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. 2016 Mar 29;113(13):3509-14.
doi: 10.1073/pnas.1602472113. Epub 2016 Mar 14.

Molecular basis for inhibition of AcrB multidrug efflux pump by novel and powerful pyranopyridine derivatives

Hanno Sjuts  1 , Attilio V Vargiu  2 , Steven M Kwasny  3 , Son T Nguyen  3 , Hong-Suk Kim  4 , Xiaoyuan Ding  3 , Alina R Ornik  1 , Paolo Ruggerone  5 , Terry L Bowlin  3 , Hiroshi Nikaido  6 , Klaas M Pos  7 , Timothy J Opperman  8
Affiliations

Molecular basis for inhibition of AcrB multidrug efflux pump by novel and powerful pyranopyridine derivatives

Hanno Sjuts et al. Proc Natl Acad Sci U S A. .

Abstract

The Escherichia coli AcrAB-TolC efflux pump is the archetype of the resistance nodulation cell division (RND) exporters from Gram-negative bacteria. Overexpression of RND-type efflux pumps is a major factor in multidrug resistance (MDR), which makes these pumps important antibacterial drug discovery targets. We have recently developed novel pyranopyridine-based inhibitors of AcrB, which are orders of magnitude more powerful than the previously known inhibitors. However, further development of such inhibitors has been hindered by the lack of structural information for rational drug design. Although only the soluble, periplasmic part of AcrB binds and exports the ligands, the presence of the membrane-embedded domain in AcrB and its polyspecific binding behavior have made cocrystallization with drugs challenging. To overcome this obstacle, we have engineered and produced a soluble version of AcrB [AcrB periplasmic domain (AcrBper)], which is highly congruent in structure with the periplasmic part of the full-length protein, and is capable of binding substrates and potent inhibitors. Here, we describe the molecular basis for pyranopyridine-based inhibition of AcrB using a combination of cellular, X-ray crystallographic, and molecular dynamics (MD) simulations studies. The pyranopyridines bind within a phenylalanine-rich cage that branches from the deep binding pocket of AcrB, where they form extensive hydrophobic interactions. Moreover, the increasing potency of improved inhibitors correlates with the formation of a delicate protein- and water-mediated hydrogen bond network. These detailed insights provide a molecular platform for the development of novel combinational therapies using efflux pump inhibitors for combating multidrug resistant Gram-negative pathogens.

Keywords: RND efflux transporters; X-ray crystallography; efflux pump inhibitors; molecular dynamics simulation; multidrug resistance.

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Conflict of interest statement

Conflict of interest statement: K.M.P. and reviewer B.L. coauthored a review article in 2015.

Figures

Fig. 1.
Fig. 1.
Structure of inhibitors and the inhibitor-bound AcrBper. (A) Chemical structures of the studied MBX compounds. (B) Inhibitor binding is observed to the hydrophobic trap and associated deep binding pocket distal from the switch-loop (red) of the T monomer (yellow-colored cartoon) as represented by the MBX3132 Fo-Fc omit map (green mesh, contoured at 4.0 σ). The L protomers of AcrBper are shown in a blue surface sphere and cartoon representation.
Fig. 2.
Fig. 2.
R6G binding to AcrBper is observed in the deep binding pocket of the T monomer. (A) Fo-Fc omit map of the R6G ligand is shown as green mesh, contoured at 3.0 σ. (B) Blue mesh (contoured at 1.0 σ) represents the R6G 2Fo-Fc electron density map after refinement of the complex structure. R6G is shown in a ball-and-stick representation (carbon, magenta; oxygen, red; nitrogen, blue). Side-chain residues of the AcrB deep binding pocket are shown as sticks (carbon, yellow; oxygen, red; nitrogen, blue).
Fig. 3.
Fig. 3.
Details of the binding of inhibitors with AcrBper. MBX2319 (A and B) and MBX2931 (C and D) mainly interact via hydrophobic stacking interactions with residues comprising the deep binding pocket and the hydrophobic trap. MBX3132 (E and F) and MBX3135 (G and H) are additionally engaged in a water-mediated hydrogen bond network, extending from the acetamide and acrylamide groups, respectively. Hydrogen bonds and water molecules are shown as red lines (with distances in angstroms) and as cyan-colored spheres, respectively. MBX compounds are shown as sticks (carbon, gray; oxygen, red; nitrogen, blue; sulfur, yellow). (Left) AcrB residues involved in inhibitor binding are shown as sticks (carbon, yellow; oxygen, red; nitrogen, blue; sulfur, gold), and the 2Fo-Fc electron density maps (blue-colored mesh) are contoured at 1.0 σ (MBX2319 and MBX2931) and at 1.5 σ (MBX3132 and MBX3135), respectively. (Right) AcrB deep binding pocket surface is colored according to its hydrophobicity (red, hydrophobic; gray, hydrophilic), and the substrate pathway is indicated with arrows. MBX compounds are shown in a ball-and-stick representation (cyan-colored carbon atoms).
Fig. 4.
Fig. 4.
Analogs of the pyranopyridine EPI MBX2319 exhibit improved efflux pump inhibition. The bactericidal activity of a minimally bactericidal dose of ciprofloxacin (0.01 μg/mL) is potentiated against E. coli by MBX2319 (A), MBX3132 (B), and MBX3135 (C). The concentrations of MBX3132 and MBX3135 used are 10-fold lower than the concentration of MBX2319. Inhibition of efflux pump activity is shown in a cell-based assay that measures accumulation of the fluorescent dye H33342 in the presence of MBX2319 (D), MBX2931 (E), MBX3132 (F), and MBX3135 (G). Effect of 10 nM MBX2319 (H), MBX3132 (I), and MBX3135 (J) on the Michaelis–Menten kinetics of AcrAB-TolC using the nitrocefin efflux assay (31). CIP, ciprofloxacin; Cmpd, MBX compound alone; Cp, periplasmic concentration of nitrocefin (micromolar); RFU, relative fluorescence unit; Ve, efflux pump velocity (in nanomoles per second per milligram).
Fig. 5.
Fig. 5.
MBX compound binding site overlaps with substrate binding sites. The superimposition of MBX3132 coordinates (carbon, cyan; oxygen, red; nitrogen, blue; sulfur, yellow) with MIN [A; carbon, green; PDB ID code 4DX5 (21)], R6G (B; carbon, magenta; this study), and doxorubicin [C; carbon, orange; PDB ID code 4DX7 (21)] is shown, indicating that the EPI sterically prevents substrate binding to the AcrB deep binding pocket. AcrB side chains involved in the binding of substrates or EPIs are indicated and shown as sticks (carbon, yellow).
Fig. 6.
Fig. 6.
Hydrophilic, water-filled cavity provides potential for MBX inhibitor improvement. The AcrB deep binding pocket surface is colored as in Fig. 1. MBX3132 is shown in a ball-and-stick representation (carbon, cyan; oxygen, red; nitrogen, blue; sulfur, yellow). Coordinated water molecules in the extended hydrophilic pocket between the acetamide moiety of MBX3132 and residues 179–181 and 271–277 (shown as sticks) are shown as orange spheres. Augmented inhibitors could potentially protrude into this solvent-filled pocket.
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