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 脓毒症现在对许多较老的抗生素具有抗药性。由此产生的 健康威胁促使人们寻找结构独特的 具有新作用模式的抗菌剂。双环素就是其中之一 这种商业上可以买到的药物。我们发现这一点 结构独特的抗生素具有一种新的活性和结合 作用机制及在大肠杆菌中发挥药物作用的主要部位 是细胞内必需的蛋白质，转录终止因子Rho。 在这项建议中，我们概述了一种综合方法，以进一步 了解双环素的表达机制。研究 目标包括(1)确定地点、地区和化学计量比 双环素-Rho键(2)结构和化学结构的测定 拜环霉素-Rho复合体的生物物理性质(3)阐明 双环素及其衍生物对Rho依赖性的影响 过程，(4)确定Rho中关键残基的作用 双环素结合和键合转化，以及(5)设计 具有改进活性的第二代双环素类似物，以及 双环素在其他革兰氏阴性菌中作用途径的测定 有机体。用于实现这些目标的方法包括MASS 化学和酶消化液的光谱分析和氨基酸分析 联环素-Rho和双环素亲和配体-Rho络合物的抗肿瘤活性 确定药物粘合的部位和区域。此外，酶制剂 将使用分析和热化学测量来确定 Rho-药物键的化学计量学及其能量学 变形。X射线晶体结构与有限胰酶消化 将进行调查，以澄清该结构和 双环素-Rho复合体的构象。灵敏的酶分析 (例如，聚(C)刺激的ATPase活性、RNA结合、Rho依赖 流程。设计了定点诱变和随机定向诱变实验 确定双环素在Rho中的关键催化部位 功能。这些集体实验将提供分子基础 为合理设计新的双环素类似物奠定基础。

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.