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

项目摘要 Apidaecin(Api)和Droocin(Dro)是蜜蜂和蜜蜂产生的富含脯氨酸的抗菌肽(PRAMP)。 果蝇，它们都有一个独特的作用机制。我们之前对Api的研究表明，在进入 革兰氏阴性细菌细胞通过SBMA转运蛋白，Api结合在刚刚释放的核糖体的出口隧道中 这种新合成的蛋白质通过捕获相关的tRNA和释放因子，在终止密码子上阻止核糖体。AS 这样，Api代表了第一个被描述的翻译终止的特异性抑制物。我们随后的全基因组 研究表明，阻止终止核糖体触发了几个下游事件，从而加剧了抑制作用 PrAMP的作用，包括核糖体排队和停止密码子的通读。我们的初步数据显示， 尽管它有不同的氨基酸序列，但也抑制了翻译的终止步骤，其机制很可能类似于 那是阿比的。它们与靶标结合的独特模式，独特的作用机制，以及 下游效应对细菌细胞有害，使这些抗菌肽成为开发新型抗菌肽的诱人模型 抗生素。此外，这些野马的生物学特性为它们的筛选和 通过在细菌细胞中直接产生数十万个多肽变体进行优化。 在目前的提案中，我们将使用三个拥有生物化学和专业知识的实验室的联合努力 核糖体抗生素基因组学，在多肽化学和核糖体-抗生素复合体的结构分析中 加深对Api和Dro样翻译终止抑制剂作用机制的基本认识 并鉴定具有良好的靶向活性和广谱抗菌作用的衍生物。为了实现 这些目标我们将通过肽基因文库的可调表达在细菌细胞中测试Api和dro变体的阵列， 确定核糖体-多肽复合体的高分辨X射线晶体结构，并采用合理的结构基础 设计通过化学合成产生具有优越性能的多肽变体。具体来说：在目标1中，我们将确定 来自革兰氏阴性和革兰氏阳性病原体的API衍生多肽对核糖体的活性提高。在目标2中， Api样肽的作用范围将通过绕过SBMA转运体摄取的必要性而扩大。 最后，在目标3，我们将分析核糖体结合和作用机制，并使用比较 分析以确定定义以翻译终止为目标的抗菌肽类别的关键特征。这个 三个目标紧密相连，但又完全相互独立。 将在拟议工作过程中产生的试剂和工具旨在作为以下方面的线索 未来的临床发展。重要的是，拟议研究的结果将大大推进 从根本上了解PRMP的性质和作用机制，并将推动 核糖体靶向抗菌肽领域，目前还处于起步阶段。

Project Summary Apidaecin (Api) and Drosocin (Dro), are proline-rich antimicrobial peptides (PrAMPs) produced by honeybees and fruit flies, respectively, which share a unique mechanism of action. Our previous studies of Api showed that upon entering Gram-negative bacterial cells through the SbmA transporter, Api binds in the exit tunnel of ribosomes that have just released the newly made protein and arrests the ribosomes at stop codons by trapping the associated tRNA and release factor. As such, Api represents the first-ever described specific inhibitor of translation termination. Our subsequent whole-genome studies revealed that arresting terminating ribosomes triggers several downstream events that accentuate the inhibitory action of this PrAMP, including ribosome queuing and readthrough of stop codons. Our preliminary data indicate that Dro, despite its distinct amino acid sequence, inhibits the termination step of translation as well, by a mechanism likely resembling that of Api. Their idiosyncratic mode of binding to the target, the unique mechanism of action, and the triggering of downstream effects harmful for the bacterial cell, make these antibacterial peptides an attractive model for developing novel antibiotics. Furthermore, the biological nature of these PrAMPs opens unique opportunities for their screening and optimization by generating hundreds of thousands of peptide variants directly in bacterial cells. In the current proposal we will use the combined effort of three laboratories with expertise in biochemistry and genomics of ribosomal antibiotics, in peptide chemistry and in structural analysis of ribosome-antibiotic complexes to advance the fundamental understanding of the mechanism of action of Api- and Dro-like translation termination inhibitors and identify derivatives with superior on-target activity and expanded spectrum of antibacterial action. In order to achieve these goals we will test arrays of Api and Dro variants in bacterial cells by the tunable expression of peptide gene libraries, determine high-resolution X-ray crystal structures of ribosome-peptide complexes, and employ rational structure-based design to generate via chemical synthesis peptide variants with superior properties. 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 analyze the ribosome binding and mechanism of action of Dro-like peptides and use comparative analysis to identify the key features that define the class of antimicrobial peptides that target translation termination. The three Aims are tightly interconnected but completely independent from each other. The reagents and tools that will be generated in the course of the proposed work are aimed to serve as leads for future clinical development. Importantly, the results obtained in the proposed studies will significantly advance the fundamental understanding of the properties and mechanisms of action of PrAMPs and will stimulate the progress of the field of ribosome-targeting antibacterial peptides, which currently is still in its infancy.