Antimycobacterial NO from KatG Activation of Isoniazid

描述(申请人提供)：异烟肼(INH)是结核病治疗的一线药物，既有效又有选择性，但在发现50年后，其作用机制尚未完全确定。异烟肼是一种被分枝杆菌过氧化物酶KatG激活的产品，它能产生一系列活性自由基，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-)特别敏感，NO-是一种已知在机体对结核病的免疫反应中起重要作用的自由基。申请者将检验这一创新假设，即自由基一氧化氮是由异烟肼的KatG氧化形成的，并在异烟肼的整体作用中发挥重要作用。因此，结核分枝杆菌对异烟肼的高度敏感性可能部分是由于它对异烟肼激活形成的NO极端敏感的结果。申请人产生的初步数据确实表明，异烟肼的KatG氧化产生NO-，NO清除剂可以保护分枝杆菌免受异烟肼的影响，以及已知的对NO-敏感的分枝杆菌酶被异烟肼抑制。然而，为了使这些发现被广泛接受，在进行深入研究之前，必须解决关于NO产生的机制及其在异烟肼行动中的总体重要性的关键细节。具体目标1将通过对异烟肼的KatG激活、EPR自旋捕获和核磁共振产物的研究，结合异烟肼的特定稳定同位素标记来表征NO形成的途径。将对与异烟肼耐药性相关的S315T KatG突变进行研究，以确定NO产量降低是否可能与该突变体的耐药性有关。具体目标2将通过使用一系列特定的NO清除剂来检查NO-对异烟肼整体作用的贡献，以确定优化NO清除为结核分枝杆菌提供对异烟肼的最大保护。我们还将使用RT-PCR和酶方法来比较分枝杆菌对NO-和异烟肼的防御反应特征，并确定分枝杆菌对异烟肼的反应与其对NO-的已知反应有关的程度。

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-.