Context-specific action of antibiotics targeting the catalytic center of the bacterial ribosome

针对细菌核糖体催化中心的抗生素的特定作用

• 批准号: 9158354
• 负责人: ALEXANDER S MANKIN
• 金额: $ 39.98万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-15 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9158354
• 关键词: Accounting; Address; Amino Acids; Amino Acyl Transfer RNA; Anti-Bacterial Agents; Antibiotic Therapy; Antibiotics; Bacterial Infections; Binding; Binding Sites; Biochemical; C-terminal; Catalytic Domain; Cells; Chloramphenicol; Codon Nucleotides; Data; Development; Drug effect disorder; Drug usage; Engineering; Future; Genes; Genomics; Goals; Individual; Initiator Codon; Linezolid; Location; Medical; Messenger RNA; Molecular; Nature; Oxazolidinones; Peptides; Peptidyltransferase; Pharmaceutical Preparations; Play; Property; Protein Biosynthesis; Protein Synthesis Inhibitors; Proteins; Proteome; Resolution; Ribosomes; Role; Site; Specific qualifier value; Specificity; Structure; Techniques; Testing; Translations; base; biophysical properties; cell growth; combat; design; genetic approach; genome-wide analysis; improved; inhibitor/antagonist; innovation; insight; novel therapeutics; ribosome profiling; single-molecule FRET; treatment strategy

Antibiotics that inhibit cell growth by interfering with protein synthesis have been among the most clinically successful antibacterials. In spite of the importance of these inhibitors, there are significant gaps in our understanding of the most fundamental principles of their action. Many of the protein synthesis inhibitors, from the classic chloramphenicol (CHL) to the newer linezolid (LZD), bind at the catalytic peptidyl transferase center (PTC) of the ribosome, where they clash with the placement of aminoacyl-tRNA. Because of the location of their binding site, it is commonly assumed that they inhibit translation by interfering with formation of every peptide bond, either at the start codon or at any of the internal codons of a gene. However, our preliminary data show that this view is principally incorrect. Instead of indiscriminately inhibiting peptide bond formation, CHL and LZD stall elongation of translation only at specific mRNA sites. The nature of the nascent peptide chain appears to play the major role in specifying the sites of translation arrest, but the general rules that define the sites of stalling and the molecular mechanisms that underlie this effect remain unknown. Therefore, the main goal of this project is to gain a detailed understanding of the context specific action of PTC-targeting antibiotics. The study will primarily focus on LZD and CHL. LZD is the first and most broadly medically used oxazolidinone. CHL is one of the oldest known ribosomal antibiotics. In spite of its reduced medical importance, inclusion of CHL in the study is crucial, not only because of the vast amount of information available for this inhibitor, but also to contrast its context specific action with that of LZD and correlate the effects with individual structural properties of the drugs. In Specific Aim 1, whole-cell ribosome profiling and quantitative biochemical testing will be used to identify the detailed requirements for the sequence context that defines the preferred sites of inhibition of translation by LZD or CHL. In Specific Aim 2, an array of biochemical, structural and genetic approaches will be employed to understand the molecular mechanisms that account for the context-specific action of the PTC- targeting inhibitors. The use of innovative techniques, such as single molecule FRET or an engineered tethered ribosome, are expected to provide principally new insights into the most fundamental aspects of action of the inhibitors of the ribosomal catalytic center. Specific Aim 3 will address a conceptually important and medically-relevant question, whether context specificity of drug action results into protein-specific inhibition of translation by the PTC-targeting antibiotics. The anticipated findings should significantly expand the understanding of the general mode of action of clinically-important antibacterials that act upon the catalytic center of the ribosome and may open new venues for rational development of protein synthesis inhibitors with superior antibiotic properties.

通过干扰蛋白质合成来抑制细胞生长的抗生素是最多的 临床上成功的抗菌作用。尽管这些抑制剂的重要性，但仍有很大的差距 我们对行动最基本原则的理解。许多蛋白质合成抑制剂， 从经典的氯霉素（CHL）到较新的线唑胺（LZD），在催化肽基转移酶时结合 核糖体的中心（PTC），在那里它们与氨基酰基-TRNA的位置相冲突。因为 其结合位点的位置，通常假定它们通过干扰形成而抑制翻译 每个肽键，无论是在开始密码子还是基因的任何内部密码子处。但是，我们的 初步数据表明，此观点主要是不正确的。而不是不加选择的抑制肽键 仅在特定mRNA位点的翻译形成，CHL和LZD失速伸长。新生的本质 肽链似乎在指定翻译逮捕的地点方面起着主要作用，但是一般规则 这定义了停滞的部位和基于这种效果的分子机制仍然未知。 因此，该项目的主要目标是详细了解特定上下文的行动 靶向PTC的抗生素。这项研究将主要关注LZD和CHL。 LZD是第一个也是最广泛的 医学使用的阿沙唑啉酮。 CHL是最古老的已知核糖体抗生素之一。尽管减少了 医学重要性，将CHL纳入研究至关重要，不仅是因为有大量信息 适用于该抑制剂，但也可以将其特定环境的作用与LZD进行对比，并将其相关联 对药物的单个结构特性的影响。 在特定的目标1中，全细胞核糖体分析和定量生化测试将用于 确定序列上下文的详细要求，该要求定义了抑制的首选站点 LZD或CHL翻译。在特定的目标2中，一系列生化，结构和遗传方法将 被用来了解解释PTC-的上下文特定作用的分子机制 靶向抑制剂。创新技术的使用，例如单分子fret或设计的 束缚核糖体有望提供有关最基本方面的主要新见解 核糖体催化中心抑制剂的作用。特定目标3将解决一个概念上重要的 以及与医学相关的问题，药物作用的背景特异性是否导致蛋白质特异性抑制作用 通过PTC靶向抗生素的翻译。 预期的发现应大大扩展对一般行动方式的理解 作用于核糖体的催化中心的临床至关重要的抗菌物，可能打开新场地 用于具有优质抗生素特性的蛋白质合成抑制剂的合理发展。