Evaluating darobactins as antimicrobial agents

评价达罗巴汀作为抗菌药物

• 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
• 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

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.

抗菌抗性危机（AMR）已被认可多年，并且该全球的意义 人类健康问题将其与其他类型的疾病区分开来，因为它不仅影响个人，而且会影响 有可能破坏社会生活。我们很明显地提醒了病原体带来的能力 随着我们经历了COVID-19的大流行，正常生命会停止。就病毒而言，我们通常可以指望 疫苗的合理发展。对于多药耐药细菌，我们没有类似的 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- 阴性细菌非常薄（刘易斯，细胞，2020年）。我们最近发现了一种新颖的化合物表演 反对重要的革兰氏阴性病原体，darobactins（Imai等，自然，2019年）。 darobactin a是7- MER修饰的肽包含两个异常融合的环。这会从肽产生刚性的β-strain 骨干。目标是巴马，这是一种必需的伴侣 膜。巴马识别出结合其β链之一的传入肽的信号序列。 具有唯一预制的β链的Darobactin是一种更好的粘合剂，可防止底物相互作用 与巴马。重要的是，巴马突变体耐darobactin a丢失的病毒。 darobactin a没有 细胞毒性和针对病原体的小鼠败血症和大腿模型的效率良好 耐多糖剂的大肠杆菌和KPC K.肺炎。 darobactin a是核糖体翻译和编码的 Ripp Operaon。对NCBI基因组数据库的生物信息学搜索导致识别8个类似物 相同的脚手架和6个类似类似的类似物。该项目的目的是评估darobactins和 确定最好的线索。通过搜索浏览的原始数据，将确定其他类似物 宏基因组学数据库。我们将合成DAR操纵子，克隆它们进入大肠杆菌并优化生产。 为此，我们将使用我们最近开发的一种方法，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螺旋体。 FACS分析允许对测试的液滴进行排序 菌株被抑制。将确定动作范围，抗性突变体将确认巴马为 目标，或指向一个新的。我们将详细分析抗性突变体的病毒。化合物将进行测试 用于细胞毒性和动物安全以及目标病原体的效率。该项目将导致潜在客户准备 进入开发以治疗关键优先级的病原体。