MODE OF ACTION OF BICYCLOMYCIN

细菌对抗生素的耐药性已成为世界性的健康问题 危机 引起腹泻、尿路感染和 脓毒症现在对许多旧的抗生素有抗药性。 所得 健康威胁促使人们寻找结构独特的 具有新作用模式的抗菌剂。 双环霉素是一种 例如市售药物。 我们发现， 结构独特的抗生素具有新的活化和结合 大肠杆菌中药物作用的主要部位 是必需的细胞蛋白质，转录终止因子rho。 在本提案中，我们概述了一种综合方法， 了解双环霉素的表达机制。 研究 目标包括（1）确定的网站，区域，和化学计量， 双环霉素-rho键合（2）确定结构和化学， bycyclomycin-rho复合物的生物物理性质，（3）阐明 双环霉素和双环霉素衍生物或rho依赖性 过程，（4）确定关键残基的作用， 双环霉素结合和键合转化，和（5）设计 具有改进活性的第二代双环霉素类似物，和 确定双环霉素在其他革兰氏阴性菌中的作用途径 有机体 实现这些目标所使用的方法包括质量 化学和酶促分解物光谱和氨基酸分析 双环霉素-rho和双环霉素亲和配体-rho复合物与 确定药物结合的位点和区域。 此外，酶 分析和热化学测量将用于确定 化学计量的rho药物键和这些能量 转变 X射线晶体结构和有限胰蛋白酶消化 将进行调查，以阐明结构， 双环霉素-rho复合物的构象。 灵敏的酶测定 (e.g., poly（C）-刺激的ATP酶活性，RNA结合，rho依赖性 流程.设计了定点突变和随机定向突变实验 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 用于新双环霉素类似物的合理设计。

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.