Inhibition of prolyl-tRNA synthetase and efflux pumps as a dual-targeting strategy against multidrug-resistant bacteria

Aminoacyl-tRNA synthetases have been widely exploited as targets for antiparasitic and antifungal inhibitors; however, they have received little attention as targets in multidrug-resistant (MDR) bacteria. Here we describe the biochemical characterisation of Prolyl-tRNA synthetase from <i>Klebsiella pneumoniae</i> (KpProRS), highlighting its ligase and proofreading activities. Distinct classes of ProRS inhibitors were evaluated against KpProRS but only halofuginone (HF) strongly modulated KpProRS activity. A new HF analog (Cpd-6) was developed and exhibited superior inhibitory activity against KpProRS relative to HF but low efficacy against MDR <i>K. pneumoniae</i>, despite good antimicrobial activity against <i>Escherichia coli</i> and <i>Staphylococcus aureus.</i> Further studies revealed that Cpd-6 resistance in <i>K. pneumonia</i> is mainly mediated by <i>AcrAB</i> efflux pump activity, which could be counteracted by efflux pump inhibitors. These findings therefore reinforce KpProRS as a target for antimicrobial development and highlight the therapeutic potential of combining HF analogues with efflux pump inhibitors to fight Gram-negative MDR pathogens.