ENZYME TARGETS OF OPPORTUNISTIC PATHOGENS IN AIDS

艾滋病中机会性病原体的酶靶标

• 批准号: 2067689
• 负责人: DANIEL V. SANTI
• 金额: $ 17.1万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-01 至 1999-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2067689
• 关键词: AIDS; Mycobacterium avium; Mycobacterium tuberculosis; Pneumocystis carinii; Toxoplasma gondii; antiAIDS agent; antibiotics; chemical binding; dihydrofolate reductase; drug design /synthesis /production; enzyme inhibitors; enzyme mechanism; folate antagonist; intermolecular interaction; methyltransferase; molecular cloning; nuclear magnetic resonance spectroscopy; opportunistic infections; protein purification; pteridines; site directed mutagenesis; thymidylate synthase; transfection

Despite advances in the therapy of opportunistic infections complicating AIDS, they remain a leading cause of morbidity and mortality. New agents are needed to combat infections by Pneumocystis carinii, Toxoplasma gondii and Mycobacterium tuberculosis and avium. We are in the process of cloning, expressing and characterizing three enzymes from these organisms to provide in vitro targets for the development of new drugs; these enzymes are dihydrofolate reductase (DHFR), thymidylate synthase (TS), and dihydropteroate synthase (DHPS). We will compare the interactions of inhibitors with P. carinii and human DHFR using binding studies and site directed mutagenesis guided by molecular modeling. The objective is to identify the molecular interactions which lead to species discrimination of inhibition. We will also prepare 15N-13C labeled Pneumocystis DHFR for NMR structural studies in collaboration with a collaborator. Toxoplasma TS-DHFR will be expressed, purified and characterized, and the RS and DHFR domains will be separated by manipulation of the gene to facilitate structural characterization. Mycobacterial TS and DHFR clones will be sequenced and the enzymes will be expressed in E. coli. Clones encoding DHPS from Toxoplasma and Mycobacterium will be isolated from cDNA and genomic DNA libraries. These and the Pneumocystis DHPS will be expressed, purified and characterized. Expression of enzymes will be achieved by subcloning coding sequences into E. coli or yeast expression systems. The enzymes will be purified using specific affinity systems when available or by combinations of conventional chromatography. DHFRs will be screened with existing inhibitors. We have established collaborations for x-ray crystallographic structural determinations. The availability of in vitro enzyme systems is providing a valuable resource for the identification and design of new inhibitors of de novo thymidylate synthesis and folate metabolism.

尽管在机会感染的治疗方面取得了进步 艾滋病，它们仍然是发病率和死亡率的主要原因。 新代理商 需要用肺炎刺激性肺炎，弓形虫弓形虫对抗感染 结核分枝杆菌和鸟类。 我们正在 克隆，表达和表征来自这些生物的三种酶 为开发新药提供体外靶标；这些酶 是二氢叶酸还原酶（DHFR），胸苷酸合酶（TS）和 二氢蛋白酶合酶（DHP）。 我们将比较抑制剂与P. carinii和人类的相互作用 DHFR使用结合研究和站点指导的诱变。 分子建模。 目的是识别分子 导致物种歧视的相互作用。 我们将 还准备15n-13c标记为肺炎藻DHFR进行NMR结构研究 与合作者合作。 弓形虫TS-DHFR将是 表达，纯化和表征，RS和DHFR域将是 通过操纵基因隔离以促进结构 表征。 分枝杆菌TS和DHFR克隆将进行测序，并且 这些酶将在大肠杆菌中表达。克隆编码DHP的克隆 弓形虫和分枝杆菌将从cDNA和基因组DNA中分离出来 库。 这些和肺炎藻DHP将被表达，纯化和 特征。 酶的表达将通过亚克隆编码来实现 序列到大肠杆菌或酵母表达系统中。 酶将是 使用特定亲和力系统纯化或组合 常规色谱。 DHFR将与现有 抑制剂。 我们已经建立了X射线晶体学的合作 结构确定。 体外酶系统的可用性正在提供有价值的 识别和设计新抑制剂的资源 胸苷酸合成和叶酸代谢。