Antimycobacterial NO from KatG Activation of Isoniazid

描述（由申请人提供）：异烟肼（INH）是结核病治疗的一线药物，具有强效和选择性，但在发现50年后，其作用机制尚未完全确定。INH是一种刺激剂，被分枝杆菌过氧化物酶KatG激活，产生一系列活性自由基，与各种分枝杆菌成分反应，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，一氧化氮是一种自由基，在人体对结核病的免疫反应中起重要作用。申请人将验证创新的假设，即自由基一氧化氮是由INH的KatG氧化形成的，并在INH的整体作用中起重要作用。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-的极端敏感性。申请人生成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，为了使这些发现被广泛接受，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。Specific Aim 1将通过KatG激活INH、EPR自旋捕获和结合INH特定稳定同位素标记的NMR产物研究来表征NO-形成的途径。研究人员将研究与INH抗性相关的S315T KatG突变，以确定降低NO产量是否有助于该突变体的抗性。特异性目的2将通过使用一系列特异性NO-清除剂来检查NO-对INH整体作用的贡献，以确定优化的NO清除为结核分枝杆菌提供的最大保护。我们还将使用RT-PCR和酶学方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的反应与其已知的NO-反应的关联程度。

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-.