GUARD: A global unbiased antimicrobial discovery platform

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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.