DIHYDRONEOPTERIN ALDOLASE, A TUBERCULOSIS DRUG TARGET

描述：本试验研究项目的目的是调查 酶二氢新蝶呤醛缩酶（DHNA，EC 4.1.2.25）作为 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， 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，用于合成还原的 叶酸从GTP。哺乳动物细胞中缺乏DHNA，使这种酶成为一种 有吸引力的化疗靶点。通过以下途径消耗还原叶酸 抑制该途径导致抑制DNA、RNA和蛋白质 合成. MTB中叶酸生物合成途径的综合研究 缺乏，但已经鉴定了编码该途径中的酶的基因 通过桑格中心的MTB基因组测序项目。一个DNA序列 MTB基因组数据库已被初步确定为编码DHNA。为 在这项初步研究中，我们建议确定被列为foIX（恩比）的基因 基因座MTCY 7 H7 B，登录号Z95557.1）和foIB（swissprot基因座FOLB MYCTU， 登录号006275）编码。我们的目标是克隆和表达 foiX/foiB在大肠杆菌中的表达，并证明该蛋白在功能上是 DHNA。我们还将通过构建 缺乏DHA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基因对结核分枝杆菌的热敏复制起点和毒性 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、分子模拟和晶体学 基于结构的药物设计

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.