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（申请人提供）：异烟肼 (INH) 是结核病治疗的一线药物，具有强效和选择性，但在其发现 50 年后，其作用机制尚未完全确定。 INH 是一种前药，被分枝杆菌过氧化物酶 KatG 激活，产生一系列反应性自由基，与各种分枝杆菌成分发生反应，从而产生治疗效果。众所周知，结核分枝杆菌对一氧化氮 (NO-) 异常敏感，一氧化氮是一种已知在人体针对结核病的免疫反应中发挥重要作用的自由基。申请人将测试这一创新假设：自由基一氧化氮是由INH的KatG氧化形成的，并且在INH的整体作用中发挥重要作用。因此，结核分枝杆菌对 INH 的高度敏感性可能部分是由于其对 INH 活化形成的 NO- 极度敏感的结果。申请人产生的初始数据确实表明INH的KatG氧化产生NO-，NO-清除剂可以保护分枝杆菌免受INH的侵害，并且已知对NO-敏感的分枝杆菌酶被INH抑制。然而，为了使这些发现得到广泛接受，在进行深入研究之前，必须解决 NO 产生机制的关键细节及其在 INH 作用中的总体重要性。具体目标 1 将通过 INH 的 KatG 激活、EPR 自旋捕获和 NMR 产物研究结合 INH 的特定稳定同位素标记来表征 NO- 形成途径。将研究与 INH 耐药性相关的 S315T KatG 突变，以确定 NO 产生降低是否可能导致该突变体的耐药性。具体目标 2 将通过使用一系列特定的 NO- 清除剂来检查 NO- 对 INH 总体作用的贡献，以确定优化的 NO 清除为结核分枝杆菌提供的针对 INH 的最大保护。我们还将使用 RT-PCR 和酶法来比较分枝杆菌对 NO- 和 INH 的防御反应特征，并确定分枝杆菌对 INH 的反应与其已知对 NO- 的反应相关的程度。