MODE OF ACTION OF PYRAZINAMIDE IN TUBERCLE BACILLUS

描述（改编自申请人的摘要）：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 这些知识对于设计新的结核病（TB）药物非常重要。两 严重的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 在这两个方面都有效。吡嗪酰胺（PZA）是一种自相矛盾的 一线结核病药物，因为虽然PZA具有强大的体内杀菌作用， 活动，通过杀死半休眠的生物种群， 因此，参与缩短结核病治疗至6个月; PZA没有 体外抗M.正常培养下的结核病 除了在酸性pH（5.5）下。PZA在M. 结核病是未知的，本项目提出解决这一问题 问题.本项目的具体目标是：（1）确定 吡嗪酸（POA），PZA的活性形式，对膜完整性 和营养物质的运输。结核(2)为了测试 POA对M.结核(3)到 鉴定和表征参与泵出POA的外排基因 分枝杆菌的感染该项目的健康相关性在于 可能设计新的抗结核药物， 目前6个月的结核病化疗的基础上了解的模式， PZA的作用。 研究人员假设，POA作为亲脂性弱酸， 分配进入M.结核病及其病因 膜的结构和功能损伤。这一假设将 在目标I中，通过EM分析，通过测量营养物质运输， 使用放射性标记的氨基酸、尿嘧啶和胸苷， POA对膜电位的可能影响。因为 POA与烟酸结构相似，他们假设POA可能 干扰M.反馈抑制型结核病 并通过作为假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前体和示踪NAD分子中的C14-POA 通过质谱和核磁共振分析。因为他们发现了 在各种分枝杆菌中挤出POA的泵变化很大， 由于他们对PZA的敏感性，他们建议识别POA 通过在桑格中心M. 结核病基因组数据库和测试分枝杆菌基因组DNA 文库的能力赋予M.结核

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.