MODE OF ACTION OF PYRAZINAMIDE IN TUBERCLE BACILLUS

描述(改编自申请者的摘要)：长期目标 这个项目的目的是提高我们对药物作用机制的理解 结核分枝杆菌的作用和耐药性，并提供 对设计新的结核病药物很重要的知识。二 重大问题严重损害了我们有效地 控制结核病：耐药菌株的出现，以及漫长的6 一个月的结核病化疗。迫切需要开发新的结核病药物 在两个方面都有效的。吡嗪酰胺(PZA)是一种矛盾的 一线结核病药物，因为虽然PZA在体内具有强大的灭菌作用 活动，通过杀死一群半休眠的生物，以及 因此参与将结核病治疗缩短到6个月；PZA没有 正常培养条件下结核分枝杆菌的体外抗菌活性 除在酸性pH(5.5)下外，其他条件。PZA在M. 结核病是未知的，这个项目建议解决这个问题 问题。项目的具体目标是：(1)确定效果 PZA的活性形式--吡嗪酸(POA)对膜完整性的影响 结核分枝杆菌的营养物质转运。(2)测试潜力 POA对结核分枝杆菌NAD代谢的抑制作用(3)至 鉴定与POA泵出有关的外排基因 分枝杆菌。这个项目与健康有关的地方在于 可能设计的新的抗结核药物可能会进一步缩短 目前的6个月结核病化疗是基于对模式的理解 PZA的行动。 研究人员假设POA是一种亲脂性弱酸性物质 结核分枝杆菌富脂膜的分割及其原因 膜的结构和功能受损。这一假说将 在目标I中通过EM分析进行测试，通过测量养分运输 使用放射性标记氨基酸、尿嘧啶和胸腺嘧啶核苷，并通过测量 POA对膜电位的可能影响。因为. POA与烟酸的结构相似性，他们假设POA可能 反馈抑制对结核分枝杆菌NAD代谢的影响 NAD生物合成和作为假NAD分子掺入 没有NAD功能。这一假设将在目标2中通过使用 C14标记的NAD前体及其分子中C14-POA的示踪 通过质谱分析和核磁共振分析。因为他们发现外排的活性 挤出POA的泵在不同的分枝杆菌中差异很大，并存在相关性 由于他们对PZA的敏感性，他们建议识别POA 通过在Sanger Centre M中测试可能的外排基因来检测外排基因。 结核病基因组数据库和分枝杆菌基因组DNA检测 图书馆有能力赋予结核分枝杆菌对PZA的耐药性。

DESCRIPTION (Adapted from the applicant's abstract): The long-term goal of this project is to improve our understanding of mechanisms of drug action and drug resistance in Mycobacterium tuberculosis and to provide knowledge important for designing new tuberculosis (TB) drugs. Two significant problems seriously compromise our ability to effectively control TB: the emergence of drug-resistant strains, and the lengthy 6 month TB chemotherapy. There is an urgent need to develop new TB drugs that are effective in both aspects. Pyrazinamide (PZA) is a paradoxical front-line TB drug, because while PZA has powerful in vivo sterilizing activity, through killing a population of semi-dormant organisms, and is thus involved in shortening the TB therapy to 6 month; PZA has no activity in vitro against M. tuberculosis under normal culture conditions except under acid pH (5.5). The mode of action of PZA in M. tuberculosis is unknown, and this project proposes to address this issue. The specific aims of the project are: (1) To determine the effect of pyrazinoic acid (POA), the active form of PZA, on membrane integrity and nutrient transport of M. tuberculosis. (2) To test the potential inhibition of POA on the NAD metabolism in M. tuberculosis. (3) To identify and characterize the efflux genes involved in pumping POA out of the mycobacteria. The health relatedness of this project lies in the possible design of new antituberculous drugs that may further shorten the current 6 month TB chemotherapy based on understanding the mode of action of PZA. The investigators hypothesize that POA as a lipophilic weak acid will partition into the lipid-rich membrane of M. tuberculosis and cause structural and functional damage to the membrane. This hypothesis will be tested in Aim I by EM analysis, by measuring the nutrient transport using radiolabeled amino acids, uracil and thymidine, and by measuring the possible effect of POA on the membrane potential. Because of structural similarity of POA to niacin, they hypothesize that POA may interfere with NAD metabolism in M. tuberculosis by feedback inhibition of NAD biosynthesis and by incorporation as a pseudo-NAD molecule without NAD function. This hypothesis will be tested in Aim 2 by using C 14-labeled precursors of NAD and by tracing C 14-POA in NAD molecule by mass spec and NMR analyses. Because they found the activity of efflux pumps extruding POA in various mycobacteria varies widely and correlates with their susceptibility to PZA, they propose to identify the POA efflux genes by testing putative efflux genes in the Sanger Centre M. tuberculosis genome database and by testing mycobacterial genomic DNA libraries for their ability to confer PZA resistance in M. tuberculosis.