Antimycobacterial NO from KatG Activation of Isoniazid

KatG 激活异烟肼的抗分枝杆菌 NO

• 批准号: 7230267
• 负责人: GRAHAM S TIMMINS
• 金额: $ 18.21万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-15 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7230267
• 关键词: Antimycobacterial; NO; KatG; Activation; Isoniazid

DESCRIPTION (provided by applicant): Isoniazid (INH) is a frontline drug in tuberculosis treatment that is both potent and selective, yet 50 years after its discovery, its mechanisms of action are not completely defined. INH is a prod rug that is activated by the mycobacterial peroxidase KatG to produce a range of reactive free radicals that react with various mycobacterial components to cause therapeutic effect. It is also known that Mycobacterium tuberculosis is exceptionally sensitive to nitric oxide (NO-) a free radical known to be important in the bodies immune response against TB. The applicant will test the innovative hypothesis that the free radical nitric oxide is formed by KatG oxidation of INH, and plays an important role in the overall action of INH. Thus, the exquisite sensitivity of M. tuberculosis to INH may be, in part, the result of its extreme sensitivity to NO- formed by INH activation. Initial data generated by the applicant does show that KatG oxidation of INH generates NO-, that an NO- scavenger can protect mycobacteria against INH, and that mycobacterial enzymes known sensitive to NO- are inhibited by INH. However, for these findings to be widely accepted, and before in-depth studies are conducted, key details on the mechanism of NO- production, and its overall importance in INH action must be addressed. Specific Aim 1 will characterize the pathway of NO- formation by KatG activation of INH, EPR spin trapping, and NMR product studies in combination with specific stable isotopic labeling of INH. The S315T KatG mutation associated with INH resistance will be studied to determine if lowered NO production may contribute to resistance in this mutant. Specific Aim 2 will examine the contribution of NO- to the overall action of INH by using a range of specific NO- scavengers to determine the maximal protection that optimized NO scavenging provides M. tuberculosis against INH. We will also use RT-PCR and enzymic approaches to compare mycobacterial defense responses signatures to NO- and INH and determine the extent to which the mycobacterial response to INH is related to its known response to NO-.

描述（由申请人提供）：Isoniazid（INH）是结核病治疗中的一线药物，既有效又有选择性，但是在发现50年后，其作用机理尚未完全定义。 INH是一种由分枝杆菌过氧化物酶KATG激活的产品地毯，可产生一系列反应性自由基，这些自由基与各​​种分枝杆菌成分反应，从而引起治疗作用。还知道结核分枝杆菌对一氧化氮（NO-）非常敏感的自由基在体内对TB的免疫反应中很重要。申请人将检验创新的假设，即自由基一氧化氮是由INH的KATG氧化形成的，并且在INH的整体作用中起着重要作用。因此，结核分枝杆菌对INH的精致敏感性可能部分是由于INH激活对NOT的极端敏感性的结果。申请人生成的初始数据确实表明，INH的KATG氧化不会产生NO-，NO-Scavenger可以保护分枝杆菌免受INH的影响，并且INH抑制了已知对无敏感的分枝杆菌酶。但是，对于这些发现被广泛接受，并且在进行深入研究之前，必须解决有关无生产机制的关键细节，并且必须解决其在INH行动中的总体重要性。特定的目标1将通过KATG激活INH，EPR旋转捕获和NMR产品研究来表征无形成的途径，并结合INH的特定稳定同位素标记。将研究与INH耐药性相关的S315T KATG突变，以确定降低的生产是否可能导致该突变体的抗性。特定的目标2将通过使用一系列特定的无清除剂来确定对INH的总体作用的贡献，以确定优化无清除的最大保护，可为INH提供结核分枝杆菌。我们还将使用RT-PCR和酶方法将分枝杆菌防御反应与NO-和INH进行比较，并确定对INH的分枝杆菌反应与其对NO-的已知反应有关的程度。