Molecular mechanisms of action of ribosome-targeting antibiotics.

核糖体靶向抗生素的分子作用机制。

• 批准号: 10376206
• 负责人: YURY POLIKANOV
• 金额: $ 31.45万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10376206
• 关键词: Alanine; Amides; Amino Acyl Transfer RNA; Anti-Bacterial Agents; Antibiotics; Bacterial Infections; Basic Science; Binding; Biochemical; Catalytic Domain; Chemicals; Chloramphenicol; Clinical; Complex; Crystallization; Data; Development; Drug Targeting; Drug usage; Erythromycin; Essential Drugs; Exhibits; FDA approved; Goals; Growth; Human; Knowledge; Linezolid; Link; Macrolides; Methodology; Molecular; Molecular Mechanisms of Action; Peptides; Peptidyltransferase; Pharmaceutical Preparations; Play; Positioning Attribute; Property; Protein Biosynthesis; Protein Synthesis Inhibitors; RNA, Transfer, Amino Acid-Specific; Ribosomal Interaction; Ribosomes; Serine; Site; Specificity; Structure; Techniques; Testing; Therapeutic Agents; Thermus thermophilus; Threonine; Transfer RNA; Translations; X-Ray Crystallography; antimicrobial drug; clinically relevant; inhibitor; insight; peptidyl-tRNA; thermophilic bacteria; tool

SUMMARY Ribosome-targeting antibiotics are indispensable both as therapeutic agents and as tools for basic research. In spite of the importance of these inhibitors, there are significant gaps in our understanding of the most fundamental principles of their action. Most of them interfere with protein synthesis by blocking the functional centers of the ribosome. Out of several functional centers, the catalytic peptidyl transferase center (PTC) and the nascent peptide exit tunnel (NPET) are the sites targeted by the broadest array of inhibitors. In the proposed project, we will explore the molecular mechanisms of action of the most basic PTC-targeting antibiotics and macrolides – chloramphenicol (CHL) and erythromycin (ERY). Recent studies yielded the unexpected conclusion that, in contrast to the general view of CHL and ERY as global and indiscriminate inhibitors, these antibiotics interfere with translation in a context-specific manner indicating that our understanding of their mechanism of action is incomplete and possibly even wrong. One way to obtain a clear explanation for the paradigm-shifting phenomenon of context-specific activity of PTC-acting inhibitors and macrolides is to directly visualize them within the ribosome complexes conducive to their action. Previous crystal structures uncovered how CHL and ERY bind to the PTC and NPET of the vacant bacterial ribosome and therefore provide information that is irrelevant for their context-specific activity. By determining the structures of CHL and ERY (as well as other PTC- acting drugs and macrolides) in functionally relevant ribosome complexes containing A-site aminoacyl-tRNA and P-site peptidyl-tRNA we will provide atomic-level view of their interactions not only with the ribosome (as before) but also with the growing peptide. Moreover, such structures could also reveal rearrangements that take place in the PTC of the ribosome upon drug binding and result in allosteric effects. Hence, in the Specific Aim 1, we will focus on obtaining the structures of 70S complexes carrying various aminoacyl-tRNAs in the A site in the presence and absence of CHL. Then, in the Specific Aim 2, we will obtain the first set of CHL-bound ribosome structures featuring dipeptidyl-tRNAs in the P site containing alanine, serine, or threonine in the penultimate position (the only sequence requirement for the efficient CHL-induced stalling). Finally, in the Specific Aim 3, we will provide structural and mechanistic insights into the context-specific activity of ERY and other macrolides. Once our proposed methodology is established and refined, we will expand it onto the newest FDA-approved clinically important drugs, such as linezolid, tedizolid, telithromycin, and solithromycin. The anticipated findings should significantly expand our understanding of the general mode of action of basic, as well as clinically- important, antibacterial drugs 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.

概括 核糖体靶向抗生素作为治疗剂和作为基础研究的工具都是必不可少的。在 这些抑制剂的重要性，我们对最多的理解存在很大的差距 他们行动的基本原则。它们中的大多数通过阻止功能来干扰蛋白质合成 核糖体的中心。在几个功能中心中，催化肽基转移酶中心（PTC）和 新生的肽出口隧道（NPET）是最广泛的抑制剂靶向的部位。在提议中 项目，我们将探讨最基本的靶向PTC抗生素的作用分子机制和 大环内酯类 - 氯霉素（CHL）和红霉素（ERY）。最近的研究得出了意外的结论 与Chl和Ery的一般观点相反，这些抗生素是全球和不加选择的抑制剂 以特定于上下文的方式干扰翻译，表明我们对它们的机制的理解 行动是不完整的，甚至可能是错误的。获取范式转移的明确解释的一种方法 PTC作用抑制剂和大环内酯类药物的上下文特异性活动的现象是直接可视化它们 在核糖体复合物中导电到其作用。以前的晶体结构发现了CHL和 ery结合空缺细菌核糖体的PTC和NPET，因此提供了信息 与其上下文特定的活动无关。通过确定CHL和ERY的结构（以及其他PTC- 作用药物和大环内酯类） p位点肽基-TRNA我们将不仅提供与核糖体相互作用的原子级别（如前） 而且随着肽的增长。此外，这样的结构也可以揭示发生的重排 在药物结合后，核糖体的PTC中产生变构作用。因此，在特定的目标1中，我们 将着重于获得70年代络合物的结构 CHL的存在和不存在。然后，在特定的目标2中，我们将获得第一组CHL结合的核糖体 在倒数第二次含有丙氨酸，丝氨酸或苏氨酸的P位置中具有二肽基-TRNA的结构 位置（有效CHL引起的失速的唯一序列要求）。最后，在特定目标3中，我们 将提供对Ery和其他大花生剂的特定于上下文活动的结构和机械见解。 一旦我们提出的方法被建立和完善，我们将将其扩展到最新的FDA批准 临床上重要的药物，例如linezolid，tedizolid，telithromycin和solidhromycin。预期的发现 应该显着扩展我们对基本以及临床上基本的一般作用方式的理解 重要的，在核糖体催化中心作用的抗菌药物，可能为 具有优质抗生素特性的蛋白质合成抑制剂的合理发展。