Overcoming fluoroquinolone resistance in Neisseria gonorrhoeae

项目摘要 氟喹诺酮类药物是全球使用最广泛的口服抗菌药物。然而，崛起 对这类抗菌药物的耐药性影响了它们的使用。氟喹诺酮类药物是一线 到2007年，每年有7800万例淋病感染病例，由淋病奈瑟菌引起。 当时，疾病控制中心改变了建议，以应对日益增长的耐药率 在细菌中。最重要的氟喹诺酮类耐药突变是靶介导的， 细菌II型拓扑异构酶、促旋酶和拓扑异构酶IV。寻找抗菌药物的新靶点是 困难的任务.然而，克服氟喹诺酮耐药性的两种替代方法是：1）改变 氟喹诺酮结构，以鉴定具有增加的效力和/或功效的衍生物，以及2）开发新的 靶向经验证的细菌II型拓扑异构酶，但与不同的 酶中的残留物。在后一种方法中的努力已经产生了一类新的化合物， 细菌拓扑异构酶抑制剂（NBTI）。 氟喹诺酮类和NBTI的酶靶点，促旋酶和拓扑异构酶IV，对于 调节细菌细胞中的DNA拓扑结构。它们调节DNA超螺旋的水平， 通过使一段DNA通过一个在单独的DNA链中产生的短暂双链断裂， 片段两种药物类别都靶向共价拓扑异构酶切割的DNA复合物（切割复合物）， 抑制断裂链的连接。如果复制或转录机制遇到共价键， 拓扑异构酶-DNA切割复合物，基因组可以被片段化，这导致SOS反应和细胞凋亡。 在细菌中死亡。本项目的主要目标是克服耐氟喹诺酮类药物的N。 通过增加我们对氟喹诺酮和NBTI与其靶点相互作用的理解， 靶介导的耐药性发展，以帮助药物设计。这一目标将通过三个具体目标实现： 在具体目标1中，我将确定水-金属离子桥是否以及如何促进拓扑异构酶- 氟喹诺酮在其他细菌物种中的相互作用，在N.淋病为此，我将使用 用野生型和抗性酶测量DNA切割、超螺旋和松弛。我还打算 产生氟喹诺酮结合数据以使用荧光各向异性进一步量化桥的影响。 在具体目标2中，我将重点讨论NBTI类，以确定NBTI如何对N起作用。淋病促旋酶 和拓扑异构酶IV，以及旋转酶和拓扑异构酶IV中的抗性突变如何消除药物作用。这 将需要与具体目标1中类似的酶活性测定。最后，在具体目标3中，我将评估 氟喹诺酮类和NBTI的细胞效应，因为它们与拓扑异构酶-DNA切割复合物相关 使用酶生物测定的体内复合物来形成。这些研究有可能有助于 开发克服抗菌药物耐药性的药物。

PROJECT SUMMARY Fluoroquinolone-based drugs are the most widely used oral antibacterials worldwide. However, the rise of resistance to this class of antibacterials has impacted their usage. The fluoroquinolones were a first-line therapy for the 78 million annual cases of gonorrheal infections, caused by Neisseria gonorrhoeae, until 2007. At that time, the Centers for Disease Control altered recommendations in response to growing resistance rates in the bacterium. The most important fluoroquinolone resistance mutations are target mediated and develop in the bacterial type II topoisomerases, gyrase and topoisomerase IV. Finding new targets for antibacterials is a difficult task. However, two alternative approaches to overcoming fluoroquinolone resistance are 1) altering fluoroquinolone structure to identify derivatives with increased potency and/or efficacy and 2) developing new classes of compounds that target the validated bacterial type II topoisomerases, but interact with different residues in the enzymes. Efforts in the latter approach have resulted in a new class of compounds called novel bacterial topoisomerase inhibitors (NBTIs). The enzyme targets of fluoroquinolones and NBTIs, gyrase and topoisomerase IV, are essential for regulating DNA topology in bacterial cells. They modulate levels of DNA supercoiling and untangle and unknot the genome by passing a segment of DNA through a transient double-stranded break made in a separate segment. Both drug classes target the covalent topoisomerase-cleaved DNA complex (cleavage complex) and inhibit ligation of the broken strands. If replication or transcription machinery encounters the covalent topoisomerase-DNA cleavage complex, the genome can be fragmented, which leads to SOS responses and cell death in the bacteria. The primary goal of this project is to overcome fluoroquinolone resistance in N. gonorrhoeae by increasing our understanding of fluoroquinolone and NBTI interactions with their targets and target-mediated resistance development to aid drug design. This goal will be addressed by three specific aims: In Specific Aim 1, I will determine if and how a water-metal ion bridge, shown to facilitate topoisomerase- fluoroquinolone interactions in other bacterial species, functions in N. gonorrhoeae. To this end, I will use assays that measure DNA cleavage, supercoiling, and relaxation with wild-type and resistant enzymes. I also intend to generate fluoroquinolone binding data to further quantify the effects of the bridge using fluorescence anisotropy. In Specific Aim 2, I will focus on the NBTI class to determine how NBTIs function against N. gonorrhoeae gyrase and topoisomerase IV, and how resistance mutations in gyrase and topoisomerase IV abrogate drug action. This will require similar enzymological activity assays as in Specific Aim 1. Finally, in Specific Aim 3, I will assess the cellular effects of fluoroquinolones and NBTIs as they correlate with topoisomerase-DNA cleavage complex formation using an in vivo complex of enzyme bioassay. These studies have the potential to aid in the development of drugs to overcome antibacterial resistance.