Evaluating darobactins as antimicrobial agents

抗菌素耐药性危机(AMR)已经被认识到多年，这一全球危机的意义 人类的健康问题使它有别于其他类型的疾病，因为它不仅影响个人，而且 有可能扰乱社会生活。我们有一个严酷的提醒，病原体有能力将 随着我们经历新冠肺炎大流行，正常生活陷入停顿。在病毒的情况下，我们通常可以指望 疫苗的相当快速的发展。对于多重耐药细菌，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化合物的流水线，MDR Gram- 负性细菌是非常薄的(Lewis，Cell 2020)。我们最近发现了一类新的化合物 针对重要的革兰氏阴性病原体，Darobactins(Imai等人，《自然》，2019年)。Darobactin A是一个7- 聚合体修饰的含有两个不寻常的融合环的多肽。这就从多肽中产生了刚性的β菌株 我的脊梁。目标是BAMA，一种将孔蛋白等蛋白质插入外层的基本伴侣 薄膜。BAMA识别与其一条β链结合的传入多肽的信号序列。 Darobactin具有独特的预先形成的β-链，是一种更好的结合剂，可以防止底物相互作用 和巴马一起。重要的是，对darobactin A具有抗性的BAMA突变体失去了毒力。达罗巴汀A没有 细胞毒性，在小鼠败血症和大腿模型中表现出良好的疗效 耐多粘菌素的大肠埃希菌和肺炎克雷伯菌。Darobactin A是由核糖体翻译和编码的 RIPP操纵子。对NCBI基因组数据库的生物信息学搜索发现了8个与 同样的支架，和6个类似Darobactin的类似物。该项目的目标是评估Darobactins和 找出最佳线索。通过搜索原始数据将识别其他类似物 元基因组学数据库。我们将合成DAR操纵子，将它们克隆到大肠杆菌中，并优化生产。 为此，我们将使用我们最近开发的一种方法，在琼脂糖中筛选诱变的生产者 含有YFP标记的测试病原体的微滴。FACS分析允许对测试中的液滴进行分类 菌株被抑制。作用范围将被确定，抗药性突变将确认巴马是一种 目标，或指向新的目标。我们将详细分析抗性突变体的毒力。化合物将被测试 针对目标病原体的细胞毒性和动物安全性和有效性。该项目将导致潜在客户做好准备 进入治疗关键优先病原体的开发阶段。

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.