Molecular mechanisms of action of ribosome-targeting antibiotics.

摘要 核糖体靶向抗生素作为治疗剂和基础研究的工具都是不可或缺的。在……里面 尽管这些抑制物很重要，但我们对大多数 他们行动的基本原则。它们中的大多数通过阻断功能性的蛋白质合成而干扰蛋白质的合成。 核糖体的中心。在几个功能中心中，催化肽转移酶中心(PTC)和 新生肽出口通道(NPET)是最广泛的一系列抑制剂的靶点。在建议的 项目中，我们将探索最基本的PTC靶向抗生素和 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 与普遍认为CHL和ERY是全球不分青红皂白的抑制剂相反，这些抗生素 以特定于语境的方式干扰翻译，表明我们对其机制的理解 行动是不完整的，甚至可能是错误的。获得对范式转换的明确解释的一种方法 PTC作用抑制剂和大环内酯类化合物的上下文特定活性现象是将它们直接可视化 在核糖体复合体内有利于它们的作用。先前的晶体结构揭示了CHL和CHL如何 ERY与空细菌核糖体的PTC和NPET结合，因此提供了 与他们特定于环境的活动无关。通过确定CHL和ERY(以及其他PTC-)的结构 作用药物和大环内酯类)在含有A位氨酰基-tRNA和 我们将提供它们相互作用的原子水平视图，而不仅仅是与核糖体的相互作用(如前所述) 而且还伴随着生长中的多肽。此外，这种结构还可能揭示发生的重新安排 在核糖体的PTC上与药物结合并导致变构效应。因此，在具体目标1中，我们 将专注于获得在A位携带各种氨基酰基-tRNA的70S复合体的结构 CHL的存在和缺失。然后，在特定的目标2中，我们将获得第一组与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位具有二肽-tRNAs的结构 位置(有效的CHL诱导失速的唯一顺序要求)。最后，在具体目标3中，我们 将提供对ERY和其他大环内酯类特定背景活动的结构性和机械性见解。 一旦我们提议的方法建立和改进，我们将把它扩展到FDA批准的最新方法 临床上重要的药物，如利奈唑胺、替地唑胺、泰红霉素和索利霉素。预期的调查结果 应该显著扩大我们对基础以及临床上的一般作用模式的理解- 重要的，作用于核糖体催化中心的抗菌药物，可能会为 合理开发具有优异抗菌性能的蛋白质合成抑制剂。

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.