Dengue and West Nile Viral Protease Inhibitors

登革热和西尼罗河病毒蛋白酶抑制剂

基本信息

批准号：
6954153
负责人：
Radhakrishnan Padmanabhan
金额：
$ 19.14万
依托单位：
GEORGETOWN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-09-30 至 2007-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6954153
关键词：
West Nile virus antiviral agents dengue virus drug design /synthesis /production drug screening /evaluation endopeptidases enzyme activity enzyme structure fluorescent dye /probe gel filtration chromatography protease inhibitor protein purification protein structure function tissue /cell culture virus infection mechanism virus protein

项目摘要

DESCRIPTION (provided by applicant): The mosquito-borne flavivirus family includes human pathogens such as yellow fever virus (YFV), West Nile virus (WNV), and the four serotypes of Dengue (DEN) viruses. These viruses, included in the NIAID list of Class A, B, or C human pathogens, cause serious illnesses associated with considerable morbidity and mortality and these diseases have emerged in recent years. Since the first cases of WNV infections in the Western hemisphere were recorded in New York city in 1999, the WNV has become a rapidly spreading major public health concern throughout the U.S. One of the long tern goals of this laboratory has been to develop antiviral therapeutics through understanding of key pathways in the viral lifecycle through development of in vitro systems that mimic processes these viruses use in their hosts. For example, polyprotein processing has been identified as a key early event that is crucial for the viral life cycle. The two-component dengue viral serine protease, required in this process has been expressed in E. coli, purified, and biochemically and kinetically characterized. The crystal structures of the protease domain, alone or in complex with a serine protease inhibitor have been solved. The overall objective of this proposal is to purify the E. coli-expressed West Nile viral protease and characterize its biochemical and kinetic properties in the presence and absence of protease inhibitors. This objective will be achieved with the following Specific Aims. Aim 1: Purification and characterization of the two-component WNV NS3-protease.The protease will be purified from E. coli and determine the kinetic and biochemical parameters of the enzyme. The WNV protease has been expressed with an N-terminal or C-terminal His tag. The enzyme will be purified to near-homogeneity in two steps using metal affinity and gel filtration chromatography steps. The enzyme activity will be assayed using radiolabeled natural polypeptide precursor having the protease sensitive site, fluorogenic peptide substrates, or internally quenched (IQ) fluorogenic substrates and the kinetic parameters will be determined. Aim 2: Synthesis of novel novel heterocyclic scaffold core structure, covalent and non-covalent inhibitors of DEN and WNV proteases will be synthesized. This strategy has been successfully employed in development of chymotrypsin-and trypsin-like serine proteases and preliminary screen of DEN protease activity shows promising results. Aim 3. Analysis of potencies of serine protease inhibitors in vitro and in vivo. The in vitro assays with fluorogenic (and IQ) substrates will be carried out in the presence and absence of inhibitors. Product-based inhibitors, inhibitors similar to those developed for HCV protease, covalent and noncovalent inhibitors synthesized using the heterocyclic scaffold will be assayed. A cell-based assay will be developed for evaluating the inhibitory potencies of these compounds. Since there is no effective vaccine available for either DEN or WNV, this antiviral strategy is likely to yield lead inhibitors useful as therapeutic agents in the control of these lethal pathogens.

描述（由申请人提供）：蚊媒黄病毒家族包括人类病原体，例如黄热病病毒（YFV）、西尼罗河病毒（WNV）和登革热（DEN）病毒的四种血清型。这些病毒被列入 NIAID A、B 或 C 类人类病原体清单中，可引起严重的疾病，并导致相当高的发病率和死亡率，并且这些疾病是近年来出现的。自 1999 年在纽约市记录到西半球第一例西尼罗河病毒感染病例以来，西尼罗河病毒已成为整个美国迅速蔓延的主要公共卫生问题。该实验室的长期目标之一是通过以下方式开发抗病毒疗法：通过开发模仿这些病毒在宿主中使用的过程的体外系统，了解病毒生命周期中的关键途径。例如，多蛋白加工已被确定为对病毒生命周期至关重要的关键早期事件。该过程所需的双组分登革热病毒丝氨酸蛋白酶已在大肠杆菌中表达、纯化，并进行了生化和动力学表征。单独或与丝氨酸蛋白酶抑制剂复合的蛋白酶结构域的晶体结构已得到解决。该提案的总体目标是纯化大肠杆菌表达的西尼罗河病毒蛋白酶，并在存在和不存在蛋白酶抑制剂的情况下表征其生化和动力学特性。这一目标将通过以下具体目标来实现。目标 1：双组分 WNV NS3 蛋白酶的纯化和表征。将从大肠杆菌中纯化该蛋白酶，并测定该酶的动力学和生化参数。 WNV 蛋白酶已用 N 端或 C 端 His 标签表达。使用金属亲和力和凝胶过滤色谱步骤，分两步将酶纯化至接近均质。将使用具有蛋白酶敏感位点的放射性标记的天然多肽前体、荧光肽底物或内部猝灭(IQ)荧光底物来测定酶活性，并确定动力学参数。目标2：合成新型杂环支架核心结构，将合成DEN和WNV蛋白酶的共价和非共价抑制剂。该策略已成功应用于胰凝乳蛋白酶和类胰蛋白酶丝氨酸蛋白酶的开发，并且 DEN 蛋白酶活性的初步筛选显示出有希望的结果。目标 3. 丝氨酸蛋白酶抑制剂的体外和体内效力分析。荧光（和 IQ）底物的体外测定将在存在和不存在抑制剂的情况下进行。将分析基于产品的抑制剂、类似于为 HCV 蛋白酶开发的抑制剂、使用杂环支架合成的共价和非共价抑制剂。将开发基于细胞的测定法来评估这些化合物的抑制效力。由于目前还没有针对 DEN 或 WNV 的有效疫苗，因此这种抗病毒策略可能会产生先导抑制剂，可用作控制这些致命病原体的治疗剂。