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: 10443921
• 负责人: ALEXANDER S MANKIN
• 金额: $ 45.43万
• 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/10443921
• 关键词: Acinetobacter baumannii; Affect; Amino Acid Sequence; Amino Acids; Anti-Bacterial Agents; Antibiotics; Bacteria; Binding; Biochemistry; Biological; Bypass; Cells; Chemistry; Clinical; Complex; Crystallization; 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; Translations; Variant; Work; antimicrobial peptide; base; 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.

项目摘要 apidaecin（API）和drosocin（DRO）是由蜜蜂和 果蝇分别具有独特的作用机理。我们先前对API的研究表明，进入 革兰氏阴性细菌通过SBMA转运蛋白，API结合刚刚释放的核糖体出口隧道 新制作的蛋白质并通过捕获相关的tRNA和释放因子来阻止终止密码子的核糖体。作为 这样的API代表了翻译终止的第一个描述的特定抑制剂。我们随后的全基因组 研究表明，逮捕终止核糖体会触发几个下游事件，这些事件突显了抑制性 该pramp的作用，包括核糖体排队和终止密码子的读取。我们的初步数据表明DRO， 尽管它具有独特的氨基酸序列，但也抑制了翻译的终止步骤，但可能类似于一种机制 API。他们与目标结合的特质模式，独特的作用机理以及触发 对细菌细胞有害有害的下游效应，使这些抗菌肽成为开发新型的有吸引力的模型 抗生素。此外，这些pramps的生物学性质为其筛查和 通过直接在细菌细胞中产生数十万个肽变体来优化。 在当前的提案中，我们将使用三个实验室的共同努力，并具有生物化学专业知识和 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至 促进对API和DRO样翻译终止抑制剂的作用机理的基本理解 并鉴定具有出色靶向活性的衍生物和抗菌作用范围扩大的衍生物。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，并采用基于理性结构 设计具有具有出色性能的化学合成肽变体。具体：在AIM 1中，我们将确定 API衍生的肽具有改善革兰氏阴性和革兰氏阴性病原体核糖体活性的活性。在AIM 2中， 通过绕过SBMA转运蛋白吸收的必要性，将扩大API样肽的作用范围。 最后，在AIM 3中，我们将分析核糖体的结合和Dro-like肽的作用机理，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 三个目标是紧密互连的，但完全独立。 在拟议的工作过程中将生成的试剂和工具的目的是作为潜在客户 未来的临床发展。重要的是，在拟议的研究中获得的结果将显着推动 对Pramps的特性和作用机理的基本了解，并将刺激 核糖体靶向抗菌肽的领域，目前仍处于起步阶段。