Dengue and West Nile Viral Protease Inhibitors

描述(申请人提供)：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。这些病毒被列入NIAID的A、B或C类人类病原体名单，可引起与相当高的发病率和死亡率相关的严重疾病，这些疾病是近年来出现的。自从1999年纽约市记录了西半球第一例西半球西尼罗河病毒感染病例以来，西尼罗河病毒已经成为在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宿主中使用的过程的体外系统，通过了解病毒生命周期中的关键途径来开发抗病毒疗法。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。在这一过程中所需的双组分登革病毒丝氨酸蛋白酶已在大肠杆菌中表达、纯化、生化和动力学特征。蛋白酶域的晶体结构已经被解决，无论是单独的还是与丝氨酸蛋白酶抑制剂的复合体。这项建议的总体目标是纯化在大肠杆菌中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性质。这一目标将通过以下具体目标来实现。 目的1：分离纯化WNV NS3-双组分蛋白，并测定其动力学和生化参数。西尼罗河病毒的蛋白水解酶已经表达了N-端或C-端的His标签。该酶将通过金属亲和层析和凝胶过滤层析两步纯化到接近均一的水平。用具有酶敏感部位的放射性标记的天然多肽前体、荧光肽底物或内部猝灭(IQ)荧光底物测定酶活性，并确定动力学参数。 目的2：合成新型杂环骨架核心结构，合成DEN和WNV蛋白水解酶的共价和非共价抑制剂。这一策略已成功地应用于胰凝乳酶和类胰蛋白酶丝氨酸蛋白酶的开发，并对DEN蛋白酶活性进行了初步筛选，结果令人满意。 目的3.丝氨酸蛋白酶抑制剂的体内外效价分析。使用荧光(和智商)底物的体外分析将在存在和不存在抑制剂的情况下进行。以产物为基础的抑制剂、类似于那些为丙型肝炎病毒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被检测。将开发一种基于细胞的分析来评估这些化合物的抑制效力。由于目前还没有针对登革热或西尼罗河病毒的有效疫苗，这种抗病毒策略可能会产生可用作治疗药物的先导抑制剂，以控制这些致命的病原体。

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.