Dengue and West Nile Viral Protease Inhibitors

描述（由申请方提供）：蚊媒黄病毒家族包括人类病原体，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。包括在NIAID A、B或C类人类病原体清单中的这些病毒引起与相当高的发病率和死亡率相关的严重疾病，并且这些疾病近年来出现。自从1999年在纽约市记录了西半球的第一例西尼罗河病毒感染以来，西尼罗河病毒已经成为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开发模拟这些病毒在其宿主中使用的过程的体外系统，通过理解病毒生命周期中的关键途径来开发抗病毒治疗。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。在此过程中所需的双组分登革热病毒丝氨酸蛋白酶已在E.大肠杆菌，纯化，生化和动力学特征。蛋白酶结构域单独或与丝氨酸蛋白酶抑制剂复合的晶体结构已被解析。本提案的总体目标是纯化E.大肠杆菌表达的西尼罗病毒蛋白酶，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实现。 目的1：从大肠杆菌中分离纯化双组分西尼罗河病毒NS 3蛋白酶，并对其性质进行研究。并测定酶的动力学和生化参数。WNV蛋白酶已表达具有N-末端或C-末端His标签。将使用金属亲和和凝胶过滤色谱步骤在两个步骤中将酶纯化至接近同质。将使用具有蛋白酶敏感位点的放射性标记天然多肽前体、荧光肽底物或内淬灭（IQ）荧光底物测定酶活性，并测定动力学参数。 目标二：合成新型杂环骨架核心结构、DEN和WNV蛋白酶的共价和非共价抑制剂。这种策略已成功地用于胰凝乳蛋白酶和胰蛋白酶样丝氨酸蛋白酶的开发，并初步筛选DEN蛋白酶活性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。将在存在和不存在抑制剂的情况下使用荧光（和IQ）底物进行体外试验。将对基于产品的抑制剂、与针对HCV蛋白酶开发的抑制剂类似的抑制剂、使用杂环支架合成的共价和非共价抑制剂进行分析。将开发一种基于细胞的试验，用于评价这些化合物的抑制效力。由于没有有效的疫苗可用于DEN或WNV，这种抗病毒策略可能产生可用作控制这些致命病原体的治疗剂的先导抑制剂。

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.