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 二氢新蝶呤醛缩酶（DHNA，EC 4.1.2.25）作为靶标 对结核分枝杆菌（MTB）引起的疾病进行治疗干预。 与哺乳动物细胞不同，哺乳动物细胞通过主动获取外源叶酸 运输、MTB 和许多其他细菌必须从头合成叶酸。 DHNA 是 一种存在于代谢途径早期的酶，用于合成还原酶 GTP 中的叶酸。哺乳动物细胞中缺乏 DHNA，使得该酶成为 有吸引力的化疗靶点。通过减少叶酸的消耗 抑制该途径会导致 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包括 该途径中缺乏但编码酶的基因已被鉴定 通过桑格中心 MTB 基因组测序项目。中的 DNA 序列 MTB基因组数据库已初步确定为DHNA的编码。为了 在这项试点研究中，我们建议确定列为 foIX (embi 基因座 MTCY7H7B，登记号 Z95557.1) 和 foIB (swissprot 基因座 FOLB MYCTU， 登记号 006275) DHNA 代码。我们的目标是克隆和表达 在大肠杆菌中检测foiX/foiB，并证明该蛋白具有功能性 DHNA。我们还将通过构建来评估该基因的重要性 DHNA 缺陷的 MTB 菌株。这将在 MTB 中通过等位基因交换完成 基于分枝杆菌的诱变和反选择方法 sacB基因对MTB的热敏复制起点和毒性 蔗糖的存在。这项试点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。它还将 为未来的药物发现研究提供纯化的 DHNA 结构-活性关系、分子建模和晶体学 基于结构的药物设计。