HYPOXANTHINE GUANINE PHOSPHORIBOSYLTRANSFERASE FROM TOXOPLASMA GONDII

来自弓形虫的次黄嘌呤鸟嘌呤磷酸核糖基转移酶

• 批准号: 6268050
• 负责人: BUDDY ULLMAN
• 金额: $ 12.88万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6268050
• 关键词: Escherichia coli; SDS polyacrylamide gel electrophoresis; Toxoplasma gondii; X ray crystallography; active sites; antiprotozoal agents; chemical kinetics; chemical models; circular dichroism; complementary DNA; computer simulation; drug design /synthesis /production; drug screening /evaluation; enzyme mechanism; enzyme structure; enzyme substrate; high performance liquid chromatography; hypoxanthine phosphoribosyltransferase; laboratory mouse; microorganism culture; protein isoforms; protein purification; purine analog; site directed mutagenesis

This grant application pertains to a critical issue in the treatment and control of toxoplasmosis, the need for better chemotherapies. Amalgamating techniques of molecular biology, biochemistry, structural biology, and computational chemistry, this proposal offers a multidisciplinary dissection of the hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) enzyme from Toxoplasma gondii, an enzyme that renders an important nutritional function for the parasite and that catalyzes the phosphoribosylation of certain cytotoxic purine base analogs that are not substrates for the human HGPRT counterpart. The proposed investigations constitute a logical step in the implementation of a rational strategy of drug discovery, and ultimately drug design, for the treatment and prevention of toxoplasmosis. Reagents available for these studies include: i. two biochemically distinct T. gondii hgxprt cDNAs ii., E. coli that overproduce each of the T. gondii HGXPRT proteins; and iii effectively unlimited amounts of the two T. gondii HGXPRT proteins that appear homogeneous by SDS-PAGE and iv. hgxprt populations of T. gondii that were generated by insertional mutagenesis. In addition, an homology-based 3-D molecular model of the T. gondii HGXPRT has been computationally constructed and serves as a cornerstone for our structural studies. The first specific aim of this project will be to perform a thorough biochemical characterization of the T. gondii HGXPRT protein. This will involve kinetic, mechanistic, and physicochemical studies on the recombinant protein and the generation of antibodies to determine which of the HGXPRT isoforms is/are physiologically relevant. Specific Aim II will be to evaluate the 3-D model of the HGXPRT protein by site-directed mutagenesis of key amino acid residues that are postulated to participate in catalytic activity or govern substrate specificity and biochemical characterization of the genetically altered proteins. The second aspect of Specific Aim II will be to introduce crystallographic methods to the structural studies for the ultimate purpose of determining the structure of the T. gondii HGXPRT protein itself. The third and final specific aim will involve computational screens of 3-D small molecule structural databases with our molecular models, and ultimately with resolved structures, to discover novel 'lead' compounds that target the active site pocket of the T. gondii HGXPRT protein. Computationally identified compounds from the database screens, as well as approximately 40 procured purine base analogs, will be evaluated as potential antitoxoplasmal compounds using a simple, yet multifaceted, screen comprising of purified recombinant HGXPRT enzymes, E. coli that overexpress hgxprt genes, and intact parasites.

该赠款申请与治疗中的关键问题有关 和控制弓形虫病，需要更好的化学疗法。 分子生物学，生物化学，结构的合并技术 生物学和计算化学，该建议提供了 低黄嘌呤 - 黄甘氨酸 - 黄斑的多学科解剖 来自弓形虫的磷酸贝糖基转移酶（HGXPRT）酶，一种 为寄生虫提供重要的营养功能的酶 并催化某些细胞毒性嘌呤的磷酸蛋白质化 不是人类HGPRT对应物的底物的基本类似物。 拟议的调查构成了逻辑步骤 实施一项合理的药物发现策略，并 最终药物设计，以治疗和预防 弓形虫病。可用于这些研究的试剂包括：i。二 生物化学上不同的T. gondii hgxprt cdnas ii。，大肠杆菌 过量生产T. gondii Hgxprt蛋白；和iii 有效的两个T. gondii HGXPRT蛋白的无限量 SDS-PAGE和IV似乎是同质的。 T.的HGXPRT人群。 由插入诱变产生的gondii。 此外， T. gondii hgxprt的基于同源的3-D分子模型已经是 计算构造并充当我们的基石 结构研究。该项目的第一个具体目的是 对T. gondii进行彻底的生化表征 HGXPRT蛋白。这将涉及动力学，机械性和 关于重组蛋白和产生的物理化学研究 确定哪种HGXPRT同工型为/是的抗体 生理上相关。特定目标II将是评估3-D 通过位置定向诱变的关键氨基的HGXPRT蛋白模型 假定参与催化活性的酸残基 或控制底物特异性和生化表征 遗传改变的蛋白质。 特定目标的第二个方面 II将将晶体学方法引入结构 为了确定结构的最终目的的研究 T. gondii HGXPRT蛋白本身。 第三个也是最后一个特定目标 将涉及3-D小分子结构的计算屏幕 具有分子模型的数据库，并最终通过解决 结构，发现针对活动的新颖的“铅”化合物 T. gondii HGXPRT蛋白的位置袋。计算 从数据库屏幕中识别出的化合物以及 大约有40个采购的嘌呤碱类似物将被评估为 潜在的抗毒性化合物使用简单但多方面的 包括纯化的重组HGXPRT酶的筛网，大肠杆菌 过表达HGXPRT基因和完整的寄生虫。