HYPOXANTHINE GUANINE PHOSPHORIBOSYLTRANSFERASE FROM TOXOPLASMA GONDII

这项拨款申请与治疗中的一个关键问题有关 以及控制弓形虫病，需要更好的化疗方法。 分子生物学、生物化学、结构学的融合技术 生物学和计算化学，这项建议提供了一个 次黄嘌呤-鸟嘌呤-黄嘌呤的多学科剖析 弓形虫磷酸核糖基转移酶(HGXPRT) 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 不是人类HGPRT类似物底物的碱基类似物。 拟议的调查构成了 实施合理的药物发现战略，以及 最终药物设计，用于治疗和预防 弓形虫病。可用于这些研究的试剂包括：I.两 生物化学特征不同的弓形虫hgxprt cDNAs II.大肠杆菌 过量生产每个弓形虫HGXPRT蛋白；以及 两种弓形虫HGXPRT蛋白的有效无限量 经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法和静脉注射法鉴定均一。T.hgxprt种群 通过插入诱变产生的弓形虫。此外, 建立了基于同源性的弓形虫HGXPRT三维分子模型 以计算方式构建，并作为我们的 结构研究。该项目的第一个具体目标将是 对弓形虫进行彻底的生化鉴定 HGXPRT蛋白。这将涉及到动力学、机械性和 重组蛋白及其产生的物理化学研究 以确定哪些HGXPRT亚型是/是 生理上相关的。具体目标II将是评估3D 关键氨基酸定点突变HGXPRT蛋白模型 被认为参与催化活性的酸性残基 或控制底物专一性和生化特性 经过基因改造的蛋白质。具体目标的第二个方面 二是将结晶学方法引入到结构 研究的最终目的是确定 弓形虫HGXPRT蛋白本身。第三个也是最后一个具体目标 将涉及三维小分子结构的计算筛选 数据库与我们的分子模型，并最终与解决 结构，以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。从计算上讲 从数据库屏幕中识别出的化合物，以及 大约40个采购的嘌呤类似物，将被评估为 潜在的抗弓形虫化合物使用简单但多方面的， 含纯化重组HGXPRT酶的大肠杆菌的筛选 过度表达hgxprt基因，以及完整的寄生虫。

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.