Advancing ribosome-targeting antibacterial peptides with a unique mechanism of action

项目摘要 蜂毒素(Apidaecin，Api)是蜜蜂产生的一种抗菌肽，具有独特的作用机制。我们以前的 研究表明，通过SBMA转运蛋白进入革兰氏阴性细菌细胞后，Api与新生细菌结合 细菌核糖体的多肽退出隧道，捕获释放因子，并在终止密码子阻止翻译。因此，阿比 表示有史以来第一个特定的翻译终止抑制器。我们随后的全基因组研究表明 阻止终止的核糖体触发了许多下游事件，包括核糖体排队和通读 终止密码子，这突出了Api的主要抑制作用。与目标绑定的特殊模式，独特的 作用机制，以及引发对细菌细胞有害的下游效应，使Api成为一个有吸引力的模型 用于开发性能更好的新型抗生素。 在目前的提案中，我们将使用三个拥有核糖体抗生素专业知识的实验室的联合努力， 多肽化学和结构分析，以促进对Api样抑制物和 鉴定具有良好的靶向活性和广谱抗菌作用的Api衍生物。我们将实现这些目标 目标是通过基因的控制表达直接在细菌细胞中产生和测试Api变异体阵列 文库，合成经过特殊化学修饰的多肽，以及测定核糖体的X射线晶体结构- API复合体。具体地说：在目标1中，我们将鉴定对核糖体具有更高活性的Api衍生多肽。 革兰氏阴性和革兰氏阳性病原体。在目标2中，Api样肽的作用范围将通过以下方式扩展 绕过了SBMA转运体摄取的必要性。最后，在目标3中，我们将拓宽天然 具有抑制翻译终止能力的抗菌肽。所有的目标都是相互独立的， 但为每个目标计划的实验的具体实施可能会受到来自 其他目标。在拟议工作过程中产生的试剂和工具将作为未来的线索 临床发展。重要的是，拟议研究的结果将大大推进一般领域的发展 核糖体靶向抗菌肽，目前仍处于起步阶段。

Project Summary Apidaecin (Api), an antimicrobial peptide produced by honeybees, has a unique mechanism of action. Our previous studies showed that after entering the cell of Gram-negative bacteria through the SbmA transporter, Api binds in the nascent peptide exit tunnel of the bacterial ribosome, traps the release factors and arrests translation at stop codons. As such, Api represents the first-ever specific inhibitor of translation termination. Our subsequent whole-genome studies have shown that arresting terminating ribosomes triggers a number of downstream events, including ribosome queuing and readthrough of stop codons, that accentuate Api's primary inhibitory action. The idiosyncratic mode of binding to the target, the unique mechanism of action, and the triggering of downstream effects harmful for the bacterial cell, make Api an attractive model for developing novel antibiotics with improved properties. In the current proposal we will use the combined effort of three laboratories with expertise in ribosomal antibiotics, peptide chemistry and structural analysis to advance the fundamental understanding of action of Api-like inhibitors and identify Api derivatives with superior on-target activity and expanded spectrum of antibacterial action. We will achieve these goals by generating and testing arrays of Api variants directly in the bacterial cell by the controlled expression of gene libraries, synthesizing peptides with specific chemical modifications, and determining X-ray crystal structures of ribosome- Api complexes. Specifically: In Aim 1, we will identify Api-derived peptides with improved activity upon ribosomes from Gram-negative and Gram-positive pathogens. In Aim 2, the spectrum of action of Api-like peptides will be expanded by bypassing the necessity for uptake by the SbmA transporter. Finally, in Aim 3, we will broaden the variety of natural antimicrobial peptides that have the ability to inhibit translation termination. All of the Aims are independent from each other, but the specific implementation of experiments planned for each aim could be influenced by the results emerging from the other aims. The reagents and tools that will be generated in the course of the proposed work will serve as leads for future clinical development. Importantly, the results obtained in the proposed studies will significantly advance the general field of ribosome-targeting antibacterial peptides, which currently is still in its infancy.