MODE OF ACTION OF BICYCLOMYCIN

细菌对抗生素的耐药性已成为世界性的健康危机。导致腹泻、尿路感染和败血症的微生物现在对许多较旧的抗生素具有抗药性。由此产生的健康威胁促使人们寻找具有新作用模式的结构独特的抗菌剂。双环素就是这样一种商业化药物。我们发现，双环素在大肠杆菌中发挥作用的主要部位是必需的细胞蛋白-转录终止因子Rho。在这项建议中，我们概述了一种综合的方法，以进一步了解双环素的表达机制。研究目标包括(1)确定Rho中的双环素结合区并阐明抗生素活性所必需的关键氨基酸的作用；(2)阐明双环素-Rho复合体的化学计量学并确定双环素结合口袋相对于Rho内其他功能区的区域分布；(3)确定药物-Rho结合过程的能量学；(4)阐明双环素抑制过程的机制；(5)将体外和体内的双环素抑制途径联系起来；(6)确定双环素抑制途径在微生物中的共性。将使用化学、生化、分子生物学和生物物理方法来实现这些目标。这些进展包括：对双环素和ATP亲和-Rho复合体进行质谱分析，以确定药物结合的部位和区域；产生和评估随机突变和位点特异性Rho突变，以确定药物功能所需的氨基酸残基；利用Biacore技术确定双环素-Rho相互作用的能量学；利用荧光光谱学来监测RNA诱导的Rho过程，并用X射线结晶学来阐明蛋白质-药物复合体的结构；使用双环素生物力学探针来确定药物作用的途径；以及建立体内试验，以关联双环霉素和双环素类似物的体外和体内作用模式。这些研究将为确定细菌控制的新途径提供分子基础。

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 the primary site for bicyclomycin 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 mechanisms of bicyclomycin expression. Research goals include (1) identifying the bicyclomycin binding domain in Rho and elucidating the role of key amino acids necessary for antibiotic activity, (2) elucidating the stoichiometry of the bicyclomycin-Rho complex and determining the regional disposition of the bicyclomycin binding pocket in relation to other functional domains within Rho, (3) determining the energetics of the drug-Rho binding process, (4) elucidating the mechanism of the bicyclomycin inhibition process, (5) correlating the in vitro and the in vivo bicyclomycin inhibitory pathways, and (6) determining the generality of the bicyclomycin inhibition pathway in microbial organisms. Chemical, biochemical, molecular biology, and biophysical methodologies will be used to meet these objectives. These include mass spectrometric analyses of bicyclomycin and ATP affinity-Rho complexes to identify the site and region of drug binding; generation and evaluation of random and site specific Rho mutations to identify amino acid residues required for drug function; use of BIAcore technology to determine the energetics for bicyclomycin-Rho interactions, fluorescence spectroscopy to monitor RNA-induced Rho processes, and X-ray crystallography to elucidate the structure of the protein-drug complex; use of bicyclomycin biomechanistic probes to determine the pathway for drug function; and development of an in vivo assay to correlate the in vitro and the in vivo mode of actions for bicyclomycin and bicyclomycin analogues. These investigations will provide the molecular basis to define novel pathways for bacterial control.