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: 5205491
• 负责人: BUDDY ULLMAN
• 金额: --
• 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/5205491
• 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.

该拨款申请涉及治疗中的一个关键问题 而控制弓形体病，需要更好的化疗。 分子生物学、生物化学、结构技术的融合技术 生物学和计算化学，该提案提供了 次黄嘌呤-鸟嘌呤-黄嘌呤的多学科解剖 来自弓形虫的磷酸核糖转移酶 (HGXPRT)，一种 为寄生虫提供重要营养功能的酶 并催化某些细胞毒性嘌呤的磷酸核糖基化 碱基类似物不是人类 HGPRT 对应物的底物。 拟议的调查是合乎逻辑的一步 实施合理的药物发现策略，以及 最终的药物设计，用于治疗和预防 弓形体病。可用于这些研究的试剂包括：二 生化上不同的弓形虫 hgxprt cDNA ii.、大肠杆菌 过量产生每种弓形虫 HGXPRT 蛋白；和三 实际上无限量的两种弓形虫 HGXPRT 蛋白 通过 SDS-PAGE 和 iv 看来均质。 T 的 hgxprt 种群。 通过插入诱变产生的弓形虫。 此外， 基于同源性的弓形虫 HGXPRT 3D 分子模型已被 通过计算构建并作为我们的基石 结构研究。该项目的第一个具体目标是 对弓形虫进行彻底的生化表征 HGXPRT 蛋白。这将涉及动力学、机械和 重组蛋白的理化研究及其产生 抗体以确定哪些 HGXPRT 同种型是/是 生理相关。具体目标 II 将是评估 3-D 通过关键氨基定点突变建立 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特征 基因改变的蛋白质。 具体目标的第二个方面 II 将晶体学方法引入结构 研究的最终目的是确定结构 弓形虫 HGXPRT 蛋白本身。 第三个也是最后一个具体目标 将涉及 3D 小分子结构的计算屏幕 数据库与我们的分子模型，并最终解决 结构，发现针对活性物质的新型“先导”化合物 弓形虫 HGXPRT 蛋白的位点口袋。计算上 从数据库筛选中识别出化合物，以及 大约 40 种采购的嘌呤碱类似物，将被评估为 潜在的抗弓形虫化合物使用简单但多方面的， 筛选由纯化的重组 HGXPRT 酶、大肠杆菌组成， 过度表达 hgxprt 基因和完整的寄生虫。