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.

项目摘要 抗菌抗性感染每年在世界各地造成超过120万人死亡，这 除非有效遏制，否则预计到2050年，数量将增长到1000万。特定的病原体 关注的是耐药的Neiserseria Gonorrhoeae，它被列为五个“紧急威胁”之一（威胁最高的威胁之一 水平）由疾病控制与预防中心（CDC）。建议使用氟喹诺酮（FQS） 自1993年以来，作为9800万例淋病病例的一线治疗，但它们的使用已停产 2007年由于目标介导的电阻水平增加。当特定突变中的突变 FQS的靶标，II型细菌拓扑酶（Gyrase and Toisomerase iv）响应于FQ 治疗。克服抗性的一种策略是开发与II型细菌相互作用的新化合物 氨基酸保留的拓扑异构酶不同于FQ。使用这种方法，一个新的类 鉴定了带有螺旋吡咪替翁酮（SPT）药效团的Gyrase/topoisomerase IV靶向剂。 回旋酶和拓扑异构酶IV调节细菌细胞中DNA的拓扑状态。这些必不可少 酶控制DNA超螺旋的水平（过度和下侧），然后从 通过完整的双螺旋通过瞬态双链DNA断裂，通过在 单独的DNA段。 FQS和SPT均稳定共价酶切割的DNA复合物（裂解 复杂）使得在进行复制和转录机械接近这些复合物时，它们 可以碎裂基因组，触发SOS反应和其他细胞死亡过程。这个主要目标 项目将通过增加我们对SPT相互作用的理解来克服淋病猪笼草中的FQ抗性 与Gyrase和topoisomerase IV以及靶标介导的抗性发展的细菌种类的抗性发展 告知设计更多潜在和有效的抗菌物质。该目标将由三个具体目标解决： 在特定的目标1中，我将评估N. N. SPT对Gyrase和Topoisomerase IV的作用。 淋病，炭疽芽孢杆菌和大肠杆菌，以确定该类别的活动如何变化 细菌物种。为此，我将使用测量DNA裂解，切割复杂稳定性和 用野生型酶催化抑制。在特定目标2中，我将评估新型SPT克服的能力 回旋酶和拓扑异构酶IV中的FQ-和SPT抗性突变，并描述突变的基础 在这些酶会议上对SPT的抗性。如所述，这将需要类似的酶学活性评估 在特定目标1中。此外，我将测量SPT-酶结合的相互作用并进行竞争研究 确定抗性突变是否影响在回旋酶/拓扑酶IV中的药物结合和/或放置活性 地点。最后，在特定的目标3中，我将评估SPT稳定裂解络合物的水平和持久性 由回旋酶和拓扑异构酶IV在淋病链球菌细胞中产生，并使用酶生物测定的体内复合物产生。 这些研究有可能告知新药的开发以克服抗菌抗性。