DIHYDRONEOPTERIN ALDOLASE, A TUBERCULOSIS DRUG TARGET

二氢蝶呤醛缩酶，结核病药物靶标

• 批准号: 6312374
• 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 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6312374
• 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：这项先导研究项目的目的是调查 酶二氢喋呤醛缩酶(DHNA，EC 4.1.2.25)作为靶标 结核分枝杆菌(MTB)引起疾病的治疗干预。 与哺乳动物细胞不同，哺乳动物细胞通过活性获得外源叶酸 运输、结核分枝杆菌和许多其他细菌必须从头合成叶酸。DHNA是 一种存在于新陈代谢途径早期的酶，用于合成还原的 来自GTP的叶酸。哺乳动物细胞中缺乏DHNA使这种酶成为一种 是有吸引力的化疗靶点。通过以下途径消耗还原的叶酸 抑制这一途径会导致DNA、RNA和蛋白质的抑制 综合。结核分枝杆菌叶酸生物合成途径的综合研究 缺乏但编码这一途径中的酶的基因已被鉴定 通过桑格中心结核分枝杆菌基因组测序项目。基因中的DNA序列 结核分枝杆菌基因组数据库已初步确定为DHNA的编码。为 在这项先导性研究中，我们建议建立列为FOIX(EMBI)的基因 基因座MTCY7H7B，注册号Z95557.1)和foIB(swissprot基因座Folb MYCTU， 加入006275)DHNA的代码。我们的目标将是克隆和表达 在大肠杆菌中表达foix/foiB，并证明该蛋白具有功能。 DHNA。我们还将通过构建 DHNA缺陷型结核分枝杆菌。这将通过等位基因交换在MTB中完成 基于分枝杆菌的诱变和反选择方法 SACB基因复制的温敏来源及对结核分枝杆菌的毒性 蔗糖的存在。这项初步研究的结果将使我们能够 更好地了解结核分枝杆菌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 构效关系、分子模拟和结晶学 基于结构的药物设计。