HEPATITIS C VIRUS NS3 PROTEASE--ACTIVE SITE

该项目的总体目标是开发新的化合物， 表现出抗丙型肝炎病毒的抗病毒活性。 实现 为此，我们将针对必需的加工酶NS 3。 基于在处理点的不寻常的底物特异性， 病毒多聚蛋白，应该有可能设计和构建选择性的 和有效化合物。 我们实现总体目标的计划包括： 具体目标如下： 1. HCV NS 3的催化结构域将被表达以产生 足够数量的蛋白质， 特征化 由于其他人观察到的问题，我们将尝试 以下变化：（a.）催化结构域将被亚克隆到 E.大肠杆菌分泌载体，将表达的蛋白引导至 周质间隙;（B.）催化区会和麦芽糖融合- 结合蛋白或谷胱甘肽-S-转移酶，在E.大肠杆菌， 通过亲和色谱法纯化，随后进行因子Xa切割;（c.） 将构建突变体序列以用以下取代表面残基： 亲水残基;我们还将在N-和C- 末端以进一步努力增加溶解度;（d.）较大 HCV基因组的区段，包括N-和/或C- 终端侧，将表示;（e.）杆状病毒表达系统 也将使用酵母毕赤酵母表达系统 以获得正确折叠的活性材料的较高产量。 2.有了功能蛋白， HCV NS 3的结构/催化性质将继续进行。 我们将：（a） 向合作者提供蛋白质以确定三种- 维度结构;（B.）用荧光光度法研究催化剂的活性 基于切割特异性设计的测定法;（c.）探索的范围 允许切割点两侧的序列变异。 这 信息将有助于抑制剂设计;（d）研究性质 不同基因型和亚型的HCV NS 3变异体。 3.有了一个确定的检测方法，我们可以设计，构建和评估 发现抗病毒先导化合物的抑制剂。 这些新化合物 可用于上述2a中的晶体学研究。 我们的系统 将探讨的问题包括：（a.）小的抑制剂如异香豆素， 氯甲基酮和硼酸;和（B.）小分子蛋白酶 抑制剂，如BPTI。 虽然我们不会使用BPTI的突变体作为 治疗剂，很可能来自这种蛋白质的信息- 蛋白质相互作用系统将产生线索来驱动抑制剂的设计。

The overall goal of this project is to develop new compounds that will exhibit anti-viral activity against the hepatitis C virus. To achieve this objective, we will target the essential processing enzyme, NS3. Based on the unusual substrate specificity at processing points in the viral polyprotein, it should be possible to design and construct selective and effective compounds. Our plan to realize the overall goal consists of the following specific aims: 1. The catalytic domain of HCV NS3 will be expressed to produce adequate amounts of protein for structural and biochemical characterization. Due to problems observed by others, we will attempt the following variations: (a.) The catalytic domain will be subcloned into an E. coli secretory vector to direct the expressed protein to the periplasmic space; (b.) The catalytic domain will be fused to maltose- binding protein or Glutathione-S-transferase, expressed in E. coli, purified by affinity chromatography followed by Factor Xa cleavage; (c.) Mutant sequences will be constructed to substitute surface residues with hydrophilic residues; We will also add blocks of lysine at the N- and C- terminal ends in a further effort to increase solubility; (d.) Larger segments of the HCV genome, including extensions on the N- and/or C- terminal sides, will be expressed; (e.) The baculovirus expression system and the yeast, pichoris pastoris, expression system will also be employed to obtain higher yields of correctly folded, active material. 2. With functional protein in hand, studies on the structural/catalytic properties of HCV NS3 will proceed. We will: (a.) Provide protein to a collaborator for determination of the three- dimensional structure; (b.) Study the catalytic activity in a fluorometric assay designed based on cleavage specificity; (c.) Explore the range of permitted sequence variation on both sides of the cleavage point. This information will contribute to inhibitor design; (d) Study the properties of variants of HCV NS3 derived from different genotypes and subtypes. 3. With an assay established, we can design, construct, and evaluate inhibitors to discover antiviral lead compounds. These new compounds could be used in the crystallographic studies in 2a above. The systems we will explore include: (a.) Small inhibitors such as isocoumarins, chloromethylketones, and boronic acids; and (b.) Small protein proteinase inhibitors, such as BPTI. Although we will not use mutants of BPTI as therapeutic agents, it is likely that information from such protein- protein interacting systems will yield clues to drive inhibitor design.