Evaluating darobactins as antimicrobial agents

评价达罗巴汀作为抗菌药物

基本信息

批准号：
10177579
负责人：
Kim Lewis
金额：
$ 62.02万
依托单位：
NORTHEASTERN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10177579
关键词：
Acute Affect Animals Antibiotics Antimicrobial Resistance Bacterial Genome Binding Bioinformatics Biological Assay COVID-19 pandemic Cells Clinic Code Data Databases Development Disease Dose Drug resistance Epidemic Escherichia coli Ethers Evaluation Fiber Future Gene Cluster Goals Gram-Negative Bacteria Health Human In Vitro Individual Infection Klebsiella pneumoniae Label Lateral Life Link Location Lung infections Lysine Membrane Metagenomics Modeling Modification Molecular Chaperones Multiple Bacterial Drug Resistance Mus Mutagenesis Nature Nematoda Operon Penetration Peptide Signal Sequences Peptides Permeability Pharmaceutical Preparations Photorhabdus Polymyxin Resistance Predisposition Probability Problem Sets Production Property Proteins Pseudomonas aeruginosa Resistance Resistance development Ribosomes Sepharose Septicemia Societies Sorting - Cell Movement Structure Surface Testing Thigh structure Thinness Time Toxic effect Translating Tryptophan VDAC1 gene Vertebral column Virulence Virus analog animal efficacy animal safety antimicrobial drug base beta pleated sheet cell envelope cytotoxicity cytotoxicity test drug candidate drug development efficacy evaluation efficacy testing experience experimental study genome database improved in vivo in vivo evaluation knock-down member mortality mouse model mutant novel pathogen periplasm pharmacokinetics and pharmacodynamics prevent resistance frequency scaffold screening symbiont vaccine development

项目摘要

The Antimicrobial Resistance Crisis (AMR) has been recognized for years, and the significance of this global human health problem sets it apart from other types of diseases, because it affects not only individuals, but has a potential to disrupt the life of society. We have a stark reminder of the ability of a pathogen to bring normal life to a halt, as we experience the COVID-19 pandemic. In the case of a virus, we can usually count on a reasonably rapid development of a vaccine. For multidrug-resistant bacteria, we do not have a similarly reliable approach, and the pipeline of novel compounds against the most problematic pathogens, MDR Gram- negative bacteria, is very thin (Lewis, Cell 2020). We recently discovered a novel class of compounds acting against important Gram-negative pathogens, the darobactins (Imai et al., Nature 2019). Darobactin A is a 7- mer modified peptide containing two unusual fused rings. This creates a rigid β-strain from the peptide backbone. The target is BamA, an essential chaperone that inserts proteins such as porins into the outer membrane. BamA recognizes a signal sequence of incoming peptides that bind to one of its β-strands. Darobactin, which has a unique preformed β-strand, is a better binder and prevents substrates from interacting with BamA. Importantly, BamA mutants resistant to darobactin A lose virulence. Darobactin A has no cytotoxicity and shows good efficacy in mouse septicemia and thigh models against such pathogens as polymyxin-resistant E. coli and KPC K. pneumoniae. Darobactin A is ribosomally translated and coded by a RiPP operon. Bioinformatics search of the NCBI genomes database resulted in identifying 8 analogs with the same scaffold, and 6 darobactin-like analogs. The goal of this project is to evaluate the darobactins and identify the best leads. Additional analogs will be identified by searching through the raw data of the metagenomics database. We will synthesize the dar operons, clone them into E. coli and optimize production. For this, we will use an approach we recently developed, screening mutagenized producers in agarose microdroplets containing a YFP-labeled test pathogen. FACS analysis allows to sort droplets in which the test strain is inhibited. Spectrum of action will be determined, resistant mutants will either confirm BamA as a target, or point to a new one. We will analyze virulence of resistant mutants in detail. Compounds will be tested for cytotoxicity and animal safety and efficacy with target pathogens. This project will result in leads ready to enter into development to treat pathogens of critical priority.

抗菌素耐药性危机 (AMR) 多年来一直受到人们的关注，这一全球性危机的重要性人类健康问题与其他类型的疾病不同，因为它不仅影响个人，而且影响病原体有可能扰乱社会生活。当我们经历 COVID-19 大流行时，我们通常可以指望正常生活停止。对于多重耐药细菌，我们还没有类似的快速开发疫苗。可靠的方法，以及针对最有问题的病原体的新型化合物的管道，MDR Gram- 阴性细菌非常稀薄（Lewis，Cell 2020）我们最近发现了一类新型化合物。 Darobactin A 是一种针对重要的革兰氏阴性病原体的药物（Imai 等人，Nature 2019）。 mer 修饰肽含有两个不寻常的融合环，这会从肽中产生刚性 β 菌株。目标是 BamA，一种将孔蛋白等蛋白质插入外层的重要伴侣。 BamA 识别与其 β 链之一结合的传入肽的信号序列。 Darobactin 具有独特的预成型 β 链，是更好的粘合剂，可防止底物相互作用重要的是，对 Darobactin A 具有抗性的 BamA 突变体不会失去毒力。细胞毒性，并在小鼠败血症和大腿模型中针对以下病原体显示出良好的功效多粘菌素耐药大肠杆菌和 KPC 肺炎克雷伯菌 Darobactin A 由核糖体翻译和编码。 RiPP 操纵子对 NCBI 基因组数据库的生物信息学搜索发现了 8 个类似物相同的支架和 6 种 darobactin 类似物该项目的目标是评估 darobactin 和 6 种 darobactin 类似物。确定最佳线索将通过搜索原始数据来确定。我们将合成 dar 操纵子，将其克隆到大肠杆菌中并优化生产。为此，我们将使用我们最近开发的方法，筛选琼脂糖中的诱变生产者含有 YFP 标记的测试病原体的微滴可以通过 FACS 分析对其中进行测试的液滴进行分类。菌株的作用谱将被确定，抗性突变体将确认 BamA 为一种。目标，或指向一个新的目标，我们将详细分析耐药突变体的毒力。该项目将产生针对目标病原体的细胞毒性和动物安全性和功效。进入开发以治疗最重要的病原体。