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，耐药性已成为主要问题。较长的治疗时间（6-24个月）尤其成问题，因为它给维持患者对治疗的依从性带来了困难。然后，零星治疗会促进耐抗生素结核分枝杆菌的出现，结核分枝杆菌是结核病的病原体。需要较长的治疗时间，可能是因为即使是活动性感染也含有一些不生长（休眠）的杆菌，这些杆菌不易被大多数抗菌剂杀死。我们的目标是获得新的氟喹诺酮类药物，迅速杀死不生长的结核分枝杆菌，从而从根本上缩短治疗时间。作为其作用机制的一部分，喹诺酮类药物捕获DNA回转酶作为药物-酶-DNA复合物，其中DNA被破坏，但由蛋白质结合在一起。这些复合物的形成被认为可以阻止细菌的生长，而镁离子的螯合作用可以逆转这些复合物的形成。最近的研究使我们提出，当喹诺酮的致死浓度高于阻断生长所需的浓度时，额外的药物结合会产生一种新的复合物，这种复合物不会被镁的螯合逆转。我们提出，新的复合体导致染色体断裂，在生长的细胞中，随后是致命的活性氧级联。只有一种喹诺酮类药物能杀死未生长的细胞。这些喹诺酮类药物被认为可以刺激染色体断裂，直接导致死亡，而不需要活性氧的级联反应。上述情况将通过设计、合成和表征新的喹诺酮类药物和喹诺酮类衍生物的结构来与gyrase相互作用并杀死非生长细胞。在目标1中，用培养细胞进行的测试将使用旋转酶突变体、体内交联、药物结构变化和分子模型来检查实验化合物和旋转酶结构之间的关系。当蛋白质合成被抑制以消除与生长细胞一起操作的致命途径时，主要读数将是细胞死亡。在目标2中，实验将集中在新发现的喹诺酮-环酶- dna复合物的生化特性上，该复合物是在杀死细菌所需的高药物浓度下形成的。注意力将集中在促进DNA断裂从回转酶介导的约束释放的药物结构上。在目标3中，药物结构与致死活性的关系将通过计算分析(例如：三维定量结构-活性关系模型)不依赖于药物-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三个目标中工作的优越化合物的致死率。预期的结果是先导化合物和开发新的喹诺酮类抑制剂的新规则，这些抑制剂可以快速杀死非生长的持久性细胞。

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.