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规模最大的一次疫情(http://www.cdc.gov))，根据世界卫生组织(WHO)的数据，截至10月19日底，已有9,936例埃博拉病毒感染病例，4,877人死亡。同样令人担忧的是，最近有关埃博拉病毒传播给医院护理人员的报道，尽管官方发出了相反的警告，称在美国等更发达的国家，传播可能不会成为问题。总而言之，这些史无前例的事件突显了埃博拉病毒可能成为大流行的严重性，以及迫切需要更全面地描述病毒的复制方式，以便采取措施阻止当前和未来可能爆发的疫情。该项目使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。表征小分子如何与埃博拉蛋白相互作用，最终将有助于更好地理解如何阻止病毒进入和控制埃博拉感染。这对于研究其他包膜病毒，如流感、艾滋病毒、SARS、MERS等也将是重要的。这项工作的更广泛影响包括增加关于哪些类型的分子在阻止埃博拉感染方面最有效的知识，这将有助于研究相关的包膜病毒，并针对类似的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、研究生和博士后各级利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群体的计划参与。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，这些探针将阻止病毒生命周期所需的早期膜融合事件。该项目将采用一种强大的新的计算对接策略，允许在原子水平上绘制和利用假定的结合界面，例如埃博拉病毒进入蛋白GP2和GP1上的结合界面。具体地说，使用按残基相互作用能量的计算足迹方法将被用来识别埃博拉前发夹和前融合模型中的目标事件，然后对接，以确定最有希望获得最高得分的化合物，以便进行进一步的深入表征和实验测试。其目标是利用大规模的高通量-商业可用化合物的虚拟筛选和实验测试，确定目标为破坏N-螺旋形成和C-螺旋结合的有利位置的化合物。在含有埃博拉病毒包膜蛋白GP2和GP1的非复制型病毒样颗粒中使用定量报告基因的实验假类型病毒系统将被用于确认来自虚拟筛选的化合物阻止病毒进入/融合。这类化合物将优先用于进一步的研究和开发。这项工作的更广泛影响包括增加关于哪些类型的化合物在阻止埃博拉感染方面最有效的知识，这既可能导致埃博拉病毒靶向疗法的开发，也有利于相关包膜病毒的研究和类似病毒蛋白介导的类似病毒进入事件的靶向。该项目还将教育本科生、研究生和博士后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为未来解决重要研究问题做好准备。

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.