Lethal action of fluoroquinolones with non-growing Mycobacterium tuberculosis

描述（由申请人提供）：该项目的长期目标是了解喹诺酮类药物如何杀死结核分枝杆菌。目前的建议侧重于这些化合物如何在喹诺酮-回转酶- dna复合物中引起染色体断裂和细胞快速死亡。目标是确定喹诺酮类药物和旋酶的结构特征，增强致命作用，特别是对不生长的细菌。初步研究已经确定了当蛋白质合成受阻时，使一些喹诺酮衍生物在杀死分枝杆菌方面异常活跃的结构部分，并与其他细菌一起表明，这种致病性可能是由喹诺酮诱导的、gyase介导的染色体断裂引起的。使用新的c -8-甲氧基氟喹诺酮类药物，染色体断裂和细胞死亡都不需要持续的蛋白质合成、DNA复制或有氧生长，这表明这些化合物可能能够杀死不生长的结核分枝杆菌。喹诺酮类药物的分子靶点结核分枝杆菌DNA回转酶将被纯化并用于研究喹诺酮类药物与分离的染色体（类核）和质粒形成的回转酶-DNA复合物的生化相互作用。旋切酶和喹诺酮类药物结构的改变将用于探讨染色体断裂的机制。Gyrase的变化将集中在影响GyrA二聚体相互作用的突变上；喹诺酮的变异将涉及喹诺酮核心环结构和连接在N-1、C-6、C-7和C-8位置的取代基。化学交联将与三元药物-酶- dna复合物一起使用，以表征药物结合的各个方面，如药物-旋酶取向。了解特定喹诺酮取代基如何破坏药物-酶-DNA复合物的稳定性并释放致命的DNA断裂，将用于设计新的喹诺酮类药物。在通过阻断蛋白质合成、逐渐去除氧气和用一氧化氮处理停止生长后，将测试最活跃的药物杀死培养细胞的能力。喹诺酮类药物在体外分裂染色体和杀死培养细胞方面异常活跃，将在小鼠感染模型中检测结核分枝杆菌的致死率，其中可以观察到结核分枝杆菌的生长和生长停止。这项工作有望为设计新一代喹诺酮类药物提供信息，其特点是快速杀死非生长的细菌细胞，这一特性可能缩短结核病的治疗时间，并有助于限制耐多药结核病。

DESCRIPTION (provided by applicant): The long-term goal of this program is to understand how the quinolones kill Mycobacterium tuberculosis. The present proposal focuses on how these compounds behave in quinolone-gyrase-DNA complexes to cause chromosome fragmentation and rapid cell death. A goal is to identify structural features of the quinolones and gyrase that enhance lethal action, particularly with non-growing bacteria. Preliminary studies have identified structural moieties that make some quinolone derivatives exceptionally active at killing mycobacteria when protein synthesis is blocked, and work with other bacteria indicates that this lethality may arise from quinolone-induced, gyrase-mediated chromosome fragmentation. With new C-8-methoxy fluoroquinolones, neither chromosome fragmentation nor cell death requires ongoing protein synthesis, DNA replication, or aerobic growth, suggesting that these compounds may be able to kill non-growing M. tuberculosis. M. tuberculosis DNA gyrase, the molecular target of quinolones, will be purified and used to study biochemical interactions of quinolones with gyrase-DNA complexes formed with isolated chromosomes (nucleoids) and plasmids. Alteration of gyrase and quinolone structure will be used to probe the mechanism of chromosome fragmentation. Gyrase changes will focus on mutations expected to affect GyrA dimer interactions; quinolone variation will involve the quinolone core ring structure and substituents attached at the N-1, C-6, C-7, and C-8 positions. Chemical cross-linking will be used with ternary drug-enzyme-DNA complexes to characterize aspects of drug binding such as drug-gyrase orientation. Knowledge of how particular quinolone substituents destabilize drug-enzyme-DNA complexes and release lethal DNA breaks will be used to design new quinolones. The most active will be tested for the ability to kill cultured cells after growth has been halted by blocking protein synthesis, by gradual removal of oxygen, and by treatment with nitric oxide. Quinolones that are exceptionally active at fragmenting chromosomes in vitro and killing cultured cells will be examined for lethality with M. tuberculosis in a murine model of infection in which M. tuberculosis growth and growth arrest can be observed. This work is expected to provide information for the design of a new generation of quinolone characterized by rapid killing of non-growing bacterial cells, a property that may shorten treatment of tuberculosis and help limit multidrug-resistant tuberculosis.