FolK, a Mycobacterium tuberculosis Drug Target

FolK，结核分枝杆菌药物靶点

• 批准号: 6590941
• 负责人: WILLIAM J SULING
• 金额: $ 12.12万
• 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 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6590941
• 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（由申请人提供）：该试验项目的目的是研究酶6-羟基甲基-7,8-二氢蛋白毒素焦磷酸激酶（Folk，HPPK，EC 2.7.6.3），作为由结核病（MTB）引起的疾病干预的目标。与脊椎动物细胞通过主动转运进行外源的叶出现不同，MTB和许多其他细菌必须合成从头开始的叶酸。 HPPK是一种在代谢途径早期存在的酶，用于合成GTP的减少叶状体。宿主中缺乏HPPK使该酶成为化学疗法的有吸引力的靶标。通过抑制这种途径减少掉落的掉落会导致DNA，RNA和蛋白质合成的抑制。缺乏对分枝杆菌中叶酸途径的全面研究，但是通过Sanger Center MTB基因组测序项目鉴定出了该途径中编码酶的基因。 MTB基因组数据库中的DNA序列已被注释为HPPK的可能民间编码。在这项试点研究中，我们建议确定该基因列为HPPK的RV3606C代码。我们的目标是克隆并表达Escherichia卷轴中的RV3606C，并证明该蛋白质在功能上是HPPK。我们还将通过构建HPPK缺陷型MTB菌株来评估基因的重要性。这将在MTB中通过等位基因交换诱变和基于分枝杆菌在蔗糖存在下对MTB的复制和毒性的反敏感性的反登录方法。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。它还将根据结构 - 活性关系，分子建模和基于晶体结构的药物设计提供纯净的HPPK，用于未来的药物发现研究。