Pyridylpiperazine-based allosteric inhibitors of RND-type multidrug efflux pumps

Abstract

Efflux transporters of the RND family confer resistance to multiple antibiotics in Gram-negative bacteria. Here, we identify and chemically optimize pyridylpiperazine-based compounds that potentiate antibiotic activity in E. coli through inhibition of its primary RND transporter, AcrAB-TolC. Characterisation of resistant E. coli mutants and structural biology analyses indicate that the compounds bind to a unique site on the transmembrane domain of the AcrB L protomer, lined by key catalytic residues involved in proton relay. Molecular dynamics simulations suggest that the inhibitors access this binding pocket from the cytoplasm via a channel exclusively present in the AcrB L protomer. Thus, our work unveils a class of allosteric efflux-pump inhibitors that likely act by preventing the functional catalytic cycle of the RND pump.

Fig. 1. Checkerboard assay evaluating pyridomycin and oxacillin antibiotic activity in the presence of different concentrations of BDM88855.HCl (9’) and PAβN.

Experiments were performed on both E. coli BW25113 and the pyridylpiperazine resistant E. coli isolate (carrying the S450P AcrB mutation) to confirm the on-target activity of BDM88855.HCl (9’). Bacterial viability was determined using the resazurin reduction assay and is expressed as a percentage resazurin reduction compared to the untreated bacteria, with this viability colour coded from yellow (100%) to blue (0%). Data represent the mean bacterial viability of at least four independent replicates. Source data are provided as a Source Data file.

Fig. 2. Structure of AcrB in complex with BDM88855 (9).

a Side view of the AcrB trimer comprising the L (marine), T (yellow), and O (red) protomers. BDM88855 (9) (green) binds to the TM domain of the L protomer. The boundaries of the inner membrane (IM) are indicated for the L protomer. b Side view on the TM domain of the L protomer. The apo-structure (dark blue) and the BDM88855 (9)-bound L protomers (marine) are superimposed and indicate subtle shifts in helix movements to accommodate the pyridylpiperazine inhibitor. The inward-open TM domain of the AcrB L protomer indicates the binding of BDM88855 (9) (green, stick representation) near the proton relay triad D407, D408, and K940. c Cytoplasmic side view into the TM domain of the AcrB L protomer (marine) and the location of BDM88855 (9) (green, stick representation) nested between TM helices 4, 5, and 10. d Enlarged view of the inhibitor binding site showing interacting residues (marine sticks) in less or equal than 4 Å distance from BDM88855 (9). The salt bridge between the piperazine ring and D408, the halogen bond between the BDM88855 (9) chlorine and the K940 main chain carbonyl oxygen, and the hydrogen-bond between the L404 main chain carbonyl oxygen and the piperazine ring are indicated by dashed lines and numbers represent the distance in Å.

Fig. 3. Putative entry of BDM88832 (8) to its binding site in AcrB.

Figure shows the preferred cumulative location (green surfaces) of BDM88832 (8) as seen in all-atom MD simulations of several compounds placed around AcrB embedded in a phospholipid bilayer. a Side view of BDM88832 (8) density on the whole AcrB tripartite efflux pump (L, T, and O protomers are shown as blue, yellow and red, respectively). b BDM88832 (8) density distribution viewed from the cytoplasmic side with only the TM domain shown for clarity. Relevant TM helices are labelled, and dashed circles on each monomer delimit the location of the experimental binding site. c Zoomed side view of the L protomer showing accumulation of BDM88832 (8). The X-ray conformation of BDM88832 (8) in AcrB is superimposed in CPK coloured by atom type (C, N, I, and Cl in green, blue, pink, and yellow, respectively). TM helix 5 is shown transparent for clarity. All data are cumulative results from 5 independent MD simulations of 2 μs each; transparent and solid green surfaces represent iso-values of 3 and 5, respectively.

Fig. 4. Comparison of BDM88855 (9) and MBX3132 binding pocket in AcrB.

Superimposition of the structures of BDM88855 (9) (green compound) bound AcrB (pdb 7OUK) and MBX3132 (red compound) bound AcrB (pdb 5ENQ), shows these efflux pump inhibitors to occupy different protomer states of AcrB (the blue T protomer for BDM88855 (9) and the yellow L protomer for MBX3132), in spatially distant binding pockets.