Actions of spiropyrimidinetriones against bacterial type II topoisomerases

螺嘧啶三酮对细菌 II 型拓扑异构酶的作用

• 批准号: 10750473
• 负责人: Jessica A Collins
• 金额: $ 3.3万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10750473
• 关键词: Active Sites; Address; Affect; Amino Acids; Anti-Bacterial Agents; Bacillus anthracis; Bacterial Drug Resistance; Bacterial Infections; Binding; Biochemical; Biological Assay; Biological Models; Cell Death Process; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Ciprofloxacin; Clinical; Comparative Study; Complex; DNA; DNA Double Strand Break; DNA Topoisomerase IV; Data; Development; Drug toxicity; Drug-resistant Neisseria Gonorrhoeae; Enzyme Interaction; Enzymes; Escherichia coli; Fluorescence Anisotropy; Fluoroquinolones; Genetic; Genetic Materials; Genetic Recombination; Genetic Transcription; Genetic study; Genome; Geometry; Goals; Gonorrhea; Growth; Human; Impairment; In Vitro; Infection; Infertility; Measures; Mediating; Methods; Monitor; Multienzyme Complexes; Mutation; Neisseria gonorrhoeae; Neurofibrillary Tangles; Pelvic Inflammatory Disease; Persons; Pharmaceutical Preparations; Positioning Attribute; Reaction; Recommendation; Resistance; Resistance development; SOS Response; Site-Directed Mutagenesis; Stress; Superhelical DNA; System; Topoisomerase; Topoisomerase II; Topoisomerase Inhibitors; Toxin; Work; antimicrobial resistant infection; cellular targeting; clinically relevant; cytotoxic; design; drug development; ds-DNA; fluoroquinolone resistance; improved; in vivo; mutant; novel; novel therapeutics; pathogen; pharmacophore; phase III trial; resistance mutation; response; targeted agent; tool; treatment guidelines

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.

项目概要 抗菌药物耐药性感染每年导致全球超过 120 万人死亡，这 除非有效遏制特定病原体的耐药性，否则预计到 2050 年这一数字将增至 1000 万。 值得关注的是耐药性淋病奈瑟菌，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) 建议使用氟喹诺酮类药物 (FQ)。 自 1993 年以来，它作为每年 9800 万淋病病例的一线治疗，但在 2017 年停止使用。 2007年，由于靶标介导的耐药性水平不断增加，这种耐药性发生在特定突变时。 FQ 的靶标，即细菌 II 型拓扑异构酶、旋转酶和拓扑异构酶 IV，是对 FQ 的反应而产生的 克服耐药性的一种策略是开发与 II 型细菌相互作用的新化合物。 拓扑异构酶的氨基酸残基与 FQ 的氨基酸残基不同，使用这种方法，产生了一类新的拓扑异构酶。 鉴定出具有螺嘧啶三酮 (SPT) 药效团的旋转酶/拓扑异构酶 IV 靶向药物。 旋转酶和拓扑异构酶 IV 调节细菌细胞中 DNA 的拓扑状态。 酶控制 DNA 超螺旋（过旋和欠旋）的水平，并消除 DNA 中的结和缠结 遗传物质通过使完整的双螺旋穿过在 FQ 和 SPT 均能稳定共价酶切割的 DNA 复合物（切割）。 复合体），这样当先进的复制和转录机器接近这些复合体时，它们 可以片段化基因组，触发 SOS 反应和其他细胞死亡过程。 该项目旨在通过增加我们对 SPT 相互作用的理解来克服淋病奈瑟菌的 FQ 耐药性 旋转酶和拓扑异构酶 IV 以及跨细菌物种的靶标介导的耐药性发展 为设计更有效的抗菌药物提供信息 该目标将通过三个具体目标来实现： 在具体目标 1 中，我将评估 SPT 对来自 N. 的旋转酶和拓扑异构酶 IV 的作用。 淋病杆菌、炭疽杆菌和大肠杆菌，以确定该类别的活性在不同环境中有何差异 为此，我将使用测量 DNA 裂解、裂解复合物稳定性和 在特定目标 2 中，我将评估新型 SPT 克服野生型酶催化抑制的能力。 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 这些酶中的酶赋予了对 SPT 的抗性，这将需要进行类似的酶活性测定。 在具体目标1中。此外，我将测量SPT-酶结合相互作用并进行竞争研究 确定抗性突变是否影响药物结合和/或在旋转酶/拓扑异构酶 IV 活性中的放置 最后，在具体目标 3 中，我将评估 SPT 稳定的裂解复合物的水平和持久性。 使用酶生物测定的体内复合物在淋病奈瑟菌细胞中由旋转酶和拓扑异构酶 IV 产生。 这些研究有可能为克服抗菌药物耐药性的新药开发提供信息。