Rational drug design of new efflux pump inhibitors of Gram-negative bacteria: novel in silico/in vitro combined approach to overcome antimicrobial resistance.

The project aims to address antimicrobial resistance in Gram-negative bacteria through efflux pump inhibitors (EPIs). Starting with berberine, a natural alkaloid, its ability to reduce the Minimal Inhibitory Concentration (MIC) of tobramycin in highly resistant strains of Pseudomonas aeruginosa was demonstrated. Berberine was found to strongly bind to the MexY protein within the MexXY-OprM efflux system, through computational techniques and in vitro microbiological assays. Derivatives of berberine were synthesized and functionalized at the C-13 position to enhance EPI activity and binding specificity. Computational analysis explored the binding modes of the synthesized derivatives. Inhibitory activity was tested through in vitro assays, using tobramycin. Results showed that chemical substitutions at C13 improve the compounds' specificity for the inhibition site more than berberine itself, aiding in reducing tobramycin MIC in laboratory and clinical strains with EP overexpression. The polymorphism of the MexY protein was considered to evaluate how ligands may be affected in their positioning, affinity, and stability within the binding pocket.