Lethal action of fluoroquinolones with non-growing Mycobacterium tuberculosis

氟喹诺酮类药物对非生长结核分枝杆菌的致死作用

• 批准号: 7821486
• 负责人: KARL A DRLICA
• 金额: $ 72.64万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-15 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7821486
• 关键词: Address; Aerobic; Affect; Amino Acids; Animal Model; Antitubercular Agents; Bacteria; Binding; Biochemical; Biological Assay; Cell Culture Techniques; Cell Death; Cell-Free System; Cells; Cessation of life; Chemicals; Chloramphenicol; Chromosomes; Chronic; Colony-forming units; Complex; Cultured Cells; DNA; DNA Crosslinking; DNA Gyrase; DNA biosynthesis; Data; Dose; Drops; Edetic Acid; Endopeptidase K; Enzymes; Escherichia coli; Excision; Feedback; Figs - dietary; Fluoroquinolones; Generations; Genus Mycobacterium; Goals; Growth; Homologous Gene; In Vitro; Incubated; Infection; Iowa; Knowledge; Lead; Life; Link; Liquid substance; Lung; Mass Spectrum Analysis; Measurement; Measures; Mediating; Methods; Modeling; Molecular Target; Moxifloxacin; Multidrug-Resistant Tuberculosis; Mus; Mutation; Mycobacterium tuberculosis; Nalidixic Acid; Nitric Oxide; Norfloxacin; Oxygen; Pharmaceutical Preparations; Plasmids; Positioning Attribute; Predisposition; Procedures; Property; Protein Biosynthesis; Protein Synthesis Inhibition; Proteins; Quinolones; Reaction; Research; Resistance; Route; Sedimentation process; Site; Solutions; Structure; System; Testing; Therapeutic; Time; Tuberculosis; Variant; Viscosity; Work; aerosolized; cell growth; chemotherapy; crosslink; design; dimer; drug mechanism; gel electrophoresis; improved; in vitro Model; in vivo; inhibitor/antagonist; killings; mutant; new growth; programs; protective effect; quinolone resistance; respiratory; tuberculosis treatment; uptake

DESCRIPTION (provided by applicant): The long-term goal of this program is to understand how the quinolones kill Mycobacterium tuberculosis. The present proposal focuses on how these compounds behave in quinolone-gyrase-DNA complexes to cause chromosome fragmentation and rapid cell death. A goal is to identify structural features of the quinolones and gyrase that enhance lethal action, particularly with non-growing bacteria. Preliminary studies have identified structural moieties that make some quinolone derivatives exceptionally active at killing mycobacteria when protein synthesis is blocked, and work with other bacteria indicates that this lethality may arise from quinolone-induced, gyrase-mediated chromosome fragmentation. With new C-8-methoxy fluoroquinolones, neither chromosome fragmentation nor cell death requires ongoing protein synthesis, DNA replication, or aerobic growth, suggesting that these compounds may be able to kill non-growing M. tuberculosis. M. tuberculosis DNA gyrase, the molecular target of quinolones, will be purified and used to study biochemical interactions of quinolones with gyrase-DNA complexes formed with isolated chromosomes (nucleoids) and plasmids. Alteration of gyrase and quinolone structure will be used to probe the mechanism of chromosome fragmentation. Gyrase changes will focus on mutations expected to affect GyrA dimer interactions; quinolone variation will involve the quinolone core ring structure and substituents attached at the N-1, C-6, C-7, and C-8 positions. Chemical cross-linking will be used with ternary drug-enzyme-DNA complexes to characterize aspects of drug binding such as drug-gyrase orientation. Knowledge of how particular quinolone substituents destabilize drug-enzyme-DNA complexes and release lethal DNA breaks will be used to design new quinolones. The most active will be tested for the ability to kill cultured cells after growth has been halted by blocking protein synthesis, by gradual removal of oxygen, and by treatment with nitric oxide. Quinolones that are exceptionally active at fragmenting chromosomes in vitro and killing cultured cells will be examined for lethality with M. tuberculosis in a murine model of infection in which M. tuberculosis growth and growth arrest can be observed. This work is expected to provide information for the design of a new generation of quinolone characterized by rapid killing of non-growing bacterial cells, a property that may shorten treatment of tuberculosis and help limit multidrug-resistant tuberculosis.

描述（由申请人提供）：该计划的长期目标是了解喹诺酮类药物如何杀死结核分枝杆菌。本提案重点关注这些化合物如何在喹诺酮-旋转酶-DNA 复合物中发挥作用，导致染色体断裂和快速细胞死亡。目标是确定喹诺酮类药物和旋转酶的结构特征，以增强致死作用，特别是对于非生长细菌。初步研究已经确定了一些结构部分，当蛋白质合成受阻时，这些结构部分使一些喹诺酮衍生物在杀死分枝杆菌方面异常活跃，并且与其他细菌的研究表明，这种致死性可能是由喹诺酮诱导的、旋转酶介导的染色体断裂引起的。使用新的 C-8-甲氧基氟喹诺酮类药物，染色体断裂和细胞死亡都不需要持续的蛋白质合成、DNA 复制或有氧生长，这表明这些化合物可能能够杀死非生长的结核分枝杆菌。结核分枝杆菌 DNA 旋转酶是喹诺酮类药物的分子靶标，将被纯化并用于研究喹诺酮类药物与分离染色体（核）和质粒形成的旋转酶-DNA 复合物的生化相互作用。旋转酶和喹诺酮结构的改变将用于探测染色体断裂的机制。 Gyrase 的变化将集中于预计会影响 GyrA 二聚体相互作用的突变；喹诺酮变异将涉及喹诺酮核心环结构和连接在N-1、C-6、C-7和C-8位置的取代基。化学交联将与三元药物-酶-DNA 复合物一起使用，以表征药物结合的各个方面，例如药物-旋转酶方向。有关特定喹诺酮取代基如何破坏药物-酶-DNA 复合物稳定性并释放致命 DNA 断裂的知识将用于设计新的喹诺酮类药物。通过阻断蛋白质合成、逐渐去除氧气以及用一氧化氮处理停止生长后，将测试最活跃的细胞杀死培养细胞的能力。在体外断裂染色体和杀死培养细胞方面异常活跃的喹诺酮类药物将在小鼠感染模型中检查结核分枝杆菌的致死率，在该模型中可以观察到结核分枝杆菌的生长和生长停滞。这项工作预计将为设计新一代喹诺酮类药物提供信息，其特点是快速杀死非生长细菌细胞，这种特性可能会缩短结核病的治疗时间并有助于限制耐多药结核病。