Discovering antimicrobials acting against MDR pathogens

摘要 我们正在经历一场抗生素耐药性危机（AMR），这是抗生素耐药性下降的直接结果。 抗生素的发现世界卫生组织指定了一份优先病原体清单，其中，耐多药革兰氏- 阴性肠杆菌科（E. coli、S.鼠伤寒杆菌、肺炎克雷伯氏菌、肠杆菌属）， 铜绿假单胞菌和鲍氏不动杆菌）为“关键优先”。这些 病原体是本提案的重点，旨在开发一个有效的 发现新的抗菌剂。该领域曾经享有发现的黄金时代， 主要通过筛选土壤放线菌。所有主要类别的广谱抗生素活性 抗革兰氏阴性病原体的药物是在20世纪60年代发现的。放线菌的过度开采 导致了探索平台的崩溃从那时起才发现新的抗生素 对抗革兰氏阳性菌我们开发了一种方法， 细菌，重点是未培养的物种，占总生物多样性的99%。一些 新的化合物来自这个来源，包括teixobactin，代表了一类新的细胞， 没有可检测的电阻的壁作用化合物（Ling等人，2015年）。泰克菌素是 正在进行IND研究;它也是一种窄谱化合物。我们建议 开发一个有效发现新型抗菌剂的平台。主要问题是 大量的有毒物质，以及在较小程度上已知的化合物。我们假设 去复制的瓶颈可以通过差异筛选来解决， 在去复制之前存在有希望的化合物。通过这种方法，我们最近 发现了具有新支架并靶向基本外膜的darobactins 蛋白BamA（Imai等，2019），以及其他一些新化合物。拟议 项目，我们将开发一种基于封装生产的超高通量筛选 细菌与不同的荧光标记的报告分子一起在微滴中产生， 微流体装置我们的初步数据表明，可以以一定的速度对液滴进行分选， 每天106个，并导致检测到理想的抗菌剂的生产商。我们将评估 这种屏幕的几种形式，旨在发现选择性以及广谱 抗革兰氏阴性菌的化合物;和抗持久性化合物。平台 很有可能在抗生素发现领域有所应用。从这个屏幕出来的线索将是 在体外和动物感染模型中进行了评价。从这个项目中产生的新线索将 准备好进入IND启动研究。

Abstract We are experiencing an antimicrobial resistance crisis (AMR), a direct result of a decline in antibiotic discovery. The WHO designated a list of priority pathogens, and of these, MDR Gram- negative Enterobacteriaceae (E. coli, S. typhimurium, Klebsiella pneumoniae, Enterobacter), Pseudomonas aeruginosa, and Acinetobacter baumannii) are of “critical priority”. These pathogens are the focus of the present proposal aimed at developing a platform for efficient discovery of novel antimicrobials. The field once enjoyed a golden era of discovery, fueled mainly by screening of soil actinomycetes. All major classes of broad-spectrum antibiotics active against Gram-negative pathogens were discovered by the 1960s. Overmining of actinomycetes resulted in the collapse of the discovery platform. Novel antibiotics discovered since then only act against Gram-positive species. We developed methods to access a broader range of bacteria, with a focus on uncultured species that make up 99% of total biodiversity. A number of novel compounds came from this source, including teixobactin, representing a new class of cell- wall acting compounds without detectable resistance (Ling et al., 2015). Teixobactin is undergoing IND-enabling studies; it is also a narrow-spectrum compound. We propose to develop a platform for efficient discovery of novel antimicrobials. The main problem is the enormous background of toxic, and to a lesser extent, known compounds. We hypothesize that the bottleneck of dereplication can be resolved by differential screening that detects the presence of a promising compound prior to dereplication. Using this approach, we recently discovered darobactins that have a novel scaffold and target the essential outer membrane protein BamA (Imai et al., 2019), and several additional novel compounds. In the proposed project, we will develop an ultra-high throughput screen based on encapsulating producing bacteria together with different fluorescently labeled reporters in microdroplets created in a microfluidics device. Our preliminary data show that sorting droplets can be performed at a rate of 106 a day, and leads to detection of producers of desirable antimicrobials. We will evaluate several modalities of this screen, aimed at discovering selective as well as broad-spectrum compounds acting against Gram-negative bacteria; and anti-persister compounds. The platform is likely to be of use to the field of antibiotic discovery. Leads that come out of this screen will be evaluated in vitro and in animal models of infection. Novel leads that come out of this project will be ready to enter IND-enabling studies.