达400万人感染。 从全球健康的角度来看，严重的小头畸形和相关的出生缺陷由感染引起 治疗寨卡病毒派拉蒙的有效药物的开发。这项提案的目标，作为回应写成 最近题为“寨卡病毒(ZIKV)并发症快速评估(R21)”(PAR-16-106)的呼吁是 识别抑制ZIKV进入和复制的小分子药物先导。我们的总目标是发展 能够阻断涉及ZIKV的膜融合所需的关键构象变化的抑制剂 包膜糖蛋白(E)。我们假设，基于结构的计算筛选使用的知识 随后进行实验鉴定的病毒接口将比随机筛查更成功。这个 提出这项工作的理由是，我们的团队有能力迅速利用之前获得的经验来确定目标 HIV和埃博拉的相似事件以及构建用于ZIKV筛查的稳健结构模型的能力 基于登革热病毒糖蛋白E的高度同源性(74%的相似性，54%的识别)， 晶体结构信息是可用的。该项目被安排为两个具体目标，根据 每个PI实验室的专业知识：(目标1)识别和利用ZIKV糖蛋白E中的靶向事件。 这一目标，我们将计算目标有利的蛋白质界面，以扰乱膜融合使用高- 吞吐量-对大约500万种商业可用化合物进行虚拟筛选。三个不同的E蛋白位点 将是有针对性的，基于从登革热病毒相关蛋白质结构衍生的ZIKV模型 哈里森和他的同事，包括前面描述的β-OG口袋(融合前状态)和我们的两个口袋 根据最近报道的一种晚期融合中间体的分析确定了。筛选将雇用 原子级足迹识别最有希望的实验得分最高的化合物(300-400) 人物刻画。(目标#2)通过量化通过计算识别的小分子探针 病毒入侵中断。得分最高的化合物(300-400)预计会通过破坏膜来阻止进入 融合将在Vero细胞和人类成纤维细胞(HFF-1)中进行测试。最初的筛选将在高位进行- 吞吐量非复制伪型病毒系统(含荧光素酶的HIV颗粒中的ZIKV M/E蛋白 记者)。阳性结果将通过活的ZIKV感染性检测(空斑检测和终点稀释)进行检测 化验)。实验测试将允许对通过虚拟筛选识别的分子进行优先排序。范围更广 这项工作的影响包括增加了对如何针对包括登革热在内的相关黄病毒的理解， 黄热病、壁虱传播脑炎和西尼罗河。

PROJECT SUMMARY/ABSTRACT The mosquito-borne Zika virus (ZIKV), which the WHO has declared a "global emergency," is "spreading explosively in the Americas" and as many as 4 million people could become infected by the end of 2016. From a global health perspective, the severe microcephaly and associated birth defects arising from infection make development of effective therapeutic agents for ZIKV paramount. The goal of this proposal, written in response to the recent call titled "Rapid Assessment of Zika Virus (ZIKV) Complications (R21)" (PAR-16-106) is to identify small molecule drug-leads that inhibit ZIKV entry and replication. Our overall objective is to develop inhibitors capable of blocking key conformational changes required for membrane fusion that involve the ZIKV envelope glycoprotein (E). We hypothesize that structure-based computational screening using knowledge of viral interfaces followed by experimental characterization will be more successful than random screening. The rationale for proposing the work, is the ability of our team to rapidly leverage prior experience gained targeting analogous events in HIV and Ebola and the ability to construct robust structural models for ZIKV screening based on the high homology of dengue virus glycoprotein E (74% similarity, 54% identify) for which abundant crystallographic structural information is available. The project is arranged as two Specific Aims according to the expertise of each PI's lab: (Aim #1) Identify and exploit targetable events in ZIKV glycoprotein E. Under this aim we will computationally target favorable protein interfaces to disrupt membrane fusion using high- throughput-virtual screening of ca. 5 million commercially available compounds. Three distinct E protein sites will be targeted, based on ZIKV models derived from related protein structures of dengue virus reported by Harrison and coworkers, include the previously described β-OG pocket (pre-fusion state) and two pockets we have identified based on analysis of a recently reported late-stage fusion intermediate. Screening will employ atomic-level footprints to identify the most promising top-scoring compounds (300-400) for experimental characterization. (Aim #2) Characterize small molecule probes identified computationally by quantifying the disruption of viral entry. Top-scoring compounds (300-400) predicted to arrest entry by disrupting membrane fusion will be tested in Vero cells and human fibroblasts (HFF-1). Initial screens will be performed in a high- throughput non-replicating pseudotyped virus system (ZIKV M/E protein in an HIV particle with a luciferase reporter). Positive hits will be tested with live ZIKV infectivity assays (plaque assay and endpoint dilution assay). Experimental testing will allow prioritization of the molecules identified by virtual screening. Broader impacts of the work include increased understanding of how to target related flaviviruses including dengue, yellow fever, tick-borne encephalitis, and West Nile.