FolK, a Mycobacterium tuberculosis Drug Target

描述（由申请人提供）：本试点项目的目的是研究酶6-羟甲基-7,8-二氢蝶呤焦磷酸激酶（FolK， HPPK, EC 2.7.6.3）作为干预结核分枝杆菌（MTB）引起的疾病的靶标。与脊椎动物细胞通过主动运输从外源获取叶酸不同，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合成还原性叶酸。宿主体内缺乏HPPK使得这种酶成为化疗的一个有吸引力的靶标。通过抑制这一途径减少叶酸的消耗导致DNA、RNA和蛋白质合成的抑制。目前还缺乏对分枝杆菌中叶酸途径的全面研究，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酶的编码基因。MTB基因组数据库中的DNA序列已被注释为可能是HPPK的folK编码。在本次中试研究中，我们建议建立Rv3606c基因编码HPPK。我们的目标是在大肠杆菌线圈中克隆和表达Rv3606c，并证明该蛋白在功能上是HPPK。我们还将通过构建缺乏hppk的MTB菌株来评估该基因的重要性。这将在MTB中通过等位基因交换诱变和基于分枝杆菌复制的热敏起源和sacB基因在蔗糖存在下对MTB的毒性的反选择方法来完成。这项初步研究的结果将使我们更好地了解结核分枝杆菌中叶酸代谢的生物化学。它还将为未来基于结构-活性关系、分子模型和基于晶体结构的药物设计的药物发现研究提供纯化的HPPK。

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.