MODE OF ACTION OF BICYCLOMYCIN

双环霉素的作用方式

• 批准号: 2179043
• 负责人: HAROLD Lewis KOHN
• 金额: $ 18.13万
• 依托单位: UNIVERSITY OF HOUSTON
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2179043
• 关键词: X ray crystallography; antibiotics; bacterial proteins; bicyclic compound; chemical structure function; drug design /synthesis /production; drug metabolism; mass spectrometry; microorganism metabolism; molecular site; protein structure; receptor binding; site directed mutagenesis

Bacterial resistance to antibiotics has become a world-wide health crisis. Organisms that cause diarrhea, urinary tract infection, and sepsis are now resistant to many of the older antibiotics. The resulting health threat has prompted the search for structurally unique antibacterial agents with novel modes of action. Bicyclomycin is one such commercially available drug. We have discovered that this architecturally distinctive antibiotic has a novel activation and bonding mechanism and that the primary site for drug function in Escherichia coli is the essential cellular protein, transcription termination factor rho. In this proposal, we outline an integrated approach to further the understanding of the mechanism of bicyclomycin expression. Research goals include (1) identifying the site, region, and stoichiometry of bicyclomycin-rho bonding (2) determining structural and chemical and biophysical properties of the bycyclomycin-rho complex, (3) elucidating the effect of bicyclomycin and bicyclomycin derivatives or rho-dependent processes, (4) determining the role of key residues in rho on bicyclomycin binding and bonding transformations, and (5) designing second generation bicyclomycin analogues with improved activity, and determining the pathway of bicyclomycin function in other Gram-negative organisms. The methodologies used to meet these objectives include mass spectrometric and amino acid analyses of chemical and enzymatic digests of bycyclomycin-rho and bicyclomycin affinity ligand-rho complexes to identify the site and region of drug bonding. In addition, enzymatic assays and thermochemical measurements will be used to determine the stoichiometry of rho-drug bonding and the energetics of these transformations. X-ray crystal structure and limited tryptic digestion investigations will be conducted to elucidate the structure and conformation of the bicyclomycin-rho complex. Sensitive enzymatic assays (e.g., poly (C)-stimulated ATPase activity, RNA binding, rho-dependent processes. Site and random-directed mutagenesis experiments are designed to identify the key catalytic sites in rho necessary for bicyclomycin function. These collective experiments will provide the molecular basis for the rational design of new bicyclomycin analogues.

细菌对抗生素的耐药性已成为世界范围内的健康问题 危机。 引起腹泻、尿路感染和 脓毒症现在对许多较旧的抗生素具有耐药性。 由此产生的 健康威胁促使人们寻找结构独特的 具有新颖作用方式的抗菌剂。 双环霉素就是其中之一 这种市售药物。 我们发现这 结构独特的抗生素具有新颖的活化和结合作用 机制以及大肠杆菌中药物功能的主要位点 是必需的细胞蛋白，转录终止因子 rho。 在本提案中，我们概述了一种综合方法，以进一步促进 了解双环霉素表达机制。 研究 目标包括（1）确定位点、区域和化学计量 双环霉素-Rho 键合 (2) 确定结构和化学以及 bycyclomycin-rho 复合物的生物物理特性，(3) 阐明 双环霉素和双环霉素衍生物的作用或依赖于 rho 过程，（4）确定rho on中关键残基的作用 双环霉素结合和键合转化，以及（5）设计 具有改进活性的第二代双环霉素类似物，以及 确定双环霉素在其他革兰氏阴性菌中的功能途径 有机体。 用于实现这些目标的方法包括质量 化学和酶消化物的光谱和氨基酸分析 双环霉素-rho 和双环霉素亲和配体-rho 复合物的 确定药物结合的位点和区域。 此外，酶促 化验和热化学测量将用于确定 rho-药物键合的化学计量及其能量学 转变。 X 射线晶体结构和有限的胰蛋白酶消化 将进行调查以阐明结构和 双环霉素-rho复合物的构象。 灵敏的酶分析 （例如，聚 (C) 刺激的 ATP 酶活性、RNA 结合、rho 依赖性 流程。设计了定点和随机定向诱变实验 确定双环霉素所需的 rho 中的关键催化位点 功能。 这些集体实验将提供分子基础 合理设计新的双环霉素类似物。