HEPATITIS C VIRUS NS3 PROTEASE--ACTIVE SITE

丙型肝炎病毒 NS3 蛋白酶——活性位点

• 批准号: 2330570
• 负责人: Ben M. Dunn
• 金额: $ 28万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 1998-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2330570
• 关键词: Baculoviridae; Escherichia coli; X ray crystallography; active sites; affinity chromatography; antiviral agents; cooperative study; drug design /synthesis /production; enzyme structure; fluorimetry; glutathione transferase; hepatitis C; hepatitis C virus; molecular cloning; peptide chemical synthesis; periplasm; protease inhibitor; protein sequence; serine proteinases

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.

该项目的总体目标是开发新化合物 对丙型肝炎病毒表现出抗病毒活性。 达到 为了实现这一目标，我们将针对必需的加工酶 NS3。 基于处理点不寻常的底物特异性 病毒多蛋白，应该可以选择性地设计和构建 和有效的化合物。 我们实现总体目标的计划包括 具体目标如下： 1. HCV NS3的催化结构域将被表达以产生 足够量的蛋白质用于结构和生化 表征。 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 大肠杆菌分泌载体将表达的蛋白质引导至 周质空间； (b.)催化结构域将与麦芽糖融合 结合蛋白或谷胱甘肽-S-转移酶，在大肠杆菌中表达， 通过亲和层析纯化，然后裂解因子 Xa； （三） 将构建突变序列以替换表面残基 亲水残基；我们还将在 N- 和 C- 处添加赖氨酸块 末端进一步努力增加溶解度； (d.) 更大 HCV 基因组片段，包括 N- 和/或 C- 上的延伸 终端两侧，将被表达； (e.) 杆状病毒表达系统 并且还将使用酵母、毕赤酵母表达系统 以获得更高产率的正确折叠的活性材料。 2. 掌握功能蛋白，研究 HCV NS3 的结构/催化特性将继续进行。 我们将：(a.) 向合作者提供蛋白质以测定三- 维度结构； (b.) 研究荧光测定中的催化活性 基于裂解特异性设计的测定； (c.) 探索范围 允许切割点两侧的序列变化。 这 信息将有助于抑制剂的设计； (d) 研究性质 源自不同基因型和亚型的 HCV NS3 变体。 3. 建立测定后，我们可以设计、构建和评估 抑制剂以发现抗病毒先导化合物。 这些新化合物 可用于上述2a中的晶体学研究。 我们的系统 将探索包括： (a.) 小抑制剂，例如异香豆素、 氯甲基酮和硼酸； (b.) 小蛋白蛋白酶 抑制剂，例如 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为驱动抑制剂设计提供线索。