Actions of spiropyrimidinetriones against bacterial type II topoisomerases

项目摘要 抗生素耐药性感染每年在全世界造成120多万人死亡， 除非有效遏制耐药性，预计到2050年这一数字将增至1 000万。一种特殊的病原体 令人担忧的是抗药性淋病奈瑟菌，它被列为五个“紧急威胁”之一（最高威胁）， 疾病控制和预防中心（CDC）。推荐使用氟喹诺酮类药物 自1993年以来，作为每年9800万淋病病例的一线治疗药物， 2007年，由于目标介导的抗性水平增加。这种耐药性发生时， FQs的靶标，即细菌II型拓扑异构酶、促旋酶和拓扑异构酶IV，在对FQs的应答中产生 治疗克服耐药性的一种策略是开发与细菌II型相互作用的新化合物 拓扑异构酶的氨基酸残基不同于FQs。使用这种方法，一类新的 鉴定了具有螺嘧啶三酮（SPT）药效团的促旋酶/拓扑异构酶IV靶向剂。 螺旋酶和拓扑异构酶IV调节细菌细胞中DNA的拓扑状态。这些基本 酶控制DNA超螺旋的水平（过度缠绕和欠缠绕），并从DNA中去除结和缠结。 通过使完整的双螺旋通过瞬时的双链DNA断裂， 一个单独的DNA片段FQs和SPT均稳定共价酶切割的DNA复合物（切割 复合物），使得当推进复制和转录机制接近这些复合物时，它们 可以使基因组片段化，引发SOS反应和其他细胞死亡过程。这个的主要目标 项目是克服N.淋病通过增加我们对SPT相互作用的理解 与促旋酶和拓扑异构酶IV和靶介导的耐药性发展， 为设计更有效的抗菌药物提供信息。这一目标将通过三个具体目标实现： 在具体目标1中，我将评估SPT对N. 淋病，炭疽杆菌和大肠杆菌，以确定这类的活动如何在不同的 细菌种类为此，我将使用测定DNA切割、切割复合物稳定性和DNA断裂的方法。 野生型酶的催化抑制。在具体目标2中，我将评估新型SPT克服 促旋酶和拓扑异构酶IV中的FQ和SPT抗性突变，并描述突变的基础 在这些酶中赋予对SPT的抗性。这将需要类似的酶学活性测定，如所述 具体目标1。此外，我将测量SPT-酶结合相互作用并进行竞争研究 以确定耐药突变是否影响药物结合和/或在促旋酶/拓扑异构酶IV活性中的放置， 绝佳的价钱最后，在具体目标3中，我将评估SPT稳定的切割复合物的水平和持久性 由N.淋病细胞，使用酶生物测定的体内复合物。 这些研究有可能为开发克服抗菌药物耐药性的新药提供信息。

PROJECT SUMMARY Antibacterial resistant infections cause more than 1.2 million deaths around the world each year, and this number is predicted to grow to 10 million by 2050 unless resistance is effectively curbed. A pathogen of particular concern is drug-resistant Neisseria gonorrhoeae, which is listed as one of five “urgent threats” (the highest threat level) by the Centers for Disease Control and Prevention (CDC). Fluoroquinolones (FQs) were recommended as frontline treatment for the 98 million annual cases of gonorrhea since 1993, but their use was discontinued in 2007 due to increasing levels of target-mediated resistance. This resistance occurs when specific mutations in the target for FQs, the bacterial type II topoisomerases, gyrase and topoisomerase IV, arise in response to FQ treatment. One strategy to overcome resistance is to develop new compounds that interact with bacterial type II topoisomerases at amino acid residues distinct from those of FQs. Using this approach, a new class of gyrase/topoisomerase IV-targeted agents with a spiropyrimidinetrione (SPT) pharmacophore was identified. Gyrase and topoisomerase IV regulate the topological state of DNA in bacterial cells. These essential enzymes control levels of DNA supercoiling (over- and underwinding) and remove knots and tangles from the genetic material by passing an intact double helix through a transient double-stranded DNA break generated in a separate DNA segment. Both FQs and SPTs stabilize the covalent enzyme-cleaved DNA complex (cleavage complex) such that when advancing replication and transcription machinery approach these complexes, they can fragment the genome, triggering the SOS response and other cell death processes. The primary goal of this project is to overcome FQ resistance in N. gonorrhoeae by increasing our understanding of SPT interactions with gyrase and topoisomerase IV and target-mediated resistance development across bacterial species to inform the design of more potent and efficacious antibacterials. This goal will be addressed by three specific aims: In Specific Aim 1, I will assess the actions of SPTs against gyrase and topoisomerase IV from N. gonorrhoeae, Bacillus anthracis, and Escherichia coli to determine how the activities of this class varies across bacterial species. To this end, I will use assays that measure DNA cleavage, cleavage complex stability, and catalytic inhibition with wild-type enzymes. In Specific Aim 2, I will evaluate the ability of novel SPTs to overcome FQ- and SPT-resistance mutations in gyrase and topoisomerase IV and describe the basis by which mutations in these enzymes confer resistance to SPTs. This will require similar enzymological activity assays as described in Specific Aim 1. In addition, I will measure SPT-enzyme binding interactions and conduct competition studies to determine if resistance mutations affect drug binding and/or placement in the gyrase/topoisomerase IV active site. Finally, in Specific Aim 3, I will assess the levels and persistence of SPT-stabilized cleavage complexes generated by gyrase and topoisomerase IV in N. gonorrhoeae cells using an in vivo complex of enzyme bioassay. These studies have the potential to inform the development of new drugs to overcome antibacterial resistance.