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

项目摘要 Apidaecin（Api）和Drosocin（Dro）是由蜜蜂产生的富含脯氨酸的抗菌肽（PrAMP）， 果蝇，它们有着独特的作用机制。我们之前对Api的研究表明， 在革兰氏阴性细菌细胞中，Api通过SbmA转运蛋白结合在刚刚释放的核糖体的出口通道中， 新产生的蛋白质，并通过捕获相关的tRNA和释放因子将核糖体捕获在终止密码子处。作为 因此，Api代表了第一个描述的翻译终止的特异性抑制剂。我们后来的全基因组 研究表明，阻止终止核糖体会触发几个下游事件， 这种PrAMP的作用，包括核糖体排队和终止密码子的通读。我们的初步数据显示德罗， 尽管其氨基酸序列不同，但也通过类似于 这是Api。它们与靶点结合的特异性模式、独特的作用机制和 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有吸引力的模型。 抗生素此外，这些PrAMP的生物学性质为它们的筛选提供了独特的机会， 通过直接在细菌细胞中产生数十万种肽变体来优化。 在目前的提案中，我们将利用三个具有生物化学专业知识的实验室的共同努力， 核糖体抗生素的基因组学，肽化学和核糖体抗生素复合物的结构分析， 推进对Api和Dro样翻译终止抑制剂作用机制的基本理解 并鉴定具有优异的靶向活性和广谱抗菌作用的衍生物。为了实现 为了这些目的，我们将通过肽基因文库的可调表达来测试细菌细胞中Api和Dro变体的阵列， 确定核糖体-肽复合物的高分辨率X射线晶体结构，并采用合理的基于结构的 设计以通过化学合成产生具有上级性质的肽变体。具体而言：在目标1中，我们将确定 对来自革兰氏阴性和革兰氏阳性病原体的核糖体具有改进活性的Api衍生肽。在目标2中， Api样肽的作用谱将通过绕过SbmA转运蛋白摄取的必要性而扩展。 最后，在目标3中，我们将分析Dro样肽的核糖体结合和作用机制，并使用比较 分析以鉴定定义靶向翻译终止的抗微生物肽类的关键特征。的 三个目标紧密相连，但又完全独立。 在拟议的工作过程中将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，在拟议的研究中获得的结果将大大推进 对PrAMP的性质和作用机制的基本理解，并将促进 核糖体靶向抗菌肽领域，目前仍处于起步阶段。

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.