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.

项目总结 抗药性感染每年导致全球120多万人死亡，而这 除非抗药性得到有效遏制，否则预计到2050年，这一数字将增长到1000万。一种特殊的病原体 令人担忧的是具有抗药性的淋病奈瑟菌，它被列为五个“紧急威胁”之一(最高威胁 由疾病控制和预防中心(CDC)提供)。推荐使用氟喹诺酮类药物(FQS) 作为第一线治疗淋病，自1993年以来每年有9800万人感染淋病，但在#年停止使用 2007年，由于目标介导的抵抗力水平不断增加。这种耐药性发生在特定的基因突变时 FQS的靶标是细菌II型拓扑异构酶、旋转酶和拓扑异构酶IV，它们是对FQ的反应 治疗。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 氨基酸残基上的拓扑异构酶与FQS的不同。使用这种方法，一类新的 以旋转酶/拓扑异构酶IV为靶标，以螺嘧啶三酮(SPT)为药效基团进行了鉴定。 旋转酶和拓扑异构酶IV调节细菌细胞中DNA的拓扑状态。这些是必不可少的 酶控制DNA超卷曲(过卷和下卷)的水平，并从 遗传物质通过一个完整的双螺旋通过一个短暂的双链DNA断裂在 一个单独的DNA片段。FQS和SPTS都稳定了共价酶裂解的DNA复合体(裂解 复合体)，以便当高级复制和转录机制接近这些复合体时，它们 可以碎片化基因组，触发SOS反应和其他细胞死亡过程。这样做的主要目标是 项目是通过增加我们对SPT相互作用的了解来克服淋病奈瑟菌对FQ的耐药性 通过旋转酶和拓扑异构酶IV和靶向介导的细菌物种间的耐药性发展 告知设计更有效、更有效的抗菌药。这一目标将通过三个具体目标来实现： 在特定的目标1中，我将评估SPTS对来自N。 淋球菌、炭疽芽孢杆菌和大肠杆菌，以确定这类细菌的活性在不同地区的差异 细菌种类。为此，我将使用测定DNA裂解、裂解复合体稳定性和 用野生型酶进行催化抑制。在具体目标2中，我将评估新型SPTS的克服能力 旋转酶和拓扑异构酶IV的FQ和SPT耐药突变，并描述突变的基础 在这些酶中，对SPTS具有抗性。这将需要类似于所述的酶活性分析 具体目标1。此外，我将测量SPT与酶的结合作用并进行竞争研究 确定耐药突变是否影响药物在旋转酶/拓扑异构酶IV活性中的结合和/或放置 地点。最后，在具体目标3中，我将评估spt稳定的切割复合体的水平和持久性。 由旋转酶和拓扑异构酶IV在淋球菌细胞中产生，使用体内酶生物测定复合体。 这些研究有可能为克服抗菌素耐药性的新药开发提供信息。