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: 9898395
• 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 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9898395
• 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/antagonist; 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.

总结 核糖体靶向抗生素作为治疗药物和基础研究工具是不可或缺的。在 尽管这些抑制剂很重要，但我们对大多数抑制剂的理解仍存在重大差距。 其行动的基本原则。它们中的大多数通过阻断蛋白质合成的功能而干扰蛋白质合成。 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 新生肽出口通道（NPET）是最广泛的抑制剂靶向的位点。拟议 项目，我们将探索最基本的PTC靶向抗生素的分子作用机制， 大环内酯类-氯霉素（CHL）和红霉素（ERY）。最近的研究得出了意想不到的结论 与CHL和ERY作为全球性和不加选择的抑制剂的一般观点相反，这些抗生素 以特定于上下文方式干扰翻译，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。一种方法来获得一个清晰的解释范式转变 PTC作用抑制剂和大环内酯类的背景特异性活性的现象是直接将它们可视化 在核糖体复合物内有助于它们的作用。以前的晶体结构揭示了CHL和 ERY与空细菌核糖体的PTC和NPET结合，因此提供信息， 与其特定环境活动无关。通过确定CHL和ERY（以及其他PTC- 作用药物和大环内酯类）在功能相关的核糖体复合物中， P-位点肽酰-tRNA我们将提供它们不仅与核糖体相互作用的原子水平视图（如前所述） 也与生长肽有关。此外，这种结构还可以揭示发生在 在药物结合时，在核糖体的PTC中，并导致变构效应。在具体目标1中，我们 将专注于获得70 S复合物的结构，该复合物在A位点携带各种氨酰-tRNA。 CHL的存在和不存在。然后，在具体目标2中，我们将获得第一组CHL结合的核糖体 在倒数第二位含有丙氨酸、丝氨酸或苏氨酸的P位点具有二肽基-tRNA的结构 位置（有效CHL诱导失速的唯一顺序要求）。最后，在具体目标3中，我们 将提供结构和机制的洞察ERY和其他大环内酯类药物的具体活动。 一旦我们提出的方法建立和完善，我们将扩大到最新的FDA批准的 临床上重要的药物，如利奈唑胺、替地唑胺、泰利霉素和索利霉素。预期结果 应该大大扩展我们对基本的一般作用模式的理解，以及临床- 重要的是，抗菌药物作用于核糖体的催化中心，并可能打开新的场所， 合理开发具有上级抗菌性能的蛋白质合成抑制剂。