Pyridylpiperazine efflux pump inhibitor boosts in vivo antibiotic efficacy against K. pneumoniae.

Antimicrobial resistance is a global problem, rendering conventional treatments less effective and requiring innovative strategies to combat this growing threat. The tripartite AcrAB-TolC efflux pump is the dominant constitutive system by which Enterobacterales like Escherichia coli and Klebsiella pneumoniae extrude antibiotics. Here, we describe the medicinal chemistry development and drug-like properties of BDM91288, a pyridylpiperazine-based AcrB efflux pump inhibitor. In vitro evaluation of BDM91288 confirmed it to potentiate the activity of a panel of antibiotics against K. pneumoniae as well as revert clinically relevant antibiotic resistance mediated by acrAB-tolC overexpression. Using cryo-EM, BDM91288 binding to the transmembrane region of K. pneumoniae AcrB was confirmed, further validating the mechanism of action of this inhibitor. Finally, proof of concept studies demonstrated that oral administration of BDM91288 significantly potentiated the in vivo efficacy of levofloxacin treatment in a murine model of K. pneumoniae lung infection.

Optimization of pyridylpiperazine-based inhibitors of the Escherichia coli AcrAB-TolC efflux pump.

Multidrug-resistant Escherichia coli is a continuously growing worldwide public health problem, in which the well-known AcrAB-TolC tripartite RND efflux pump is a critical driver. We have previously described pyridylpiperazines as a novel class of allosteric inhibitors of E. coli AcrB which bind to a unique site in the protein transmembrane domain, allowing for the potentiation of antibiotic activity. Here, we show a rational optimization of pyridylpiperazines by modifying three specific derivatization points of the pyridine core to improve the potency and the pharmacokinetic properties of this chemical series. In particular, this work found that the introduction of a primary amine to the pyridine through ester (29, BDM91270) or oxadiazole (44, BDM91514) based linkers allowed for analogues with improved antibiotic boosting potency through AcrB inhibition. In vitro studies, using genetically engineered mutants, showed that this improvement in potency is mediated through novel interactions with distal acidic residues of the AcrB binding pocket. Of the two leads, compound 44 was found to have favorable physico-chemical properties and suitable plasma and microsomal stability. Together, this work expands the current structure-activity relationship data on pyridylpiperazine efflux pump inhibitors, and provides a promising step towards future in vivo proof of concept of pyridylpiperazines as antibiotic potentiators.