Folk, a Mycobacterium tuberculosis Drug Target

Folk，结核分枝杆菌的药物靶点

• 批准号: 6734205
• 负责人: WILLIAM J SULING
• 金额: $ 12.08万
• 依托单位: SOUTHERN RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-15 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6734205
• 关键词: Escherichia coli; Mycobacterium tuberculosis; SDS polyacrylamide gel electrophoresis; drug design /synthesis /production; enzyme activity; enzyme mechanism; folate; gene expression; genetic manipulation; matrix assisted laser desorption ionization; microorganism disease chemotherapy; microorganism metabolism; molecular cloning; other phosphotransferase; phenotype; protein purification; protein quantitation /detection; recombinant proteins; vitamin biosynthesis; western blottings

DESCRIPTION (provided by applicant): The purpose of this pilot project is to investigate the enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (FolK, HPPK, EC 2.7.6.3) as a target for intervention in disease caused by Mycobacterium tuberculosis (MTB). Unlike vertebrate cells, which acquire folates exogenously through active transport, MTB and many other bacteria must synthesize folate de novo. HPPK is an enzyme present early in the metabolic pathway for the synthesis of reduced folates from GTP. The absence of HPPK in the host 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 pathway in mycobacteria are lacking but genes coding for enzymes in the pathway have been identified through the Sanger Centre MTB genome sequencing project. A DNA sequence in the MTB genome database has been annotated as a probable folK coding for HPPK. For this pilot study, we propose to establish that the gene listed as Rv3606c codes for HPPK. Our objectives are to clone and express Rv3606c in Escherichia coil, and prove that the protein is functionally HPPK. We will also assess the essentiality of the gene by construction of HPPK-deficient MTB strains. This will be done in MTB by allelic exchange mutagenesis and a counterselection 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 HPPK for future drug discovery studies based upon structure-activity relationships, molecular modeling and crystallographic structure-based drug design.

描述(申请人提供)：这项试点项目的目的是研究6-羟甲基-7，8-二氢蝶呤焦磷酸激酶(FLOCK，HPPK，EC 2.7.6.3)作为干预结核分枝杆菌(MTB)引起的疾病的目标。与通过主动运输外源获得叶酸的脊椎动物细胞不同，结核分枝杆菌和许多其他细菌必须从头合成叶酸。HPPK是一种存在于代谢途径早期的酶，用于从GTP合成还原型叶酸。宿主中没有HPPK，这使得这种酶成为有吸引力的化疗靶点。通过抑制这一途径来耗尽还原的叶酸会导致DNA、RNA和蛋白质合成的抑制。对分枝杆菌中的叶酸途径缺乏全面的研究，但通过桑格中心结核分枝杆菌基因组测序项目已经确定了该途径中的酶的编码基因。结核分枝杆菌基因组数据库中的一个DNA序列已被注释为HPPK的可能编码基因。在这项初步研究中，我们建议确定被列为Rv3606c的基因编码HPPK。我们的目标是克隆并在大肠杆菌中表达Rv3606c，并证明该蛋白具有HPPK功能。我们还将通过构建HPPK缺陷的结核分枝杆菌菌株来评估该基因的重要性。这将在结核分枝杆菌中通过等位基因交换突变和基于分枝杆菌热敏感复制起点和蔗糖存在下对结核分枝杆菌的毒性的反选择方法来完成。这项先导性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。它还将为基于构效关系、分子建模和基于晶体结构的药物设计的未来药物发现研究提供纯化的HPPK。