GUARD: A global unbiased antimicrobial discovery platform

摘要 抗菌素耐药性（AMR）微生物的出现已被世界卫生组织（WHO）确定为对人类健康和安全的全球威胁。滥用抗生素与自然进化相结合，使我们处于传统抗生素不再有效的境地。为了解决这一全球危机，必须开发新的分子来对抗AMR微生物。不幸的是，常规筛选方法中的显著差距阻碍了有效发现所需的新型抗菌或宿主菌依赖性抗感染（HDAI）治疗剂。该提案将致力于开发一种创新，能够同时筛选具有抗微生物和/或HDAI治疗活性的化合物，从而极大地提高阻止AMR细菌的靶向空间。具体而言，一种名为GUARD（全球无偏见抗菌剂回收和发现平台）的新型微流体系统，支持高通量，低成本筛选环境微生物，这些微生物产生直接杀死病原体或激活宿主HDAI活性的天然产物（NP）。值得注意的是，GUARD系统将以单细胞（数字）分辨率执行这些测定。该项目的目标是进一步开发和利用GUARD来鉴定直接杀死或预防耐甲氧西林金黄色葡萄球菌（MRSA）以及多重耐药鲍曼不动杆菌和铜绿假单胞菌感染的新类型分子，这些分子已被归类为全球最高优先级（即，“关键”）。此外，本项目将查明其行动机制。目标1：开发GUARD平台，并利用它筛选来自不同栖息地的超过108种微生物，以鉴定合成NP的分离株，这些NP（a）杀死多重耐药A。鲍曼不动杆菌、铜绿假单胞菌和MRSA，或诱导保护宿主细胞的HDAI活性，和（B）对宿主细胞无毒;目的2：利用测序和最先进的分析化学去复制、先导物优先化和小分子纯化策略来鉴定具有所需特征的新型NP支架;目的3：评价最有前途的NP抗菌剂和/或HDAI靶向的病原体谱，以及我们最有前途的HDAI NP的宿主靶标。这项工作的重要成果将是开发和实施一种新的平台和工作流程，用于发现击败AMR微生物的分子，并提供几种新型NP，可用于进一步的临床前测试和临床评估。

ABSTRACT The emergence of antimicrobial resistance (AMR) microbes has been identified by the World Health Organization (WHO) as global threats to human health and safety. Indiscriminate use of antibiotics conjoined with natural evolution has now put us in a situation where conventional antibiotics are no longer effective. To address this global crisis, new molecules to combat AMR microbes must be developed. Unfortunately, significant gaps in conventional screening approaches have prevented the efficient discovery of desired novel antibacterial or host­ dependent anti-infective (HDAI) therapeutics. This proposal will pursue the development of an innovation that enables the simultaneous screening of compounds with antimicrobial and/or HDAI therapeutic activities, thereby dramatically enhancing the target space for thwarting AMR bacteria. Specifically, a novel microfluidic system called GUARD (a Global Unbiased Antimicrobial Recovery and Discovery platform), which supports the high­ throughput, low-cost screening of environmental microbes that produce natural products (NP) that either directly kill pathogens, or that activate host HDAI activities, has been prototyped. Notably, the GUARD system will perform these assays at single-cell (digital) resolution. The goal of this project is to further develop and utilize GUARD to identify new classes of molecules that either directly kill or prevent infection by methicillin-resistant Staphylococcus aureus (MRSA) as well as multi-drug resistant Acinetobacter baumannii and Pseudomonas aeruginosa, which have been categorized as the highest global priority (i.e., "critical") by the WHO. In addition, this project will discern their mechanisms of action. With these ideas in mind, this proposal aims: Aim 1: To develop the GUARD platform and utilize it to screen more than 108 microbes from diverse habitats to identify isolates that synthesize NPs that (a) kill multidrug resistant A. baumannii, P. aeruginosa, and MRSA or induce HDAI activities that protect host cells, and (b) are non-toxic to host cells; Aim 2: To utilize sequencing and state­ of-the-art analytical chemical dereplication, lead prioritization, and small molecule purification strategies to identify novel NP scaffolds with desirable profiles; Aim 3: To evaluate the spectrum of pathogens targeted by the most promising NP antimicrobials and/or HDAIs, and the host targets of our most promising HDAI NPs. Important outcomes of this work will be the development and implementation of a novel platform and workflow for the discovery of molecules that defeat AMR microbes, and the delivery of several novel NPs that can be advanced for further pre-clinical testing and clinical evaluation.