BIOCHEMICAL DISSECTION OF QUINOLONE TARGETS IN MYCOBACTERIUM TUBERCULOSIS

最近出现的耐多药结核病增加了 对于一种曾经是致命的疾病来说，可能会出现一种高度致命的流行病 被认为基本上从美国人口中消失了。一种组合 一系列因素将这个问题带到了医学关注的前沿。 在感染艾滋病毒的人中，结核病是一种日益严重的流行病。这个 在过去的一半里成功地与结核病作斗争的战略 世纪之交在美国一直给予多种药物治疗。然而， 世界范围内艾滋病患者人口的爆炸性增长提供了 用于产生新的多药耐药形式的巨大且不断增长的蓄水池 几种细菌病原体。随着结核分枝杆菌的出现 对所有标准药物都有抗药性的菌株，迫切需要 为了新的方法和新的药物来阻止病毒的传播 肺结核引起的有机体。 DNA旋转酶是一种普遍存在的细菌酶，对 维护存储在数据库中的遗传信息的结构和功能 细菌细胞基因组。旋转酶在DNA的各个方面都有作用 代谢，包括DNA复制、DNA修复、重组和 基因转录。旋转酶是由两个亚基组成的复合酶。 而且它也是喹诺酮类抗生素的靶标。的总体目标 该RFA将开发新的喹诺酮类药物，目标是杀死多种药物 耐药的结核分枝杆菌。 项目I共分4个部分，第1部分着重于基因克隆， 测序，并获得M。 结核分支杆菌gyrA和gyrB蛋白。这些目标将会实现 使用PCR克隆和测序策略并通过工程实现 大量表达结核分枝杆菌旋转酶(m- 旋转酶)。项目I的第二部分是开发一种喹诺酮类药物 M-旋转酶的图谱。喹诺酮环有许多潜在的位置 化学修饰与帕克-戴维斯药物研究小组 已经合成了2300种喹诺酮类药物的库存。我们会放映 本收集采用旋转酶超螺旋和体外裂解试验 寻找最有效的化合物，用于在细胞培养和 动物试验。第三部分将确定最适合M。 TB旋转酶结合和喹诺酮类药物最有效地诱导DNA的部位 通过捕获共价旋转酶-DNA复合体造成的损伤。第4部分将探讨 超级卷曲辅助蛋白与旋转酶共同作用形成 折叠的细菌核仁。

The recent emergence of multidrug resistant tuberculosis raises the ominous possibility of a high lethal epidemic for a disease that was once considered essentially eliminated from the US population. A combination of factors have brought this problem to the forefront of medical concern. In HIV infected individuals, tuberculosis is a growing epidemic. The strategy that was successful in battling tuberculosis over the last half century in the US has been to give multidrug treatment. However, the world wide explosion in the population of AIDS patients has provided a large and growing reservoir for generating new multidrug resistant forms of several bacterial pathogens. With the appearance of M. tuberculosis strains resistant to all of the standard drugs, there is an urgent need for new approaches and new pharmaceuticals to stop the spread of tuberculosis causing organisms. DNA gyrase is a ubiquitous bacterial enzyme that is essential for maintaining the structure and function of genetic information stored in bacterial cell genome. Gyrase has roles in all aspects of DNA metabolism, including DNA replication, DNA repair, recombination, and gene transcription. Gyrase is a complex enzyme composed of two subunits and it is the target of the quinolone antibiotics. The overall aim of this RFA is to develop new quinolones targeted to kill multidrug resistant forms of M. tuberculosis. There are 4 parts of Project I. Part 1 is focused on gene cloning, sequencing, and obtaining biochemical amounts of the subunits for M. tuberculosis gyrA and gyrB proteins. These aims will be accomplished using PCR cloning and sequencing strategies and by engineering recombinant plasmids that express large amounts of the M. tb gyrase (m- gyrase) in E. coli cells. Part 2 of Project I is developing a quinolone profile of m-gyrase. The quinolone ring has many sites for potential chemical modification and the Parke-Davis Pharmaceutical Research group has synthesized an inventory of 2,300 quinolone drugs. We will screen this collection using gyrase supercoiling and cleavage assays in vitro to find the most potent compounds for testing in cell culture and in animal trials. Part 3 will identify DNA sites that are optimal for M. tb gyrase binding and sites where quinolones most efficiently induce DNA damage by trapping the covalent gyrase-DNA complex. Part 4 will explore the role of supercoil accessory proteins that work with gyrase to form the folded bacterial nucleoid.