FolK, a Mycobacterium tuberculosis Drug Target

描述（由申请方提供）：本试验项目的目的是研究6-羟甲基-7，8-二氢蝶呤焦磷酸激酶（FolK，HPPK，EC 2.7.6.3）作为结核分枝杆菌（MTB）引起疾病的干预靶点。与脊椎动物细胞通过主动转运外源获得叶酸不同，MTB和许多其他细菌必须从头合成叶酸。HPPK是存在于用于从GTP合成还原叶酸的代谢途径早期的酶。宿主中HPPK的缺乏使得这种酶成为化疗的有吸引力的靶标。通过抑制该途径消耗还原叶酸导致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MTB基因组测序项目已经鉴定出了该途径中编码酶的基因。MTB基因组数据库中的DNA序列已被注释为可能编码HPPK的folK。对于这项初步研究，我们建议确定列为Rv 3606 c的基因编码HPPK。我们的目的是克隆Rv 3606 c并在大肠杆菌中表达，证明其功能为HPPK。我们还将通过构建HPPK缺陷型MTB菌株来评估该基因的必要性。这将在MTB中通过等位基因交换诱变和基于分枝杆菌热敏性复制起点和在蔗糖存在下sacB基因对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能够更好地了解MTB中叶酸代谢的生物化学。它还将为未来基于结构活性关系、分子建模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.