Stereochemical insight for MexXY-OprM efflux system inhibition in Pseudomonas aeruginosa from a pool of dihydro and tetrahydro berberine derivatives

: Pseudomonas aeruginosa is a Gram-negative pathogen responsible for severe infections, particularly in immunocompromised patients. Its widespread antibiotic resistance is a growing concern, primarily due to the overexpression of efflux pumps (EPs) such as the MexXY-OprM system, which plays a major role in aminoglycoside resistance. Targeting EPs with specific inhibitors (EPIs) represents a promising strategy to restore antibiotic efficacy. In this study, we performed High-Throughput Virtual Screening (HTVS) of a library of synthetic berberine derivatives (tetrahydro and dihydro) targeting MexY, the inner membrane protein of the MexXY-OprM system. Based on affinity data, four racemic compounds were selected for further investigation, but only two exhibited in vitro activity against P. aeruginosa laboratory and clinical strains. Subsequent experimental and computational analyses identified compound 2f as the most effective inhibitor, selectively binding to the allosteric pocket ALP. Notably, the differences in activity between its enantiomers highlighted the importance of the chiral environment at the binding cleft, with the (S)-enantiomer showing significantly higher efficacy than the (R)-enantiomer, particularly against PA7 and CF48 clinical strain. Structural investigations comparing all previously identified berberine derivatives revealed key interactions at the ALP site, mainly involving apolar and aromatic residues, providing a valuable framework for rational drug design. These findings led to the rationalization of a 3D-pharmacophoric model for berberine-derived EPIs, offering a foundation for further optimization.