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，每年感染全世界数百万人。当前 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（Dengvaxia）去年获得批准，但对四种病毒的总体疗效仅为56%。 登革热血清型。黄病毒双组分NS 2B/NS 3蛋白酶是病毒复制所需的，因此， 有吸引力的抗病毒靶点。然而，广泛的筛选和合理的设计努力未能确定任何 临床上可行的抑制剂两个关键因素可能促成了这一挑战。一是传统 筛选工作主要依靠结合亲和力来预测药物功效。然而，越来越多的证据表明， 显示药物-靶点相互作用的停留时间是体内靶点释放的更可靠的预测因子。 药理活性这些动力学速率参数通常在药物治疗的早期阶段不可用。 的发现第二，NS 2B/NS 3蛋白酶在功能期间显示复杂的构象动力学， 抑制，这仍然是知之甚少。本项目旨在开发一种新的无标记单分子 方法来解决NS 2B/NS 3蛋白酶的构象状态。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抑制剂结合期间的变化。分析 的电流迹线将提供结合亲和力和动力学速率以及分布的完整概况 构象状态。具体地说，我们将首先建立一个纳米孔镊子工具集，它很容易调整， 捕获各种黄病毒蛋白酶。其次，我们将跟踪和分析NS 2B/NS 3的功能状态 在各种底物存在下的蛋白酶。关键残留物、底物、结构设计对 “开放”和“封闭”状态之间的动态平衡将被评估，以提供对 蛋白酶活性机制最后，纳米孔镊子将被部署，以确定结构 NS 2B/NS 3与各种抑制剂相互作用的动力学和结合热力学和动力学曲线。 一旦建立了抑制曲线，将测试纳米孔镊子限制的NS 2B/NS 3系统 用于筛选不同的化合物库以鉴定具有改善的药物样性质的新型变构抑制剂 与活性位点抑制剂相比。这项工作将提供前所未有的动力学信息的功能- NS 2B/NS 3复合物结构动力学关系以及底物结合和抑制机制， 并建立了一个新的范例，高通量药物筛选是独立的酶活性。

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.