Discovery of Ebselen Analogs as Novel Inhibitors of Polyphosphate Metabolism to Attenuate Virulence and Show Combination Benefit with Meropenem in Pseudomonas aeruginosa

Abstract Pseudomonas aeruginosa is a major opportunistic pathogen responsible for severe hospital-acquired infections. The challenge in treating these infections is exacerbated by increasing antibiotic resistance. Polyphosphate kinases (PPKs), specifically PPK1 and PPK2, play a critical role in the virulence, stress response, and antibiotic tolerance of P. aeruginosa, making it a compelling target for therapeutic intervention. In this study, we describe the discovery and development of ebselen analogs as novel inhibitors against PPK1Pa and PPK2Pa. Biochemical and cellular assays revealed that ebselens interfere with the polyphosphate homeostasis within the bacterium. EB-1 and EB-18 treatment significantly reduced intracellular polyP levels, mirroring the phenotypic effects observed in Δppk strains, including impaired biofilm formation, reduced motility, and attenuated production of pyoverdine and pyocyanin. Notably, EB-1 and EB-18 showed combination benefit with meropenem against P. aeruginosa in the Galleria model, supporting their potential as an adjunctive agent to improve the antibacterial efficacy of meropenem. Most representative compounds, including EB-1 and EB-18, showed no detectable cytotoxicity in the tested mammalian cells. These findings underscore ebselen analogs as promising dual-specific PPK inhibitors, establishing a valuable strategy for the development of adjunct therapies via the PPK-mediated anti-virulence pathways.