DIHYDRONEOPTERIN ALDOLASE, A TUBERCULOSIS DRUG TARGET

二氢蝶呤醛缩酶，结核病药物靶标

• 批准号: 6511430
• 负责人: WILLIAM J SULING
• 金额: $ 12.03万
• 依托单位: SOUTHERN RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6511430
• 关键词: Mycobacterium tuberculosis; aldehyde lyase; antitubercular agents; bacterial proteins; drug design /synthesis /production; folate; microorganism metabolism; molecular cloning; protein purification; recombinant proteins; tuberculosis

DESCRIPTION: The purpose of this pilot research project is to investigate the enzyme dihydroneopterin aldolase (DHNA, EC 4.1.2.25) as a target for therapeutic intervention in disease caused by Mycobacterium tuberculosis (MTB). Unlike mammalian cells, which acquire folates exogenously through active transport, MTB and many other bacteria must synthesize folates de novo. DHNA is an enzyme present early in the metabolic pathway for the synthesis of reduced folates from GTP. The absence of DHNA in mammalian cells makes this enzyme an attractive target for chemotherapy. Depletion of reduced folates through inhibition of this pathway leads to inhibition of DNA, RNA and protein synthesis. Comprehensive studies of the folate biosynthetic pathway in MTB are lacking but genes coding for enzymes in this pathway have been identified through the Sanger Centre MTB genome sequencing project. A DNA sequence in the MTB genome data base has been identified tentatively as coding for DHNA. For this pilot study, we propose to establish that the gene listed as foIX (embi locus MTCY7H7B, accession Z95557.1) and foIB (swissprot locus FOLB MYCTU, accession 006275) codes for DHNA. Our objectives will be to clone and express the foiX/foiB in Escherichia coli, and prove that the protein is functionally DHNA. We will also assess the essentiality of the gene by construction of DHNA-deficient MTB strains. This will be done in MTB by allelic exchange mutagenesis and a counter selection method based upon a mycobacterial thermosensitive origin of replication and toxicity of the sacB gene to MTB in the presence of sucrose. The results of this pilot study will enable us to better understand the biochemistry of folate metabolism in MTB. It will also provide purified DHNA for future drug discovery studies based upon structure-activity relationships, molecular modeling and crystallographic structure-based drug design.

描述：该试点研究项目的目的是调查 酶二氢蛋白酶醛酸酶（DHNA，EC 4.1.2.25）作为目标 由结核分枝杆菌（MTB）引起的疾病的治疗干预。 与哺乳动物细胞不同，后者通过活跃的 运输，MTB和许多其他细菌必须合成从头开始。 DHNA是 在代谢途径早期存在的一种酶，用于合成还原 GTP的叶子。在哺乳动物细胞中不存在DHNA使该酶A 化学疗法的有吸引力的靶标。减少叶的消耗 该途径的抑制会导致DNA，RNA和蛋白质的抑制 合成。 MTB中叶酸生物合成途径的综合研究是 缺乏但已经鉴定出在此途径中编码酶的基因 通过Sanger Center MTB基因组测序项目。 DNA序列 MTB基因组数据库已被暂时确定为DHNA的编码。为了 这项试验研究，我们建议确定该基因被列为泡沫（Embi 基因座MTCY7H7B，登录Z95557.1）和FOIB（瑞士语locus folb myctu， 登录006275）DHNA的代码。我们的目标是克隆并表达 大肠杆菌中的泡沫/泡沫，证明该蛋白质在功能上是 DHNA。我们还将通过构造来评估基因的本质 缺乏DHNA的MTB菌株。这将由等位基因交换在MTB中完成 诱变和基于分枝杆菌的反选择方法 SACB基因对MTB的复制和毒性的热敏性起源 蔗糖的存在。这项试验研究的结果将使我们能够 更好地了解MTB中叶酸代谢的生物化学。它也会 根据 结构活性关系，分子建模和晶体学 基于结构的药物设计。