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重要的抗生素之一。 临床上成功的抗菌药物。尽管这些抑制剂很重要，但在这些方面仍存在显着差距 我们对他们行动的最基本原则的理解。许多蛋白质合成抑制剂， 从经典的氯霉素 (CHL) 到较新的利奈唑胺 (LZD)，与催化肽基转移酶结合 核糖体中心 (PTC)，它们与氨酰基-tRNA 的位置发生冲突。因为 由于它们的结合位点的位置，通常认为它们通过干扰形成来抑制翻译 每个肽键，无论是在基因的起始密码子还是任何内部密码子。然而，我们的 初步数据表明，这种观点基本上是错误的。而不是无差别地抑制肽键 形成时，CHL 和 LZD 仅在特定 mRNA 位点阻止翻译延伸。新生的本质 肽链似乎在指定翻译停滞位点方面发挥着主要作用，但一般规则 定义失速位点以及造成这种效应的分子机制仍然未知。 因此，该项目的主要目标是详细了解上下文特定操作 PTC 靶向抗生素。该研究将主要关注 LZD 和 CHL。 LZD是第一个也是最广泛的 医用恶唑烷酮。 CHL 是已知最古老的核糖体抗生素之一。尽管其减少 医学重要性，将 CHL 纳入研究至关重要，这不仅是因为信息量巨大 可用于该抑制剂，还可以将其与 LZD 的特定作用进行对比，并将 药物的个体结构特性的影响。 在具体目标 1 中，将使用全细胞核糖体分析和定量生化测试来 确定序列背景的详细要求，该序列背景定义了优选的抑制位点 由 LZD 或 CHL 翻译。在具体目标 2 中，一系列生化、结构和遗传学方法将 用于理解 PTC 特定环境作用的分子机制 靶向抑制剂。使用创新技术，例如单分子 FRET 或工程化技术 拴系核糖体，预计将为最基本的方面提供主要的新见解 核糖体催化中心抑制剂的作用。具体目标 3 将解决概念上重要的问题 以及医学相关的问题，药物作用的背景特异性是否会导致蛋白质特异性抑制 PTC 靶向抗生素的翻译。 预期的发现将显着扩大对一般作用模式的理解 临床上重要的抗菌药物，作用于核糖体催化中心，并可能开辟新的场所 合理开发具有优异抗生素特性的蛋白质合成抑制剂。