THE CYSTEINE PROTEASE OF TRYPANOSOMA CRUZI

克氏锥虫的半胱氨酸蛋白酶

• 批准号: 6099782
• 负责人: MARY MCGRATH
• 金额: $ 12.49万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6099782
• 关键词: Trypanosoma cruzi; X ray crystallography; active sites; antiprotozoal agents; chemical kinetics; conformation; cysteine endopeptidases; drug design /synthesis /production; enzyme structure; enzyme substrate; papain; protease inhibitor; site directed mutagenesis

X-ray crystallography will be used to determine high-resolution structures for cruzain, cruzain mutants, and their complexes with a variety of inhibitors and their derivatives. Our goal is to develop a therapeutic agent for Chagas' disease by inhibition of the major cysteine protease of Trypanosoma cruzi. Structure analysis will be used in conjunction with enzyme kinetics (Dr. Craik) and computational methods (Dr. Cohen) to find, test (Dr. Craik, Dr. McKerrow, Dr. Engel) and synthesize (Dr. Roush) tight-binding and highly-specific inhibitors for cruzain. To this end, we have recently solved the 2.35Angstroms X-ray structure of cruzain by molecular replacement methods. The structure will be refined to convergence and used as a model for computational methods including DOCK3.0 to begin the search for possible inhibitors. A higher resolution structure (2.0Angstroms or better) will be obtained and will provide a more accurate and informative model for the inhibitor search. Methods will be developed for crystallization of a wide variety of complexes between the cruzains and putative inhibitors. A panoply of structures will be determined, involving cruzain bound to different types of inhibitors and their derivatives, and cruzain mutants with inhibitors and substrates. This will provide a detailed map of the substrate binding site. Our in-depth analysis, in which binding at each subsite is carefully evaluated by site-directed mutagenesis, kinetic analyses, theoretical calculations including molecular dynamics simulations and correlations with atomic temperature factors, should yield an effective therapeutic for Chagas' disease as well as elucidating nuances of cysteine protease function.

X射线晶体学将用于确定高分辨率 cruzain、cruzain 突变体及其与 a 的复合物的结构 多种抑制剂及其衍生物。 我们的目标是开发一个 通过抑制主要半胱氨酸治疗南美锥虫病的药物 克氏锥虫蛋白酶。 结构分析将用于 结合酶动力学（Craik 博士）和计算方法 （科恩博士）寻找、测试（克雷克博士、麦凯罗博士、恩格尔博士）和 合成（Roush 博士）紧密结合且高度特异性的抑制剂 克鲁扎因。 为此，我们最近解决了2.35埃X射线 通过分子置换方法确定 cruzain 的结构。 结构 将被细化为收敛并用作计算模型 包括DOCK3.0在内的方法开始寻找可能的抑制剂。 将获得更高分辨率的结构（2.0埃或更好） 将为抑制剂提供更准确、信息更丰富的模型 搜索。 将开发多种结晶方法 cruzains 和假定的抑制剂之间的复合物。 一整套 结构将被确定，涉及与不同类型结合的cruzain 抑制剂及其衍生物，以及带有抑制剂的 cruzain 突变体 和基材。 这将提供基材的详细地图 结合位点。 我们的深入分析，其中每个子网站的绑定 通过定点诱变、动力学分析仔细评估， 理论计算，包括分子动力学模拟和 与原子温度因素的相关性，应该产生有效的 恰加斯病的治疗方法以及阐明其细微差别 半胱氨酸蛋白酶的功能。