A novel approach to combat <i>Pseudomonas aeruginosa</i> : repurposing pharmaceuticals for inhibition of phospholipase A

Phospholipase A (PLA) plays critical roles in cellular physiology, making human PLAs established drug targets. On the other hand, the potential of bacterial PLAs as targets for antimicrobial drug development remains underexplored. In this study, we curated a library of 23 approved and investigational pharmaceuticals, some of which inhibit human PLA-like enzymes, through a combination of ligand structure-based searches and textual mining in literature and compound databases. Experimental screening identified that compounds GW4869, darapladib, and rilapladib significantly inhibit <i>Pseudomonas aeruginosa</i> growth by more than 50%. While these compounds did not reduce biofilm formation, GW4869 increased the proportion of dead cells in established biofilms, suggesting its role in compromising biofilm cell viability. Biochemical assays revealed that all three compounds inhibited the enzymatic activity of PlaF, a PLA virulence factor of <i>P. aeruginosa</i>, by decreasing the affinity of a model substrate. Molecular dynamics simulations and binding free energy analyses indicate that GW4869 binds to the substrate-binding and product-release tunnels of PlaF, suggesting GW4869 as a non-covalent competitive inhibitor. Notably, the mutant strain <i>P. aeruginosa</i> Δ<i>plaF</i> proved to be GW4869 resistant and did not display differential growth upon GW4869 treatment, further indicating PlaF as the primary GW4869 target. Furthermore, GW4869 and rilapladib significantly enhanced the efficacy of the last-resort antibiotic imipenem in combination treatments. Additionally, rilapladib exhibited broad-spectrum antibacterial activity by inhibiting the growth of both <i>Escherichia coli</i> and <i>Staphylococcus aureus</i>, while several other pharmaceuticals demonstrated species-specific effects, highlighting their potential for targeted antimicrobial applications beyond <i>P. aeruginosa</i>. These findings highlight the potential of GW4869, darapladib, and rilapladib to act as repurposed inhibitors of PlaF or PLA-dependent mechanisms in bacterial pathogens and underscore the promise of combination therapies against intracellular PLAs to combat antimicrobial resistance.IMPORTANCEThis study explores how existing drugs could be repurposed to fight <i>Pseudomonas aeruginosa</i>, a hospital-associated bacterial pathogen notorious for its strong antimicrobial resistance. By targeting intracellular phospholipase A, which are key to maintaining membrane balance, these drugs, originally developed for non-infectious diseases, may provide a fresh approach to tackling infections that are becoming harder to treat with standard antibiotics. The findings not only highlight the potential of phospholipases as promising antimicrobial targets but also uncover unexpected ways human drugs can interact with bacterial physiology. One standout compound, a preclinically studied drug called GW4869, both slows bacterial growth and boosts the effectiveness of the last-resort antibiotic imipenem, suggesting better outcomes with combination treatments. Overall, this research points to the exciting possibility of repurposing human-focused medicines as new antimicrobial agents to help combat the escalating crisis of antibiotic resistance while deepening our insight into how these drugs can influence microbes.