Discovering antimicrobials acting against MDR pathogens

摘要 我们正在经历一场抗菌素耐药性危机(AMR)，这是由于 抗生素的发现。世卫组织指定了一份优先病原体清单，其中，耐多药革兰氏菌- 阴性肠杆菌科(大肠杆菌、鼠伤寒沙门氏菌、肺炎克雷伯氏菌、肠杆菌)， 铜绿假单胞菌和鲍曼不动杆菌)是“关键优先事项”。这些 病原体是本提案的重点，旨在开发一个有效的 新型抗菌剂的发现。该领域曾经历过一段发现的黄金时代， 主要通过对土壤放线菌的筛选。所有主要类别的广谱抗生素都是有效的 抗革兰氏阴性病原体是在20世纪60年代发现的。过度挖掘放线菌 导致了探索平台的崩溃。从那时起才发现了新的抗生素 对革兰氏阳性物种采取行动。我们开发了各种方法来访问更广泛的 细菌，重点是占总生物多样性99%的未培养物种。一批 新的化合物来自这个来源，包括teixobactin，代表了一种新的细胞类别- 没有检测到耐药性的壁效化合物(Ling等人，2015年)。替克生是 正在进行支持IND的研究；它也是一种窄谱化合物。我们建议 开发高效发现新型抗菌剂的平台。主要的问题是 有毒化合物的巨大背景，在较小程度上，是已知化合物。我们假设 取消复制的瓶颈可以通过差异筛选来解决，该差异筛选检测 在去碱基之前存在一种有希望的化合物。使用这种方法，我们最近 发现了具有新的支架并以必要的外膜为靶点的Darobactins 蛋白质巴马(Imai等人，2019年)和其他几个新化合物。在建议的 项目，我们将开发一种基于封装生产的超高通量屏幕 细菌和不同荧光标记的报告在微滴中产生 微流控装置。我们的初步数据显示，对液滴进行分类的速度可以 每天106种，并导致检测到理想的抗菌剂生产商。我们将评估 该屏幕的几种模式，旨在发现选择性和广谱 抗革兰氏阴性细菌的化合物；以及抗过敏性化合物。站台 很可能对抗生素发现领域有所帮助。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评估。从这个项目中产生的新线索将 准备好进入支持IND的学习。

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.