ENZYME TARGETS OF OPPORTUNISTIC PATHOGENS IN AIDS

尽管机会性感染的治疗取得了进展， 艾滋病仍然是发病和死亡的主要原因。 新药剂 是用来对抗卡氏肺孢子虫、弓形虫 和结核分枝杆菌和鸟。 我们正在 从这些生物体中克隆、表达和表征三种酶 为新药的开发提供体外靶点;这些酶 是二氢叶酸还原酶（DHFR）、胸苷酸合成酶（TS），和 二氢蝶酸合酶（DHPS）。 我们将比较抑制剂与卡氏肺孢子虫和人的相互作用 DHFR结合研究和定点诱变 分子模拟 目的是鉴定 相互作用，导致物种歧视的抑制。 我们将 还制备了15 N-13 C标记的肺孢子虫DHFR用于NMR结构研究 in collaboration合作with a collaborator合作者. 弓形虫TS-DHFR将是 表达，纯化和表征，RS和DHFR结构域将被 通过操纵基因来分离， 特征化 将对分枝杆菌TS和DHFR克隆进行测序， 这些酶将在E.杆菌编码DHPS的克隆， 将从cDNA和基因组DNA中分离弓形虫和分枝杆菌 图书馆. 这些和肺孢子虫DHPS将被表达、纯化并 表征了 酶的表达将通过亚克隆编码 序列插入E.大肠杆菌或酵母表达系统。 这些酶将 当可用时，使用特定亲和系统或通过组合纯化 常规色谱法。 将使用现有的 抑制剂的 我们已经建立了合作， 结构决定。 体外酶系统的可用性提供了有价值的 用于识别和设计新的从头抑制剂的资源 胸苷酸合成和叶酸代谢。

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.