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

针对包膜糖蛋白的寨卡病毒抑制剂的基于结构的设计 (E)

• 批准号: 9262681
• 负责人: Amy Lynn Jacobs
• 金额: $ 25.27万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-18 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9262681
• 关键词: Americas; Biological Assay; Biophysics; Botulinum Toxin Type A; Buffaloes; Cellular biology; Chimeric Proteins; Computer Assisted; Computers; Congenital Abnormality; Culicidae; Custom; Dengue; Dengue Virus; Development; Drug Design; E protein; Ebola virus; Emergency Situation; Event; Fibroblasts; Flavivirus; Glycoproteins; Goals; HIV; Health; Homology Modeling; Human; Infection; Knowledge; Life; Ligands; Luciferases; Maps; Membrane; Membrane Fusion; Methodology; Microcephaly; Modeling; Molecular; Outcome; Pharmaceutical Preparations; Plaque Assay; Procedures; Proteins; Protocols documentation; Publishing; Reporter; Reporting; Research; Resolution; Running; Site; Staging; Structural Models; Structure; System; Testing; Therapeutic Agents; Tick-Borne Encephalitis; Vero Cells; Viral; Virus; Virus Diseases; Virus Inhibitors; West Nile virus; Work; Writing; Yellow Fever; Zika Virus; analog; base; combat; design; drug discovery; experience; global health; inhibitor/antagonist; innovation; particle; protein structure; public health emergency; response; screening; small molecule; small molecule inhibitor; therapeutic development; virology; virtual; virus envelope

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.

项目概要/摘要 世界卫生组织宣布“全球紧急状态”的蚊媒寨卡病毒（ZIKV）正在“传播” “在美洲呈爆炸式增长”，到 2016 年底可能有多达 400 万人被感染。 从全球健康角度来看，感染引起的严重小头畸形和相关出生缺陷使得 开发针对 ZIKV 的有效治疗药物至关重要。该提案的目标是对此作出回应。 最近题为“寨卡病毒 (ZIKV) 并发症的快速评估 (R21)”(PAR-16-106) 的电话会议是 确定抑制 ZIKV 进入和复制的小分子药物先导物我们的总体目标是开发。 能够阻断涉及 ZIKV 的膜融合所需的关键构象变化的抑制剂 我们利用以下知识捕获了基于结构的计算筛选。 病毒界面随后进行实验表征将比随机筛选更成功。 提出这项工作的理由是我们的团队有能力快速利用先前获得的目标经验 HIV 和埃博拉病毒中的类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％相似性，54％同一性），其中丰富 该项目根据两个具体目标安排。 每个 PI 实验室的专业知识：（目标 #1）识别和利用 ZIKV 糖蛋白 E 中的目标事件。 为了实现这一目标，我们将通过计算瞄准有利的蛋白质界面，以使用高通量破坏膜融合。 大约 500 万种市售化合物的通量虚拟筛选。 将根据由登革热病毒相关蛋白质结构衍生的 ZIKV 模型来确定目标 Harrison 和同事，包括先前描述的 β-OG 口袋（融合前状态）和我们的两个口袋 根据最近报道的后期融合中间体的分析确定了筛选将采用的方法。 原子级足迹，以确定最有希望进行实验的最高得分（化合物300-400） （目标#2）通过量化计算鉴定的小分子探针的特征。 预测得分最高的化合物（300-400）会通过破坏膜来阻止病毒进入。 融合将在 Vero 细胞和人成纤维细胞 (HFF-1) 中进行测试。 吞吐量非复制假型病毒系统（带有荧光素酶的 HIV 颗粒中的 ZIKV M/E 蛋白 阳性结果将用活 ZIKV 感染性进行测试（噬菌斑测定和测定终点稀释）。 实验测试将允许通过更广泛的筛选确定分子的优先级。 这项工作的影响包括加深对如何针对相关黄病毒（包括登革热）的了解， 黄热病、蜱传脑炎和西尼罗河。