A substrate-dependent mechanism of inhibition of the polyspecific multidrug efflux pump MexF from Pseudomonas aeruginosa

Pseudomonas aeruginosa presents a significant challenge in both clinical and antibiotic discovery settings due to the low permeability of its cell envelope. Efflux pumps acting synergistically with the permeability barrier of the outer membrane provide efficient protection against various antibacterial agents. Among 12 P. aeruginosa efflux pumps from the resistance-nodulation-division (RND) superfamily, the MexEF-OprN efflux system—and its transporter component MexF—remains relatively underexplored. MexEF-OprN is not expressed under laboratory conditions but plays an important role during P. aeruginosa infections as well as in clinical antibiotic resistance, particularly against fluoroquinolones and trimethoprim. To gain mechanistic insights into MexF functions, we identified and characterized SLU-1642, a small-molecule inhibitor featuring a 2-aminobenzothiazole core that selectively targets MexF and its closest homologs, but not major efflux pumps such as MexB with overlapping substrate specificities. Using a combination of experimental and computational approaches, we found that SLU-1642 specifically potentiates the activity of antibiotic substrates of MexF in P. aeruginosa hyperporinated strains overexpressing MexF. Our results indicate that small chemical modifications of SLU-1642 modulate efflux inhibition in a substrate-specific manner. Furthermore, only some of the substrates, such as doxycycline, but not trimethoprim, are sensitive to the SLU-1642 action against MexF variants, with amino acid substitutions changing the transport efficiency of the pump. Taken together, our findings establish SLU-1642 as a MexF-selective efflux inhibitor with a mechanism of action dependent on the potentiated substrate.