Enhancing antibiotic activity against Escherichia coli and Klebsiella pneumoniae with optimised pyridylpiperazine AcrAB-TolC efflux pump inhibitors.

Multidrug-resistant Enterobacterales, including Escherichia coli and Klebsiella pneumoniae, are classified as critical priority pathogens by the World Health Organization. A major contributor to resistance in these Gram-negative bacteria is the overexpression of the broad-spectrum RND-efflux pump AcrAB-TolC. To counteract efflux-mediated resistance, a series of pyridylpiperazine inhibitors was previously reported and we demonstrated that these compounds bind to a novel allosteric pocket within the transmembrane domain of AcrB in both E. coli and K. pneumoniae. Here, we synthesised 28 new analogues to explore structure-activity relationships around the benzylamine moiety, with the aim of improving the drug-like properties of the starting compound 4 (BDM91531). Replacement of the benzylamine group at position 6 of the quinoline scaffold resulted in compounds with slightly lower potency, but with the beneficial absence of intrinsic antibacterial activity and reduced cytotoxicity. Studies using E. coli AcrB mutants confirmed the previously proposed binding mode of this chemical series. Evaluation of physico-chemical properties and microsomal stability enabled the identification of compound 35 (BDM92641), bearing a thiazole ring, which displayed favourable physico-chemical and in vitro pharmacokinetic properties. Notably, this compound potentiates the activity of several antibiotic classes against E. coli and K. pneumoniae. Overall, this work provides deeper insight into the structure-activity relationships of pyridylpiperazine-based efflux pump inhibitors and represents a significant step toward their future in vivo validation as antibiotic adjuvants.