Leveraging protein dynamics to drug filovirus protein-nucleic acid interactions using simulations and experiments

项目摘要 许多蛋白质被归类为“不可药用”，特别是那些参与蛋白质-蛋白质和蛋白质- 核酸相互作用，因为它们通过平坦界面与它们的结合配偶体相互作用。这是特别 在许多与宿主因子和病毒核酸相互作用且缺乏酶促活性的病毒蛋白质的情况下， 活动在高致死性丝状病毒家族1蛋白中，病毒蛋白35（VP 35）主要负责病毒的免疫应答。 病毒的免疫逃避在这个家族中，埃博拉病毒是最致命的，最近的病死率为66%。 爆发因此，非常需要在该病毒家族中发现新的治疗靶标。 虽然蛋白质是动态的，但直到最近才考虑蛋白质动力学用于药物设计 通过分子动力学方法的进步变得易于处理。因此，发现“神秘”的口袋 在现有结构中不存在但由于蛋白质动力学而开放的蛋白质可以提供新的可药用位点。 在这里，我建议整合原子详细的模拟，生物物理和细胞实验，以了解 病毒蛋白35（VP 35）中的隐蔽口袋，来自高致死性丝状病毒。VP 35发挥多种重要作用 在这些病毒的复制周期中的作用，包括结合病毒RNA基因组以阻断宿主的先天性 免疫力和作为聚合酶辅助因子。然而，VP 35的可用晶体结构缺乏吸引力， 药物发现的口袋和蛋白质迄今为止仍然不可药用。我们最近申请了 自适应采样模拟，以优先对具有大口袋体积的构象进行采样。这揭示 一个潜在的可下药的秘密口袋虽然口袋并不直接符合关键的接口， 结合RNA，我们已经表明，口袋可以变构调节RNA结合，是一个很好的药物 目标为了进一步验证这一点，我将使用巯基标记实验来直接测试VP 35中的这个隐蔽口袋 同系物。然后，为了确定这个神秘的口袋是否与功能变构偶联，我将测试是否稳定 使用荧光各向异性测定，开放形式变构地破坏RNA结合。然后我会筛选 RNA结合的小分子抑制剂，与隐蔽口袋结合，并通过X射线证实 晶体学和突变测试最后，我将评估稳定开放口袋对VP 35的影响。 干扰素拮抗作用和使用体外ATP酶测定和细胞微基因组的病毒复制活性 比色法这些实验的成功完成将进一步促进国家综合医学研究所 科学的使命是增加我们对生物过程的理解，为推进 疾病治疗。这些结果将证明融合模拟和实验的力量， 表征隐藏的构象和动力学，揭示隐藏的口袋和变构， 治疗机会。

Project Summary Many proteins are classified as ‘undruggable,’ especially those that engage in protein-protein and protein- nucleic acid interactions as they interact with their binding partners through flat interfaces. This is particularly true in the case of many viral proteins that interact with host factors and viral nucleic acids and lack enzymatic activity. In the highly lethal filovirus family one protein, viral protein 35 (VP35) is primarily responsible for the viruses’ immune evasion. In this family the ebolavirus is the most fatal with a case fatality rate at 66% in recent outbreaks. Thus, there is a great need for discovering new therapeutic targets in this family of viruses. Although proteins are known to be dynamic, only recently has considering protein dynamics for drug design become tractable through advances in molecular dynamics methods. As such, discovering ‘cryptic’ pockets that are absent in available structures but open due to protein dynamics could provide new druggable sites. Here, I propose integrating atomically-detailed simulations, biophysical, and cellular experiments to understand the cryptic pocket in viral protein 35 (VP35) from the highly lethal filoviruses. VP35 plays multiple essential roles in these viruses’ replication cycles, including binding the viral RNA genome to block hosts’ innate immunity and acting as a polymerase co-factor. However, available crystal structures of VP35 lack appealing pockets for drug discovery and the protein has so far remained undruggable. We recently have applied adaptive sampling simulations to preferentially sample conformations with large pocket volumes. This revealed a potentially druggable cryptic pocket. While the pocket does not directly coincide with the crucial interface for binding RNA, we have shown that the pocket can allosterically modulate RNA binding and is a good drug target. To further test this, I will use a thiol labeling experiment to directly test for this cryptic pocket in VP35 homologs. Then, to determine if this cryptic pocket is allosterically coupled to function, I will test if stabilizing the open form allosterically disrupts RNA binding using a fluorescence anisotropy assay. I will then screen for small-molecule inhibitors of RNA binding that bind to the cryptic pocket and confirm this by X-ray crystallography and mutational tests. Finally, I will assess the effect of stabilizing the open pocket on VP35’s interferon antagonism, and viral replication activities using an in-vitro ATPase assay and cellular minigenome assay. Successful completion of these experiments will further the National Institute of General Medical Sciences’ mission to increase our understanding of biological processes for laying the foundation of advancing disease treatments. These results will demonstrate the power of fusing simulations and experiments to characterize hidden conformations and dynamics, uncovering cryptic pockets and allostery that present new therapeutic opportunities.