ENZYME TARGETS OF OPPORTUNISTIC PATHOGENS IN AIDS

尽管机会性感染的治疗取得了进展，但 尽管如此，艾滋病仍然是发病率和死亡率的主要原因。新代理商 是对抗卡氏肺孢子虫、弓形虫感染所需的 结核分枝杆菌和空肠杆菌。我们正在进行 这些生物中三种酶的克隆、表达和性质研究 为新药的开发提供体外靶点；这些酶 二氢叶酸还原酶(DHFR)、胸苷合成酶(TS)和 二氢翼酸合成酶(DHPS)。 我们将比较抑制剂与卡氏肺孢子虫和人类的相互作用。 DHFR使用结合研究和指导的定点突变 分子建模。我们的目标是鉴定分子 导致抑制的物种歧视的相互作用。我们会 同时制备~(15)N-~(13)C标记肺孢子虫DHFR用于核磁共振结构研究 与合作者合作。弓形虫TS-DHFR将被 表达、纯化和鉴定，RS和DHFR结构域将被 通过操纵分离的基因来促进结构 人物刻画。分枝杆菌TS和DHFR克隆将被测序并 这些酶将在大肠杆菌中表达。编码来自以下位置的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 图书馆。这些和肺孢子虫DHP将被表达、纯化和 特色化的。酶的表达将通过亚克隆编码实现 序列送入大肠杆菌或酵母表达系统。这些酶将会是 使用特定亲和系统(如果可用或通过组合)进行纯化 传统的层析技术。将对DHFRs进行筛查 抑制剂。我们已经在x射线结晶学方面建立了合作。 结构决定。 体外酶系统的可用性提供了一种有价值的 用于鉴定和设计新的从头算抑制剂的资源 胸苷合成和叶酸代谢。

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.