ANTIFOLATES AGAINST MYCOBACTERIAL INFECTIONS IN AIDS

针对艾滋病分枝杆菌感染的抗叶酸剂

• 批准号: 2887447
• 负责人: William W Barrow
• 金额: $ 37.55万
• 依托单位: SOUTHERN RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2001-03-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2887447
• 关键词: AIDS; Actinomycetales infection; Mycobacterium avium; Mycobacterium tuberculosis; Vero cells; antitubercular agents; bacterial proteins; chemical synthesis; dihydrofolate reductase; drug screening /evaluation; enzyme activity; folate antagonist; laboratory mouse; microorganism disease chemotherapy; molecular cloning; nonhuman therapy evaluation; oxidoreductase inhibitor; recombinant proteins; respiratory disorder chemotherapy

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.

描述：（改编自申请人的摘要）两种分枝杆菌构成了一种分枝杆菌。 对感染人体免疫机能丧失病毒者的主要威胁 (HIV)、鸟分枝杆菌（Mycobacterium avium）和结核分枝杆菌（Mycobacterium tuberculosis）。 发展 新药M. avium和M.结核病是一个迫切需要， 以控制其对获得性免疫缺陷综合征结果的影响 （艾滋病）是成功的。 该项目的目的是评估 一类独特的药物的抗分枝杆菌有效性， 分枝杆菌二氢叶酸还原酶（DHFR），一种关键酶， 叶酸的生物合成途径是RNA，DNA和蛋白质所必需的 合成. 这种酶代表了一个战略目标，因为它能够 开发出选择性抑制剂， 对细菌DHFR的活性高于哺乳动物DHFR。 通过努力 NIAID赞助的结核病抗菌药物采购协调 设施（TAACF），已确定了几种亲脂性DHFR抑制剂 来自2，4-二氨基-5-甲基-5-脱氮杂蝶啶衍生物的集合 南方研究所（SRI）。 在TAACF筛选中， 这些新型亲脂性抗叶酸剂表现出较低的最小抑制作用 浓度（MIC）。结核病H37 Rv，低毒性 Vero细胞。 在SRI中，衍生物显示出选择性抑制， 无细胞DHFR测定来自M.禽流感血清型4，艾滋病中发现的常见血清型 患者 评价这些新的抗分枝杆菌活性 亲脂性DHFR抑制剂将针对M在三个水平下进行。 avium和M.结核病：1）筛查 2，4-二氨基-5-甲基-5-脱氮杂喋啶衍生物将与 体外肉汤微量稀释试验和无细胞分枝杆菌DHFR试验 并与对人类细胞系的毒性和对 重组人DHFR，2）所选化合物的细胞内活性将 在鼠和人单核细胞系模型中进行评价，3）在体内 候选化合物的活性将在感染的小鼠模型中测定。 M. avium和M.结核病H37 Ra DHFR将被纯化并测序， 重组DHFR的比较和克隆。 DHFR基因将 克隆自M. avium和M.结核病H37 Ra。 重组DHFR将 表达和纯化，以获得足够的数量， 抑制研究用于开发具有改进的 选择性 根据这项研究的信息， 场分析（CoMFA）将用于预测更有效和选择性 DHFR抑制剂。 这些研究最终将导致 使用X-射线晶体学的定点亲脂性DHFR抑制剂， 分子图像技术