GENETIC ANALYSIS OF PURINE METABOLISM IN LEISHMANIA DONO

利什曼原虫嘌呤代谢的遗传分析

• 批准号: 7208083
• 负责人: BUDDY ULLMAN
• 金额: $ 35.79万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-06-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7208083
• 关键词: 3-Dimensional; Active Sites; Affinity; Allopurinol; Amino Acids; Applications Grants; Biochemical; Biochemistry; Catalysis; Chagas Disease; Databases; Dissection; Drug Design; Enzymes; Escherichia coli; Evaluation; Exhibits; Genes; Genetic; Homologous Gene; Hypoxanthine Phosphoribosyltransferase; Lead; Leishmania; Leishmania donovani; Leishmaniasis; Metabolism; Methods; Modeling; Molecular Biology Techniques; Nutritional; Parasites; Parasitic Diseases; Pharmaceutical Preparations; Photoaffinity Labels; Population; Prevention; Protein Overexpression; Proteins; Purines; Purpose; Reagent; Recombinants; Role; Site-Directed Mutagenesis; Structure; Substrate Specificity; Techniques; Testing; Treatment Efficacy; Trypanosoma brucei brucei; Trypanosoma cruzi; Virulence; analog; base; chemotherapy; computational chemistry; drug discovery; gene function; gene replacement; genetic analysis; molecular modeling; multidisciplinary; mutant; novel; purine; purine metabolism; small molecule; structural biology

This grant application pertains to a critical issue in the treatment and control of parasitic diseases, the need for better chemotherapies. Amalgamating techniques of molecular biology, biochemistry, genetics, structural biology, and computational chemistry this proposal offers a multidisciplinary dissection of the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme from Leishmania donovani, an enzyme that renders an important, if not essential, nutritional function for the parasite, and that initiates the metabolism of allopurinol, a drug that exhibits therapeutic efficacy in both leishmaniasis and Chagas disease. These studies constitute a logical step in the implementation of a rational, structure-based strategy of drug discovery, and ultimately drug design, for the treatment and prevention of leishmaniasis and other diseases of parasitic origin. Reagents available for these studies include: i. the L. donovani, T. brucei, T. cruzi and C. fasciculata hgprt genes; ii., the L. donovani aprt gene; iii., hgprt- populations of L. donovani that were generated by targeted gene replacement;' iv., E. coli that overproduce each of the trypanosomatid HGPRTs and v., effectively unlimited amounts of L. donovani, T. brucei, T. cruzi, and C. fasciculata HGPRT proteins that appear homogeneous by SDS-PAGE. In addition, an homology-based 3-D molecular model of the L. donovani HGPRT has been computationally constructed and serves as a cornerstone for our structural studies. The first objective of this application is to evaluate the contributions of HGPRT and APRT to purine metabolism in L. donovani promastigotes by phenotypic characterization of hgprt- and aprt- null mutants that will be created by homologous gene replacement. Whether hgprt and/or aprt gene function is essential for infectivity and virulence will also be tested by generating null mutants in infective Leishmania strains. The second specific aim entails the structural characterization of the L. donovani HGPRT. The first component of this specific aim will be to evaluate the 3-D model of the 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 either refining the 3-D molecular model or determining the structure of the L. donovani HGPRT protein itself. The penultimate specific aim involves the identification of key active site residues of the L. donovani HGPRT that participate in catalysis using affinity and photoaffinity labeling techniques and further evaluation of the functional role of these amino acids in catalysis by site-directed mutagenesis. Lastly, we will perform 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 L. donovani HGPRT. Computationally identified compounds from the database screens, as well as 40 procured purine base analogs, will be evaluated as potential antileishmanial compounds using a simple, yet multifaceted, screen comprising of purified recombinant HGPRT enzymes, E. coli that overexpress hgprt genes, and intact parasites.

该赠款申请与治疗中的关键问题有关 控制寄生疾病，需要更好的化学疗法。 分子生物学，生物化学，遗传学，融合技术 结构生物学和计算化学这一建议提供了 低黄嘌呤瓜氨酸的多学科解剖 来自利什曼原虫Donovani的磷酸贝糖基转移酶（HGPRT）酶 使营养功能重要的，即使不是必需的酶 对于寄生虫，并启动了植物的代谢，一种药物 这在利什曼病和chagas中都表现出治疗功效 疾病。 这些研究构成了实施的逻辑步骤 一种理性的，基于结构的药物发现策略，最终 药物设计，用于治疗和预防利什曼病和其他 寄生虫疾病。这些研究可用于这些研究 包括：i。 L. Donovani，T。Brucei，T。Cruzi和C. fasciculata Hgprt 基因； II。，Donovani Aprt基因； iii。 由靶向基因置换产生的Donovani； iv。，大肠杆菌 过量生产了每个锥虫的HGPRT和v。有效地生产 L. Donovani，T。Brucei，T。Cruzi和C. fasciculata的无限量 通过SDS-PAGE看起来同质的HGPRT蛋白。另外，一个 Donovani Hgprt的基于同源性的3-D分子模型已经 计算构建并充当我们的结构的基石 研究。该应用程序的第一个目的是评估 HGPRT和APRT对L. Donovani的嘌呤代谢的贡献 hgprt和aprt-null的表型表征的前差。 由同源基因替代产生的突变体。是否HGPRT 和/或APRT基因功能对于感染性和毒力至关重要 也可以通过在感染性利什曼原虫菌株中产生无效突变体来测试。 第二个特定目的需要L。 Donovani Hgprt。该特定目标的第一个组成部分是 通过钥匙的位置定向诱变评估蛋白质的3-D模型 假定参与催化的氨基酸残基 活动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II的第二个方面 将向结构研究介绍晶体学方法 出于完善3-D分子模型或 确定多诺瓦乳杆菌HGPRT蛋白本身的结构。这 倒数第二特定的目的涉及识别关键活动站点 L. donovani Hgprt的残留物，用于参与使用的催化 亲和力和光性标签技术，并进一步评估 这些氨基酸在催化中的功能作用通过位置定向 诱变。最后，我们将执行3-D小的计算屏幕 分子数据库与我们的分子模型，最终 使用已解决的结构，发现靶向的新型“铅”化合物 Donovani Hgprt的活跃场地袋。 计算 从数据库屏幕中识别出的化合物，以及40种采购的化合物 嘌呤碱基类似物将被评估为潜在的抗抗精神 使用简单但多方面的屏幕包含纯化的化合物 重组HGPRT酶，过表达HGPRT基因的大肠杆菌和 完整的寄生虫。