Evaluating darobactins as antimicrobial agents

抗生素耐药性危机（AMR）多年来一直受到重视，这一全球性的危机的重要性也日益突出。 人类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， 有可能扰乱社会生活。我们有一个明确的提醒病原体的能力， 正常生活陷入停顿，因为我们经历了COVID-19大流行。在病毒的情况下，我们通常可以指望 疫苗的快速发展。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型化合物的管道，MDR Gram- 阴性细菌，非常薄（刘易斯，细胞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，Nature 2019）。Darobactin A是7- 含有两个不寻常的稠合环的聚体修饰的肽。这从肽产生刚性β-应变 骨干靶点是BamA，一种将孔蛋白等蛋白质插入细胞外膜的必需分子伴侣。 膜的BamA识别与其β链之一结合的传入肽的信号序列。 Darobactin具有独特的预制β链，是一种更好的结合剂，可防止底物相互作用 关于BamA重要的是，对darobactin A具有抗性的BamA突变体丧失了毒力。Darobactin A没有 细胞毒性，并在小鼠败血症和大腿模型中显示出对诸如病原体的良好功效， 抗多粘菌素E. coli和KPC K.肺炎。Darobactin A是核糖体翻译和编码的， RiPP操纵子。NCBI基因组数据库的生物信息学搜索导致鉴定了8种类似物， 相同的支架和6个darobactin样类似物。该项目的目标是评估darobactins和 找出最好的线索将通过搜索原始数据来识别其他类似物。 宏基因组学数据库。我们将合成dar操纵子，克隆到E.大肠杆菌，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生产者 含有YFP标记的测试病原体的微滴。流式细胞仪分析允许对其中测试的液滴进行分类， 应变被抑制。作用谱将被确定，抗性突变体将证实BamA是一种有效的抑制剂。 目标，或者指向一个新的目标。我们将详细分析抗性突变体的毒力。将对化合物进行检测 针对靶病原体的细胞毒性和动物安全性和有效性。该项目将导致准备 进入开发阶段，以治疗至关重要的病原体。

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.