Probing Functional States and Inhibition of Flaviviral Proteases Using Nanopore Tweezers

项目摘要 黄病毒是主要的蚊媒病原体，每年感染全球数百万人。目前在那里 目前还没有抗病毒疗法可用于治疗西尼罗河、登革热和寨卡病毒感染。第一个疫苗CyD- 针对DENV的TDV(登革热)去年被批准，但对这四种病毒的总体疗效只有56% 登革热的血清型。黄病毒双组分NS2B/NS3蛋白酶是病毒复制所必需的，因此 诱人的抗病毒靶标。然而，广泛的筛选和合理的设计努力未能找到任何 在这一点上临床可行的抑制剂。有两个关键因素可能是造成这一挑战的原因。第一，传统 筛选工作主要依靠结合亲和力来预测药物疗效。然而，越来越多的证据表明 研究表明，药物-靶点相互作用的滞留时间是体内更可靠的预测因子 药理活性。这些动力学速率参数在药物的早期阶段通常是不可用的 发现号。第二，NS2B/NS3蛋白在功能和功能过程中表现出复杂的构象动力学。 抑制，这一点仍然知之甚少。本项目旨在开发一种新的无标记单分子 一种解析NS2B/NS3酶构象状态的方法。该方法的关键是使用 创新的纳米孔镊子，其中蛋白酶被限制在孔腔中，允许动态结构 通过电流波动信号连续监测底物或缓蚀剂结合过程中的变化。分析 将提供结合亲和力和动力学速率的完整轮廓以及分布 构象状态。具体地说，我们将首先构建一个纳米孔镊子工具集，该工具集可以很容易地调整 捕获各种黄病毒蛋白酶。其次，我们将跟踪和分析NS2B/NS3的功能状态 在各种底物存在的情况下的蛋白酶。关键残留物、底物、结构设计对 “开放”和“关闭”状态之间的动态平衡将被评估，以提供对 蛋白水解酶活性机制。最后，将部署纳米孔镊子来确定结构 NS2B/NS3与不同缓蚀剂相互作用的动力学、结合热力学和动力学特征。 一旦建立了抑制曲线，将测试限制在NS2B/NS3体系中的纳米孔镊子 用于筛选不同的化合物文库以鉴定具有改进的类药物特性的新型变构抑制剂 与活性部位抑制剂相比。这项工作将提供关于功能的前所未有的动力学信息- NS2B/NS3复合体的结构动力学关系及底物结合和抑制机制 以及建立一种不依赖于酶活性的高通量药物筛选的新范式。

Project Summary Flavivirus are major mosquito-borne pathogens infecting millions of people worldwide each year. Currently there is no antiviral therapy available for treating West Nile, Dengue and Zika viral infections. The first vaccine CYD- TDV (Dengvaxia) against DENV was approved last year but shows only 56% overall efficacy against the four dengue serotypes. The flaviviral two-component NS2B/NS3 protease is required for viral replication and thus an attractive antiviral target. However, extensive screening and rational design efforts have failed to identify any clinically viable inhibitors at this point. Two key factors have likely contributed to the challenge. First, traditional screening efforts rely primarily on binding affinity to predict the drug efficacy. Yet, increasing evidence has emerged to show that the residence time of drug-target interaction is a more reliable predictor of in vivo pharmacological activity. These kinetic rate parameters are generally not available during early stages of drug discovery. Second, NS2B/NS3 proteases display complex conformational dynamics during function and inhibition, which is still poorly understood. This project aims to develop a new label-free single molecular approach to resolve the conformational states of NS2B/NS3 proteases. Key to the approach is the use of an innovative nanopore tweezers where the protease is confined with the pore lumen, allowing dynamic structural changes during substrate or inhibitor binding to be continuously monitored by current fluctuation signals. Analysis of the current traces will provide a complete profile of binding affinity and kinetic rates as well as the distribution of conformational states. Specifically, we will first build a nanopore tweezers tool set that is readily tunable for trapping various flaviviral proteases. Secondly, we will track and analyze the functional states of the NS2B/NS3 protease in the presence of various substrates. Influence of critical residues, substrate, construct design on the dynamic equilibrium between the “open” and “closed” states will be assessed to provide insight into the mechanism of protease activity. Finally, the nanopore tweezers will be deployed to determine the structural dynamics and binding thermodynamics and kinetics profiles of NS2B/NS3 interacting with various inhibitors. Once the inhibition profiles are established, the nanopore tweezers confined NS2B/NS3 system will be tested for screening a diverse compound library to identity novel allosteric inhibitors with improved drug-like properties compared to active-site inhibitors. This work will provide unprecedented kinetic information on the function- structural dynamics relationship of NS2B/NS3 complex and mechanisms of substrate binding and inhibition, as well as establish a new paradigm for high-throughput drug screening that is independent of enzymatic activity.