Inhibitors of multidrug efflux pumps of Pseudomonas aeruginosa from natural sources: An in silico high-throughput virtual screening and in vitro validation

Pseudomonas aeruginosa is resistant to a wide range of antibiotics, thus making troublesome the infection treatment. Efflux systems are the main mechanisms involved; among these, MexAB-OprM is a tripartite efflux pump responsible for resistance to ciprofloxacin, aztreonam, gentamicin, tetracycline and tobramycin. In an attempt to contrast antibiotic efflux, databases of natural compounds were tested for their ability to bind MexB, the inner membrane channel, using a high-throughput virtual screening approach. The comparison of their common pharmacophoric features was the basis for inhibitor identification and selection process. In silico screening against the MexB protein was performed by Autodock/Vina and further refined using a minimization/focused docking protocol on the obtained complexes. The compounds showing the best docking and resulting potentially active at nanomolar concentration have been selected and used in combination with antibiotics usually exported by MexAB-OprM in antimicrobial in vitro synergy tests (checkerboard and time kill assays) against multidrug-resistant P. aeruginosa clinical isolates. The combinations morelloflavone and pregnan-20-one-derivative/ciprofloxacin showed a four-fold MIC decrease and 100-fold increase of the bacterial killing compared to the antibiotic alone. The two chosen hits were validated by ethidium bromide accumulation assay for their efflux inhibition potency. These compounds showed the ability to increase the accumulation of ethidium bromide inside the bacterial cells as evidenced by the increase of its fluorescence in the presence of the each of them. Finally, their toxicity has been preliminary tested through hemolysis assay. The observed good correlation between in silico docking and in vitro synergy tests, indicates these two compounds as promising drugs to be used in combination therapy against MDR and MexAB-OprM overexpressing P. aeruginosa