Mechanism of Quinolone Resistance

淋病是由淋病奈瑟氏菌引起的，是一种性传播疾病， 被疾病控制和预防中心归类为四个“紧急级别”耐药 威胁美国。这种疾病在现役军人中很流行， 退伍军人群体。虽然喹诺酮类药物从1993年开始被常规用于治疗淋病，但它们作为 2006年，由于耐药性发生率高，停止了一线治疗。的细胞靶标 喹诺酮是细菌II型拓扑异构酶、促旋酶和拓扑异构酶IV。确定和 表征针对这些充分验证的酶靶点起作用的新型试剂，但克服了 相关的耐药性，可能对淋病的临床治疗有重要的影响。 促旋酶和拓扑异构酶IV是调节DNA欠缠绕和过缠绕的必需酶， 通过在遗传物质中产生短暂的双链断裂来去除DNA结和缠结。 喹诺酮类药物通过增加这些促旋酶和拓扑异构酶IV产生的双螺旋酶的水平来杀死细菌。 DNA链断裂，将这些酶转化为致命的蛋白质，使基因组片段化。两 酶是喹诺酮类药物的靶点，但它们对药物作用的重要性是依赖于物种和药物的。初始 喹诺酮类耐药最常与促旋酶和/或拓扑异构酶IV的特定突变相关， 存在于位于下游4个残基的高度保守的Ser残基或Glu/Asp。根据一份出版的 Osheroff实验室的一系列功能研究，描述了 这些药物和来自炭疽杆菌、大肠杆菌和结核分枝杆菌的酶， 残基锚水-金属离子桥，作为喹诺酮类药物和 II酶。通过表征喹诺酮-拓扑异构酶相互作用，PI设计了新型药物， 克服由于M中的突变引起的抗性。结核螺旋酶和B.炭疽螺旋酶和拓扑异构酶IV。 最近，一类新的基于萘啶酮/氨基哌啶的药物，"新型细菌拓扑异构酶 抑制剂"（NBTI）。NBTI靶向细菌II型拓扑异构酶，但显示很少或没有交叉- 在促旋酶或拓扑异构酶IV中的临床相关喹诺酮耐药突变的耐药性。不像 喹诺酮类药物，这些药物要么作为催化抑制剂，要么诱导酶介导的单链DNA 休息.然而，尚未报告该药物类别任何成员的其他机制信息。 Gepotidacin是一种正在进行抗淋病临床试验的NBTI， 喹诺酮类耐药N.淋病培养物。然而，无论是其行动，也没有任何其他NBTI反对 n.淋病促旋酶或拓扑异构酶IV。 迫切需要开发新的药物来治疗耐药淋病（以及其他耐药淋病）。 细菌感染）。这项研究的前提是， 与它们的酶靶点相互作用使我们处于更有利的地位来开发克服耐药性的药物。 因此，本提案的具体目的是：1）确定喹诺酮类药物作用的机制基础， n.淋病螺旋酶和拓扑异构酶IV，确定了靶向介导的喹诺酮耐药性的基础， 利用这些发现来确定克服最常见形式的耐药性的喹诺酮类药物;以及2） 确定NBTI对N.淋病促旋酶和拓扑异构酶IV。 虽然本研究的主要研究模型将是N。淋病促旋酶和拓扑异构酶IV， 也计划进行细胞研究。此外，一些拟议的研究可能会利用M。tuberculosis，E.大肠杆菌， 或B。炭疽模型进行比较。最后，本文的研究从前人的研究成果中得到了很大的启示。 Osheroff实验室对细菌和真核生物II型拓扑异构酶机制的研究， 这些酶与喹诺酮和其他药物的相互作用。

Gonorrhea, which is caused by Neisseria gonorrhoeae, is a sexually transmitted disease that currently is categorized by the Centers for Disease Control and Prevention as one of the four “urgent level” drug-resistant threats to the United States. The disease is prevalent in active Military and rates appear to be elevated in Veteran populations. Although quinolones were used routinely to treat gonorrhea starting in 1993, their use as front-line therapy was discontinued in 2006 due to the high incidence of resistance. The cellular targets of quinolones are the bacterial type II topoisomerases, gyrase and topoisomerase IV. The identification and characterization of novel agents that act against these well-validated enzyme targets, but overcome the associated resistance, could have important ramifications for the clinical treatment of gonorrhea. Gyrase and topoisomerase IV are essential enzymes that regulate DNA under- and overwinding and remove DNA knots and tangles by generating transient double-stranded breaks in the genetic material. Quinolones kill bacteria by increasing the levels of these gyrase- and topoisomerase IV-generated double- stranded DNA breaks, which converts these enzymes into lethal proteins that fragment the genome. Both enzymes are targets for quinolones, but their importance to drug action is species- and drug-dependent. Initial quinolone resistance is most often associated with specific mutations in gyrase and/or topoisomerase IV that occur at a highly conserved Ser residue or a Glu/Asp located 4 residues downstream. Based on a published structure and a series of functional studies from the Osheroff laboratory that delineated interactions between drugs and the enzymes from Bacillus anthracis, Escherichia coli, and Mycobacterium tuberculosis, these residues anchor a water-metal ion bridge that serves as the primary conduit between quinolones and the type II enzymes. By characterizing quinolone-topoisomerase interactions, the PI has designed novel drugs that overcome resistance due to mutations in M. tuberculosis gyrase and B. anthracis gyrase and topoisomerase IV. Recently, a new class of naphthyridone/aminopiperidine-based agents, “novel bacterial topoisomerase inhibitors” (NBTIs), was reported. NBTIs target bacterial type II topoisomerases but display little or no cross- resistance to clinically relevant quinolone resistance mutations in gyrase or topoisomerase IV. Unlike the quinolones, these agents either act as catalytic inhibitors or induce enzyme-mediated single-stranded DNA breaks. However, no additional mechanistic information has been reported for any member of this drug class. Gepotidacin, an NBTI that is in clinical trials against gonorrhea, displays activity against wild-type and quinolone-resistant N. gonorrhoeae cultures. However, neither its actions, nor those of any other NBTI against N. gonorrhoeae gyrase or topoisomerase IV have been described. There is an urgent need to develop new drugs to treat resistant gonorrhea (as well as other resistant bacterial infections). The premise that underlies the proposed research is that understanding how drugs interact with their enzyme target places us in a far better position to develop drugs that overcome resistance. Thus, the specific aims of this proposal are to 1) determine the mechanistic basis for quinolone action against N. gonorrhoeae gyrase and topoisomerase IV, define the basis for target-mediated quinolone resistance, and utilize the findings to identify quinolones that overcome the most common forms of resistance; and 2) determine the mechanistic basis for the actions of NBTIs against N. gonorrhoeae gyrase and topoisomerase IV. Although the primary research models for this study will be N. gonorrhoeae gyrase and topoisomerase IV, cellular studies also are planned. In addition, some of the proposed studies may utilize M. tuberculosis, E. coli, or B. anthracis models for comparative purposes. Finally, the proposed research benefits greatly from previous studies from the Osheroff laboratory on the mechanism of bacterial and eukaryotic type II topoisomerases and the interaction of these enzymes with quinolones and other drugs.