Mechanism of Quinolone Resistance

淋病是由淋球菌引起的一种性传播疾病，目前 被美国疾病控制和预防中心列为四种“紧急级”抗药性之一 对美国的威胁。这种疾病在现役军人中流行，发病率似乎在#年有所上升。 退伍军人。虽然从1993年开始，喹诺酮类药物被常规用于治疗淋病，但它们被用作 由于耐药性的高发生率，一线治疗于2006年停止。的细胞靶标 喹诺酮类是细菌的II型拓扑异构酶、旋转酶和拓扑异构酶IV。 针对这些经过充分验证的酶靶标作用的新型制剂的表征，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。 旋转酶和拓扑异构酶IV是调节DNA欠卷和过卷和 通过在遗传物质中产生瞬时双链断裂来消除DNA结和缠结。 喹诺酮类药物通过增加这些旋转酶和拓扑异构酶IV产生的双重酶的水平来杀菌. 链状DNA断裂，将这些酶转化为致命的蛋白质，使基因组碎片化。两者都有 酶是喹诺酮类药物的靶标，但它们对药物作用的重要性取决于物种和药物。首字母 喹诺酮类耐药最常与旋转酶和/或拓扑异构酶IV的特定突变有关 发生在高度保守的Ser残基或位于下游4个残基的Glu/Asp处。基于已发布的 结构和来自Osheroff实验室的一系列功能研究描绘了 来自炭疽杆菌、大肠杆菌和结核分枝杆菌的药物和酶，这些 残基锚定水-金属离子桥，作为喹诺酮类药物和 II酶。通过表征喹诺酮-拓扑异构酶的相互作用，PI设计了新的药物 克服结核分枝杆菌旋转酶和炭疽杆菌旋转酶和拓扑异构酶IV突变造成的耐药性。 最近，一类新的以萘乙酮/氨基比林为基础的药物--“新型细菌拓扑异构酶” 抑制剂“(NBTIs)，被报道。NBTI靶向细菌II型拓扑异构酶，但很少或没有显示交叉- 对临床相关的旋转酶或拓扑异构酶IV的喹诺酮类耐药突变的耐药性。 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 休息一下。然而，没有关于这类药物的任何成员的额外机制信息的报道。 吉泊达星是一种正处于临床试验中的淋病药物，具有抗野生型和 耐喹诺酮类淋病奈瑟菌培养。然而，无论是它的行动，还是任何其他NBTI针对 淋球菌旋转酶或拓扑异构酶IV已被描述。 迫切需要开发新的药物来治疗耐药性淋病(以及其他耐药性淋病 细菌感染)。这项拟议研究的前提是理解药物是如何 与他们的酶靶标相互作用，使我们在开发克服耐药性的药物方面处于更有利的地位。 因此，这项建议的具体目标是：1)确定喹诺酮类药物对 淋球菌旋转酶和拓扑异构酶IV定义靶向介导的喹诺酮类耐药的基础，以及 利用这些发现确定克服最常见耐药形式的喹诺酮类药物；以及2) 确定NBTIs对淋球菌旋转酶和拓扑异构酶IV的作用的机制基础。 虽然这项研究的主要研究模型是淋球菌旋转酶和拓扑异构酶IV， 细胞研究也在计划中。此外，一些拟议的研究可能使用结核分枝杆菌、大肠杆菌、 或用于比较目的的炭疽杆菌模型。最后，所提出的研究成果在很大程度上得益于前人的研究。 Osheroff实验室对细菌和真核生物II型拓扑异构酶和 这些酶与喹诺酮类药物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.