Evaluating darobactins as antimicrobial agents

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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.