Abstract
The natural alkaloid berberine has been demonstrated to inhibit the Pseudomonas aeruginosa multidrug efflux system MexXY-OprM, which is responsible for tobramycin extrusion by binding the inner membrane transporter MexY. To find a structure with improved inhibitory activity, we compared by molecular dynamics investigations the binding affinity of berberine and three aromatic substituents towards the three polymorphic sequences of MexY found in P. aeruginosa (PAO1, PA7, and PA14). The synergy of the combinations of berberine or berberine derivatives/tobramycin against the same strains was then evaluated by checkerboard and time-kill assays. The in silico analysis evidenced different binding modes depending on both the structure of the berberine derivative and the specific MexY polymorphism. In vitro assays showed an evident MIC reduction (32-fold and 16-fold, respectively) and a 2–3 log greater killing effect after 2 h of exposure to the combinations of 13-(2-methylbenzyl)- and 13-(4-methylbenzyl)-berberine with tobramycin against the tobramycin-resistant strain PA7, a milder synergy (a 4-fold MIC reduction) against PAO1 and PA14, and no synergy against the ΔmexXY strain K1525, confirming the MexY-specific binding and the computational results. These berberine derivatives could thus be considered new hit compounds to select more effective berberine substitutions and their common path of interaction with MexY as the starting point for the rational design of novel MexXY-OprM inhibitors.
Highlights
Pseudomonas aeruginosa is an opportunistic pathogen and a frequent cause of lifethreatening infections in high-risk patients [1,2,3], including those with a compromised immune system due to underlying diseases such as cancer or cystic fibrosis [4]
The synergistic effect of the three berberine derivatives o-CH3, p-CH3, and p-CF3 in combination with tobramycin was tested against the P. aeruginosa strains PAO1, Molecules 2021, 26, x FOR PEER REVIEW Molecules 2021, 26, 6644
We evaluated the interaction between berberine and its three aromatic derivatives with the access to the periplasmic site between the periplasmic loop of TM7 and TM8 of the MexY protein, the inner transmembrane channel of the P. aeruginosa MexXY-OprM efflux pump, which is responsible for aminoglycoside extrusion
Summary
Pseudomonas aeruginosa is an opportunistic pathogen and a frequent cause of lifethreatening infections in high-risk patients [1,2,3], including those with a compromised immune system due to underlying diseases such as cancer or cystic fibrosis [4]. MexXY-OprM, belonging to the resistance–nodulation–cell division (RND) family, is considered the main resistance mechanism towards aminoglycosides in P. aeruginosa; mutations in its regulatory gene mexZ [6] can lead to overexpression and hamper antibiotic treatments. Three different pathways can be described for the access of the substrates to the MexY transporter (channel CH) in analogy to AcrB EP [17]: (i) the entrance above TM7/TM8 helices from the outer leaflet of the inner membrane to the proximal and binding pocket (CH1); (ii) periplasmic, through the cleft via hydrophilic compounds between subdomains PC1 and PC2 (CH2); and (iii) through the vestibule between protomers into the central cavity (CH3) (Figure 1)
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