Probing Functional States and Inhibition of Flaviviral Proteases Using Nanopore Tweezers

使用纳米孔镊子探测黄病毒蛋白酶的功能状态和抑制

• 批准号: 10426354
• 负责人: Min Chen
• 金额: $ 44.12万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-10 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10426354
• 关键词: Active Sites; Affinity; Allosteric Site; Antiviral Agents; Antiviral Therapy; Binding; Biochemical; Biological Assay; Biological Availability; Biophysics; Charge; Clinical; Complex; Computer Models; Cryoelectron Microscopy; Cytolysins; Data; Dengue; Dengvaxia; Disease Outbreaks; Drug Interactions; Drug Targeting; Epidemic; Equilibrium; Flavivirus; Foundations; Health; Human; Infection; Investigational Drugs; Kinetics; Label; Lead; Libraries; Measurement; Measures; Methods; Molecular; Molecular Conformation; Monitor; Motion; Nature; Peptide Hydrolases; Peptides; Persons; Pharmaceutical Preparations; Pharmacology; Property; Protein Dynamics; Protein Engineering; Proteins; Reaction; Recurrence; Resolution; Roentgen Rays; Rotation; Serine Protease; Serotyping; Signal Transduction; Substrate Cycling; Substrate Interaction; System; Techniques; Technology; Testing; Thermodynamics; Time; Vaccines; Viral Proteins; Virus Replication; West Nile virus; Work; ZIKA; ZIKV infection; Zika Virus; biophysical model; cryptography; design; design and construction; drug development; drug discovery; drug efficacy; enzyme activity; experience; experimental study; guided inquiry; high throughput screening; high-throughput drug screening; improved; in vivo; inhibitor; innovation; insight; maltose-binding protein; molecular modeling; mosquito-borne pathogen; nanopore; novel; rational design; residence; screening; simulation; single molecule; sugar; tool

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.

项目摘要 黄病毒是每年在全球范围内感染数百万人的主要蚊子传播病原体。目前在那里 没有可用于治疗西尼罗河，登革热和寨卡病毒感染的抗病毒疗法。第一个疫苗Cyd- 去年，针对DENV的TDV（Dengvaxia）被批准，但仅显示56％的总体效率 登革热血清型。黄素两组分NS2B/NS3蛋白酶是病毒复制需要的，因此需要 有吸引力的抗病毒靶标。但是，广泛的筛选和合理的设计工作未能识别任何 此时，临床上可行的抑制剂。两个关键因素可能导致了挑战。首先，传统 筛选工作主要依赖于结合亲和力来预测药物效率。然而，越来越多的证据 出现以表明药物目标相互作用的停留时间是体内更可靠的预测因子 药理活性。这些动力学率参数通常在药物的早期阶段不可用 发现。第二，NS2B/NS3蛋白酶在功能和 抑制作用，这仍然很少理解。该项目旨在开发一个新的无标签单分子 解决NS2B/NS3蛋白酶的构象状态的方法。方法的关键是使用 创新的纳米孔镊子，其中蛋白质限制在孔管腔中，允许动态结构 底物或抑制剂结合期间的变化将通过当前波动信号连续监测。分析 当前的痕迹将提供结合亲和力和动力学率的完整曲线以及分布 构象状态。特别是，我们将首先构建一个纳米透明镊子套件，很容易调整 捕获各种黄素蛋白酶。其次，我们将跟踪和分析NS2B/NS3的功能状态 蛋白酶在存在各种底物的情况下。关键残差，基材，构造设计对 将评估“公开”和“封闭”状态之间的动态等效物 蛋白酶活性的机理。最后，将部署纳米孔镊子来确定结构 NS2B/NS3的动力学和结合热力学和动力学分布与各种抑制剂相互作用。 一旦建立了抑制概况，将测试纳米孔镊子限制的NS2B/NS3系统 用于筛选潜水员复合库，以具有改善类似药物的特性的新型变构抑制剂 与活性位点抑制剂相比。这项工作将提供有关该功能的前所未有的动力学信息 - NS2B/NS3复合物与底物结合和抑制的机制的结构动力学关系，如 并建立了一个与酶活性无关的高通量药物筛查的新范式。