Mechanistic Studies of Gyrase/Topoisomerase IV-Targeted Antibacterials

氟喹诺酮类，如环丙沙星，是最有效和广谱的口服给药途径之一。 抗菌药物在临床上的应用世界卫生组织将其列为五个“最高优先级 重要的抗菌药物”，这些药物是全球处方量最大的抗菌药物。 氟喹诺酮类药物的细胞靶点是细菌II型拓扑异构酶、促旋酶和拓扑异构酶IV。 这些必需的酶调节DNA的欠缠绕和过缠绕，并从基因组中去除结和缠结 通过在遗传物质中产生短暂的双链断裂。氟喹诺酮类药物通过增加 由促旋酶和拓扑异构酶IV产生的双链DNA断裂，将这些酶转化为 分裂基因组的细胞毒素虽然促旋酶和拓扑异构酶IV都是生理靶点， 对于氟喹诺酮类药物，它们对药物作用的相对重要性似乎是种属和药物依赖性的。 抗菌药物耐药危机日益严重，氟喹诺酮类药物耐药现象日益普遍。这 耐药性威胁着氟喹诺酮类药物的临床疗效。最初的氟喹诺酮类耐药最常见于 与发生在丝氨酸残基（最初是丝氨酸残基）的促旋酶和/或拓扑异构酶IV中的特定突变相关 描述为大肠杆菌促旋酶的GyrA亚基中的Ser 83）和谷氨酸/天冬氨酸残基4个氨基酸 酸下游根据Osheroff实验室发表的结构和功能研究，这些 残基被提议用于锚水-金属离子桥，该离子桥用作氟- 喹诺酮类和促旋酶/拓扑异构酶IV。 鉴定和表征针对这些经过充分验证的酶靶点的新型药物 并克服氟喹诺酮耐药性可能对健康产生重要影响。最近，两个新班级 已经描述了旋转酶/拓扑异构酶IV靶向剂的组合物，其似乎克服了这种耐药性， 细菌拓扑异构酶抑制剂（NBTI）和螺嘧啶三酮（SPT）。这些阶级的成员， Gepotidacin（NBTI）和zoliflodacin（SPT）已进入3期临床试验。NBTI是独一无二的，因为它们 诱导单链而不是双链酶产生DNA断裂。然而，人们对此知之甚少。 NBTI和SPT对促旋酶/拓扑异构酶IV的作用或耐药机制。 迫切需要鉴定对氟喹诺酮耐药细菌显示活性的药物。因此，在本发明中， 本项目的目标是进一步确定氟喹诺酮类、NBTI和SPT的作用机制， 促旋酶和拓扑异构酶IV在体内和细胞中的作用，以表征靶介导的耐药性的基础， 并鉴定克服耐药性的新化合物。研究将受益于广泛的野生图书馆- 型和耐药促旋酶/拓扑异构酶IV，其包括酶 从炭疽芽孢杆菌（Bacillus anthracis）、E.大肠杆菌，金黄色葡萄球菌，结核分枝杆菌，淋病奈瑟菌， 土拉热弗朗西斯菌和鲍曼不动杆菌。这些病原体对人类健康有重大影响。

Fluoroquinolones, such as ciprofloxacin, are among the most efficacious and broad-spectrum oral antibacterials in clinical use. The World Health Organization lists them in their five “Highest Priority Critically Important Antimicrobials,” and these drugs are the most heavily prescribed antibacterials worldwide. The cellular targets of fluoroquinolones are the bacterial type II topoisomerases, gyrase and topoisomerase IV. These essential enzymes regulate DNA under- and overwinding and remove knots and tangles from the genome by generating transient double-stranded breaks in the genetic material. Fluoroquinolones act by increasing levels of double-stranded DNA breaks generated by gyrase and topoisomerase IV, which converts these enzymes into cellular toxins that fragment the genome. Although gyrase and topoisomerase IV are both physiological targets for fluoroquinolones, their relative importance to drug action appears to be species- and drug-dependent. There is a growing crisis in antibacterial resistance and fluoroquinolone resistance is becoming prevalent. This resistance is threatening the clinical efficacy of fluoroquinolones. Initial fluoroquinolone resistance is most often associated with specific mutations in gyrase and/or topoisomerase IV that occur at a serine residue (originally described as Ser83 in the GyrA subunit of Escherichia coli gyrase) and a glutamic/aspartic acid residue 4 amino acids downstream. Based on a published structure and functional studies from the Osheroff laboratory, these residues are proposed to anchor a water-metal ion bridge that serves as the primary conduit between fluoro- quinolones and gyrase/topoisomerase IV. The identification and characterization of novel agents that act against these well-validated enzyme targets and overcome fluoroquinolone resistance could have important health ramifications. Recently, two new classes of gyrase/topoisomerase IV-targeted agents have been described that appear to overcome this resistance, Novel Bacterial Topoisomerase Inhibitors (NBTIs) and Spiropyrimidinetriones (SPTs). Members of these classes, gepotidacin (NBTI) and zoliflodacin (SPT), have advanced to Phase 3 clinical trials. NBTIs are unique, as they induce single- rather than double-stranded enzyme-generated DNA breaks. However, little is known about the actions of NBTIs and SPTs against gyrase/topoisomerase IV or the mechanism of drug resistance. There is an urgent need to identify drugs that display activity against fluoroquinolone-resistant bacteria. Thus, the goals of this project are to further define the mechanism of action of fluoroquinolones, NBTIs, and SPTs against gyrase and topoisomerase IV in vivo and in cells, to characterize the basis of target-mediated drug resistance, and to identify novel compounds that overcome resistance. Research will benefit from the broad library of wild- type and drug-resistant gyrase/topoisomerase IV available in the Osheroff laboratory, which includes enzymes from Bacillus anthracis, E. coli, Staphylococcus aureus, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Francisella tularensis, and Acinetobacter baumannii. These pathogens have substantial effects on human health.