MODE OF ACTION OF BICYCLOMYCIN

细菌对抗生素的耐药性已经成为一个全球性的健康危机。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耐药性。由此产生的健康威胁促使人们寻找具有新型作用模式的结构独特的抗菌剂。双环霉素就是这样一种市售药物。我们已经发现，在大肠杆菌中，双环霉素功能的主要位点是必需的细胞蛋白，转录终止因子Rho。在这一建议中，我们概述了一种综合的方法来进一步了解双环霉素表达的机制。研究目标包括(1)鉴定Rho中的双环霉素结合域，阐明抗生素活性所需的关键氨基酸的作用；(2)阐明双环霉素-Rho复合物的化学计量学，确定与Rho内其他功能域相关的双环霉素结合袋的区域配置；(3)确定药物-Rho结合过程的能量学；(4)阐明双环霉素抑制过程的机制。(5)比较体外和体内双环霉素抑制途径的相关性；(6)确定微生物体内双环霉素抑制途径的普遍性。化学，生物化学，分子生物学和生物物理方法将被用来满足这些目标。其中包括对双环霉素和ATP亲和rho复合物的质谱分析，以确定药物结合的位点和区域；产生和评估随机和位点特异性Rho突变，以确定药物功能所需的氨基酸残基；利用BIAcore技术测定双环霉素-Rho相互作用的能量学，荧光光谱监测rna诱导的Rho过程，x射线晶体学阐明蛋白质-药物复合物的结构；利用双环霉素生物力学探针确定药物作用途径；并开发体内试验，以关联双环霉素和双环霉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.