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

以独特的作用机制推进核糖体靶向抗菌肽

• 批准号: 10569098
• 负责人: ALEXANDER S MANKIN
• 金额: $ 75万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-08 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10569098
• 关键词: Acinetobacter baumannii; Affect; Amino Acid Sequence; Amino Acids; Anti-Bacterial Agents; Antibiotics; Bacteria; Binding; Biochemistry; Biological; Bypass; Cells; Chemistry; Clinical; Complex; Data; Development; Drosophila genus; Escherichia coli; Event; Future; Gene Library; Genome; Genomics; Goals; Gram-Negative Bacteria; Gram-Positive Bacteria; Growth; Hybrids; Infection; Laboratories; Modeling; Nature; Penetration; Peptide Synthesis; Peptides; Pharmaceutical Preparations; Proline; Property; Protein Biosynthesis; Protein Synthesis Inhibitors; Proteins; Publishing; Reagent; Research; Resolution; Ribosomes; Roentgen Rays; Specificity; Staphylococcus aureus; Structure; Terminator Codon; Testing; Transfer RNA; Translational Repression; Translations; Variant; Work; antimicrobial peptide; cell growth; clinical development; clinically relevant; comparative; design; fly; human pathogen; improved; infancy; inhibitor; knowledge base; novel; pathogen; peptide chemical synthesis; peptide drug; prevent; rational design; release factor; screening; therapeutic development; tool; uptake; whole genome

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.

项目概要 Apidaecin (Api) 和 Drosocin (Dro) 是由蜜蜂产生的富含脯氨酸的抗菌肽 (PrAMP) 果蝇分别具有独特的作用机制。我们之前对 Api 的研究表明，进入后 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通道中 新制造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。我们随后的全基因组 研究表明，阻止终止核糖体会触发一些下游事件，从而加剧抑制作用 PrAMP 的作用，包括核糖体排队和终止密码子的通读。我们的初步数据表明，Dro， 尽管其氨基酸序列不同，但也通过可能类似于的机制抑制翻译终止步骤 是 API 的。它们与目标结合的特殊模式、独特的作用机制以及触发 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 的生物学性质为其筛选和筛选提供了独特的机会。 通过直接在细菌细胞中生成数十万种肽变体来进行优化。 在当前的提案中，我们将利用三个具有生物化学和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 促进对 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扩大的抗菌作用谱的衍生物。为了达到 为了实现这些目标，我们将通过肽基因库的可调表达来测试细菌细胞中的 Api 和 Dro 变体阵列， 确定核糖体-肽复合物的高分辨率 X 射线晶体结构，并采用基于合理结构的方法 设计通过化学合成产生具有优异特性的肽变体。具体来说：在目标 1 中，我们将确定 API 衍生肽对革兰氏阴性和革兰氏阳性病原体的核糖体具有改善的活性。在目标 2 中， Api 样肽的作用范围将通过绕过 SbmA 转运蛋白摄取的必要性而得到扩展。 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 分析以确定定义靶向翻译终止的抗菌肽类别的关键特征。这 三个目标紧密相连，但又完全独立。 在拟议工作过程中将产生的试剂和工具旨在作为线索 未来的临床发展。重要的是，拟议研究中获得的结果将显着推进 对 PrAMP 的性质和作用机制的基本了解，将促进该领域的进展 核糖体靶向抗菌肽领域目前仍处于起步阶段。