ANTIFOLATES AGAINST MYCOBACTERIAL INFECTIONS IN AIDS

描述：(改编自申请者摘要)两个分枝杆菌构成 对感染人类免疫缺陷病毒的个人的主要威胁 (HIV)、禽型分枝杆菌和结核分枝杆菌。发展中的 如果努力，治疗禽类分枝杆菌和结核分枝杆菌的新药是迫切需要的 控制它们对获得性免疫缺陷综合征预后的影响 (艾滋病)是成功的。本项目的目的是评估 一类独特的药物抑制分枝杆菌的有效性 分枝杆菌二氢叶酸还原酶(DHFR)是植物生长发育过程中的关键酶 RNA、DNA和蛋白质必需的叶酸生物合成途径 综合。这种酶代表了一个战略目标，因为它有能力 为了开发选择性抑制剂，可以超过数千倍 对细菌DHFR的活性高于哺乳动物DHFR。通过……的努力 NIAID发起的结核病抗菌药物采购协调 设施(TAACF)，已经确定了几种亲脂性DHFR抑制剂 从2，4-二氨基-5-甲基-5-去氮杂蝶啶衍生物的集合中 在南方研究所(SRI)综合。在TAACF筛查中， 这些新型的亲脂性抗叶酸盐表现出低的最低限度的抑制。 对结核分枝杆菌H37Rv的最低抑菌浓度(MIC)，以及对 Vero细胞。在SRI，这些衍生物在一种 艾滋病常见血清型--禽分枝杆菌第4型的无细胞DHFR检测 病人。这些新化合物的抗分枝杆菌活性评价 亲脂性的DHFR抑制剂将在三个水平上针对M。 禽类和结核分枝杆菌：1)筛查 2，4-二氨基-5-甲基-5-去氮杂蝶啶衍生物将在 体外微量肉汤稀释法和无细胞分枝杆菌DHFR法 并与对人类细胞株的毒性和抗肿瘤活性进行比较 重组人DHFR，2)选定化合物的胞内活性将 在小鼠和人单核细胞系模型中进行评估，以及，3)体内 候选化合物的活性将在感染的小鼠模型中确定。 将对禽类分枝杆菌和结核分枝杆菌H37Ra DHFR进行纯化和测序 重组dhfr的比较和克隆。DHFR基因将是 从禽流感分枝杆菌和结核分枝杆菌H37Ra克隆。重组DHFR将被 表达和纯化，以获得足够量的特定 抑制研究将用于开发具有改进的衍生物 选择性。根据这项研究的信息，一种比较分子 现场分析(CoMFA)将被用来预测更有效和更有选择性 脱氢表雄酮受体抑制剂。这些研究最终将导致 用X-射线结晶学和X射线衍射法研究亲脂性DHFR抑制剂 分子图形技术。

DESCRIPTION: (Adapted from Applicant's Abstract) Two mycobacteria pose a major threat to individuals infected with the human immunodeficiency virus (HIV), Mycobacterium avium and Mycobacterium tuberculosis. Development of new drugs for M. avium and M. tuberculosis is an urgent necessity if efforts to control their effect on the outcome of acquired immunodeficiency syndrome (AIDS) are to be successful. The purpose of this project is to evaluate the antimycobacterial effectiveness of a unique class of drugs that inhibit mycobacterial dihydrofolate reductase (DHFR), a key enzyme in the biosynthetic pathway of folates that are necessary for RNA, DNA and protein synthesis. This enzyme represents a strategic target because of the ability to develop selective inhibitors that can be several thousand times more active against bacterial DHFR than mammalian DHFR. Through the efforts of the NIAID sponsored Tuberculosis Antimicrobial Acquisition Coordinating Facility (TAACF), several lipophilic DHFR inhibitors have been identified from a collection of 2,4-diamino-5-methyl-5-deazapteridine derivatives synthesized at Southern Research Institute (SRI). In the TAACF screening, these novel lipophilic antifolates exhibited low minimal inhibitory concentrations (MICs) against M. tuberculosis H37Rv, and low toxicity to Vero cells. At SRI, the derivatives have shown selective inhibition in a cell-free DHFR assay from M. avium serovar 4, a common serovar found in AIDS patients. Evaluation of the antimycobacterial activity of these novel lipophilic DHFR inhibitors will be conducted at three levels against M. avium and M. tuberculosis: 1) screening of 2,4-diamino-5-methyl-5-deazapterdine derivatives will be performed with an in vitro broth microdilution assay and cell-free mycobacterial DHFR assays and compared with toxicity to human cell lines and activity against recombinant human DHFR, 2) intracellular activity of selected compounds will be evaluated in murine and human monocytic cell line models and, 3) in vivo activity of candidate compounds will be determined in infected mouse models. M. avium and M. tuberculosis H37Ra DHFR will be purified and sequenced for comparison with, and cloning of, recombinant DHFR. The DHFR gene will be cloned from M. avium and M. tuberculosis H37Ra. Recombinant DHFR will be expressed and purified in order to obtain sufficient quantities for specific inhibition studies to be used to develop derivatives with improved selectivity. With information from this study, a comparative molecular field analysis (CoMFA) will be used to predict more effective and selective DHFR inhibitors. These studies will eventually lead to the development of site directed lipophilic DHFR inhibitors using X-ray crystallographic and molecular graphic techniques.