DNA GYRASE AND QUINOLONE RESISTANCE IN TUBERCULOSIS

描述（改编自申请人摘要）：耐药性 是抗结核化疗的一个主要问题， 氟喹诺酮类药物不是一线药物的主要原因 剂.最近的两项进展，利用各种细菌，鼓励 氟喹诺酮的改进。 首先，阻力是逐步产生的， 两个独立靶点DNA促旋酶和DNA拓扑异构酶的突变 四. 因此，第一步抗性促旋酶突变体可用于筛选新的 氟喹诺酮类衍生物，需要两个突变赋予 阻力 由于双突变发生的频率低于单突变， 许多数量级的突变，对新化合物的抗性 应该很少出现。第二，双突变体的概念是 实验验证：C8-甲氧基减少了获得 至少提高了三个数量级 两种观点 建议定义这些原则如何适用于氟喹诺酮 结核分枝杆菌的耐药性。 一种是氟喹诺酮类 将检查结构变异体的预防能力 阻力 预防耐药性将与积极的 中等抗性菌株的杀灭1）在生长条件下纯培养 和非生长条件，和2）在培养的人巨噬细胞内。 结论的一般性将使用各种各样的测试。 M.结核 在另一种方法中， 将通过检查基因的核苷酸序列来研究耐药性 编码促旋酶和拓扑异构酶IV。 将选择突变体用于 对各种氟喹诺酮类同源物的耐药性，其中一些是 已知会引发不同的等位基因 选择的突变体将被 测试1）交叉抗性，2）适应变化的环境， 和3）人巨噬细胞的侵袭。 已经有许多新的 化合物，两种新的测定（杀死抗性突变体和预防 的抵抗力），人类巨噬细胞感染系统，和一个大的 收集M.结核 显著 有望在寻找氟喹诺酮类药物方面取得进展， 在M.结核

DESCRIPTION (Adapted from the applicant's abstract): Drug resistance is a major problem for anti-tuberculous chemotherapy and is one of the main reasons why compounds such as fluoroquinolones are not first line agents. Two recent advances, using a variety of bacteria, encourage refinements of fluoroquinolones. First, resistance arises stepwise from mutations in two independent targets, DNA gyrase and DNA topoisomerase IV. Thus a first step resistant gyrase mutant can be used to screen new fluoroquinolones for derivatives that require two mutations to confer resistance. Since double mutations occur less frequently that single mutations by many orders of magnitude, resistance to the new compounds should arise very rarely. Second, the double mutant concept was experimentally validated: a C8-methoxyl group reduced the acquisition of resistance by at least three orders of magnitude. Two approaches are proposed to define how these principles apply to fluoroquinolone resistance in Mycobacterium tuberculosis. In one, fluoroquinolone structural variants will be examined for their ability to prevent resistance. Prevention of resistance will be correlated with avid killing of moderately resistant strains 1) in pure culture under growing and non-growing conditions, and 2) inside cultured human macrophages. The generality of the conclusions will be tested using a wide variety of clinical isolates of M. tuberculosis. In the other approach, resistance will be studied by examining nucleotide sequences of genes encoding gyrase and topoisomerase IV. Mutants will be selected for resistance to various fluoroquinolone congeners, some of which are already known to elicit different alleles. Selected mutants will be tested for 1)cross-resistance, 2) adaptation to changing environment, and 3) invasion of human macrophages. Already available are many new compounds, two novel assays (killing resistant mutants and prevention of resistance), a human macrophage infection system, and a large collection of clinical isolates of M. tuberculosis. Significant progress is expected toward finding fluoroquinolones that will prevent resistance from arising in M. tuberculosis.