Collaborative Research: RAPID: Computationally designed probes to experimentally characterize mechanisms of Ebola virus membrane fusion

2014年埃博拉疫情是历史上最大的爆发（http：www.cdc.gov），根据世界卫生组织（WHO），“截至10月19日，已有9936例埃博拉病毒病病例，4877人死亡。“同样令人震惊的是，最近有报告称，埃博拉病毒传播给医院护理人员，尽管官方警告说，在美国等较发达国家，传播可能不是一个问题。 总之，这些前所未有的事件突出了埃博拉病毒成为大流行病的可能性的严重性，以及更全面地描述病毒如何复制的迫切需要，以便采取措施阻止当前和未来可能的爆发。 该项目采用原子级计算机建模（对接，虚拟筛选）来预测和表征小分子化合物探针如何与位于埃博拉病毒表面的特定病毒蛋白结合并相互作用。 得分最高的化合物将进行实验测试，以确定哪些分子可以阻止病毒进入。 小分子如何与埃博拉蛋白相互作用的表征将最终使人们能够更好地了解如何阻止病毒进入并控制埃博拉感染。 这对于其他包膜病毒如流感、HIV、SARS、MERS等的研究也很重要。 这项工作的更广泛影响包括增加了关于什么类型的分子在阻止埃博拉感染方面最有效的知识，这将有利于研究相关的包膜病毒和靶向由类似病毒蛋白介导的类似病毒进入事件。 该项目的一个重要组成部分是在本科生、研究生和博士后水平上进行关于使用尖端计算和实验工具进行病毒研究的教育培训，其中包括妇女和代表性不足的少数民族的计划参与。 技术摘要PI将进行计算和实验研究，以确定和设计小分子探针，将逮捕病毒的生命周期所必需的早期膜融合事件。 该项目将采用一种强大的新计算对接策略，允许在原子水平上绘制和利用推定的结合界面，例如埃博拉病毒进入蛋白GP 2和GP 1上的结合界面。 具体而言，采用每残基相互作用能的计算足迹方法将用于识别埃博拉前发夹和融合前模型中的靶向事件，然后进行对接以识别最有希望的得分最高的化合物，以进行额外的深入表征和实验测试。 我们的目标是确定目标的有利位置的N-螺旋线圈的形成和C-螺旋协会的破坏的化合物，使用大规模的高通量-虚拟筛选的市售化合物和实验测试。 将使用在含有埃博拉病毒包膜蛋白GP 2和GP 1的非复制病毒样颗粒中使用定量报告基因的实验假型病毒系统来确认来自虚拟筛选的化合物阻止病毒进入/融合。 这些化合物将优先进行进一步的研究和开发。 这项工作的更广泛影响包括增加了关于什么类型的化合物在阻止埃博拉感染方面最有效的知识，这可能会导致埃博拉病毒靶向治疗的发展，也有利于相关包膜病毒的研究和靶向类似病毒蛋白介导的类似病毒进入事件。 该项目还将教育本科生，研究生和博士后学生使用和分析创新的计算对接和实验生物物理方法，以帮助下一代科学家准备在未来攻击重要的研究问题。

Lay AbstractThe 2014 Ebola epidemic is the largest outbreak in history (http://www.cdc.gov) and according to the World Health Organization (WHO), "There have been 9936 Ebola virus disease cases, and 4877 deaths, up to the end of 19 October." Just as alarming are the recent reports of Ebola transmission to hospital caregivers despite official warning to the contrary that spread would likely not be an issue in more developed countries such as the United States. Together, these unprecedented events highlight the severity of the potential of Ebola virus to become pandemic and the urgent need to more fully characterize how the virus replicates so that steps can be made to stop the current and likely future outbreaks. This project employs atomic-level computer modeling (docking, virtual screening) to predict and characterize how small molecule compound probes bind to and interact with specific viral proteins located on the surface of the Ebola virus. Top-scoring compounds will be experimentally tested to determine which molecules can stop viral entry. Characterization of how small molecules interact with Ebola proteins will ultimately enable a better understanding of how viral entry can be stopped and Ebola infection controlled. This will also be important for the study of other enveloped viruses such as influenza, HIV, SARS, MERS, among others. Broader impacts of the work include increased knowledge as to what types of molecules are most effective at stopping Ebola infection which will benefit study of related enveloped viruses and targeting of similar viral entry events mediated by analogous viral proteins. An important component of the project is educational training in use of cutting-edge computational and experimental tools for viral research at the undergraduate, graduate, and postdoctoral levels that includes the planned participation of women and underrepresented minorities. Technical AbstractThe PI will conduct computational and experimental studies to identify and design small molecular probes that will arrest early membrane fusion events necessary for the life cycle of the virus. The project will employ a powerful new computational docking strategy that allows putative binding interfaces, such as those on Ebola viral entry proteins GP2 and GP1, to be mapped and exploited at the atomic level. Specifically, computational footprinting methods employing per-residue interaction energies will be used to identify targetable events in the Ebola pre-hairpin and pre-fusion models followed by docking to identify the most promising top-scoring compounds for additional in-depth characterization and experimental testing. The goal is to identify compounds that target favorable positions for disruption of N-helical coil formation and C-helix association, using large-scale high-throughput-virtual screening of commercially available compounds and experimental testing. An experimental pseudotyped virus system that uses a quantitative reporter gene in a non-replicating virus-like particle containing Ebola virus envelope proteins GP2 and GP1 will be used to confirm that compounds from the virtual screen arrest viral entry/fusion. Such compounds will be prioritized for additional study and development. Broader impacts of the work include increased knowledge as to what types of compounds are most effective at stopping Ebola infection which may both lead to development of Ebola virus-targeted therapeutics and also benefit study of related enveloped viruses and targeting of similar viral entry events mediated by analogous viral proteins. The project will also educate undergraduate, graduate, and postdoctoral students in the use and analysis of innovative computational docking and experimental biophysical methods to help prepare the next generation of scientists to attack important research problems in the future.