Novel fluoroquinolones for killing dormant Mycobacterium tuberculosis

描述（由申请人提供）：结核病是一种严重的空气传播疾病，耐药性已成为一个主要问题。长的治疗时间（6-24个月）是特别成问题的，因为它在维持患者对治疗的依从性方面产生了困难。然后，零星的治疗促进了耐抗生素结核分枝杆菌的出现，这是结核病的病原体。需要长时间的治疗，大概是因为即使是活动性感染也含有一些不生长（休眠）的杆菌，这些杆菌不容易被大多数抗菌药物杀死。我们的目标是获得新的氟喹诺酮类药物，快速杀死非生长型M。结核病，从而从根本上减少治疗时间。作为其作用机制的一部分，喹诺酮类药物捕获DNA旋转酶作为药物-酶-DNA复合物，其中DNA被破坏但被蛋白质保持在一起。这些复合物的形成被认为是阻止细菌生长的，通过镁离子的螯合作用而逆转。最近的工作使我们提出，在致命的喹诺酮浓度，这是高于所需的阻断生长，额外的药物结合产生一个新的复合物，不逆转螯合镁。我们提出，新的复合物导致染色体断裂，在生长的细胞中，随后是致命的活性氧级联反应。只有喹诺酮亚类杀死非生长细胞。这些喹诺酮类被提议刺激直接导致死亡的染色体断裂，而不需要活性氧的级联反应。上述方案将通过设计、合成和表征新的喹诺酮和喹诺酮衍生结构来测试，用于与促旋酶相互作用和用于杀死非生长细胞。在目标1中，使用培养细胞的测试将使用促旋酶突变体、体内交联、药物结构变化和分子建模来检查实验化合物与促旋酶结构之间的关系。当蛋白质合成被抑制以消除与生长细胞一起操作的致死途径时，主要读出将是细胞死亡。在目标2中，实验将集中在新发现的喹诺酮-促旋酶-DNA复合物的生物化学性质上，该复合物在杀死细菌所需的升高的药物浓度下形成。注意力将集中在药物结构，促进释放的DNA断裂从回旋酶介导的约束。在目标3中，药物结构-致死活性关系将通过计算分析来确定（例如，3-三维定量结构-活性关系模型），其不依赖于药物-促旋酶相互作用的知识。将用三种分枝杆菌生长抑制系统检查在三个目标中的每一个目标上工作的上级化合物的致死性。预期的结果是开发新的喹诺酮类抑制剂的先导化合物和新规则，这些抑制剂可以快速杀死非生长的持久细胞。

DESCRIPTION (provided by applicant): Tuberculosis is a serious airborne disease for which drug resistance has become a major issue. The long treatment time (6-24 months) is particularly problematic because it creates difficulties in maintaining patient adherence to therapy. Sporadic treatment then fosters the emergence of antimicrobial-resistant Mycobacterium tuberculosis, the causative agent of tuberculosis. Long treatment times are needed, presumably because even active infections contain some non-growing (dormant) bacilli that are not readily killed by most antimicrobials. Our goal is to obtain new fluoroquinolones that rapidly kill non-growing M. tuberculosis and would thereby reduce treatment time radically. As part of their mechanism of action, quinolones trap DNA gyrase as drug-enzyme-DNA complexes in which the DNA is broken but held together by protein. Formation of these complexes, which are thought to block bacterial growth, is reversed by chelation of magnesium ion. Recent work leads us to propose that at lethal quinolone concentrations, which are higher than required to block growth, additional drug binding generates a new complex that is not reversed by chelation of magnesium. We propose that the new complex leads to chromosome fragmentation, which in growing cells is followed by a lethal cascade of reactive oxygen species. Only a quinolone subset kills non-growing cells. These quinolones are proposed to stimulate chromosome fragmentation that causes death directly, without requiring a cascade of reactive oxygen species. The above scenario will be tested by designing, synthesizing, and characterizing new quinolones and quinolone- derived structures for interactions with gyrase and for killing non-growing cells. In aim 1, tests with cultured cells will examine relationships between experimental compounds and gyrase structure, using gyrase mutants, in-vivo crosslinking, drug structure variation, and molecular modeling. The primary readout will be cell death when protein synthesis is inhibited to eliminate the lethal pathway operating with growing cells. In aim 2, experiments will focus on biochemical properties of the newly discovered quinolone-gyrase-DNA complex formed at the elevated drug concentrations required to kill bacteria. Attention will focus on drug structures that facilitate release of DNA breaks from gyrase-mediated constraint. In aim 3, drug structure- lethal activity relationships will be determined with computational analyses (eg. 3-dimensional quantitative structure-activity relationship models) that do not rely on knowledge of drug-gyrase interactions. Superior compounds from work on each of the three aims will be examined for lethality with three mycobacterial growth-arrest systems. The expected outcomes are lead compounds and novel rules for developing new quinolone-type inhibitors that rapidly kill non-growing, persister cells.