The hydrophobic trap-the Achilles heel of RND efflux pumps

Abstract

Resistance-nodulation-division (RND) superfamily efflux pumps play a major role in multidrug resistance (MDR) of Gram-negative pathogens by extruding diverse classes of antibiotics from the cell. There has been considerable interest in developing efflux pump inhibitors (EPIs) of RND pumps as adjunctive therapies. The primary challenge in EPI discovery has been the highly hydrophobic, poly-specific substrate binding site of the target. Recent findings have identified the hydrophobic trap, a narrow phenylalanine-lined groove in the substrate-binding site, as the "Achilles heel" of the RND efflux pumps. In this review, we will examine the hydrophobic trap as an EPI target and two chemically distinct series of EPIs that bind there.

Keywords: Adjunctive therapy; Efflux pump; Efflux pump inhibitor; Hydrophobic trap; RND superfamily.

Fig. 1

Structures of the compounds discussed in this review.

Fig. 2

Structure and transport mechanism of the AcrABZ-TolC RND efflux pump. A) A cryo-EM structure of AcrABZ-TolC bound to MBX-3132 with a resolution of 3.6 Å. The entire complex is comprised of 3 AcrB, 3 AcrZ, 6 AcrA and 3 TolC subunits. The components of a single functional unit of the trimeric pump are labeled. This image was reproduced with permission from Wang et al. [33]. B) A cartoon depicting the current model of the RND efflux pump transport mechanism. In the absence of substrate (right panel), the pump is in a resting (apo) state, in which the AcrB trimer is in the LLL conformation and the central channel of TolC is closed. In the presence of efflux substrates (left panel), the pump switches to a transport activated state characterized by changes in AcrB that result in three distinct conformation states (L, T and O), which represent discreet stages of the transport mechanism. Each subunit cycles from the L to T to O conformation, a process that is driven by the translocation of protons through the transmembrane domain (TM) of AcrB and is essential for unidirectional transport. When the substrates have been depleted, the pump reverts to the apo state. The cartoons depict cross-section views through the length of the pump in which two protomers of each of the pump components are shown. The cartoons below depict a view of the molecular axis of the AcrB trimer, which shows conformations of the AcrB protomers in the apo and transport activated states. Key: OM, outer membrane; IM, inner membrane; TM, AcrB transmembrane domain; P, AcrB pore domain; D, AcrD docking domain; L, T and O, Loose, Tight and Open conformations, respectively, of AcrB. This cartoon was adapted from a figure published by Wang et al. [33].

Fig. 3

Potent efflux pump inhibitors D13-9001 and MBX-3132 bound to the hydrophobic trap of AcrB. A) D13-9001 (green, carbon; red, oxygen; blue, nitrogen; PDB entry 3W9H [29]). B) MBX-3132 (dark red, carbon; PDB entry 5ENQ [30]). The surfaces have been colored green and pink to highlight residues within 5 Å of D13-9001 and MBX-3132, respectively. Images were generated with Pymol [51]. C) Superimposition of MBX-3132 (purple, PDB entry 5ENO [30]) and D13-9001 (green, PDB entry 5ENO [30]) bound to the hydrophobic trap of AcrB shows the differences and similarities of their binding sites. Amino acid side chains of residues comprising the hydrophobic trap are shown as yellow sticks. Image was generated with Pymol [51]. D) The relative per-residue contributions to the free energy of binding (ΔGb, kilocalories per mole) were calculated for D13-9001 and MBX-3132 bound to AcrB, and are compared with estimates calculated for MIN [43]. Residues comprising the hydrophobic trap [29] are shown in bold. Residues in the switch loop are highlighted in yellow. The data for MBX-3132 were reprinted from Sjuts et al. [30] with permission and the data for D13-9001 were kindly provided by Attilio Vargiu (University of Cagliari).

Fig. 4

A multi-sequence alignment of the major RND superfamily pumps of E. coli and P. aeruginosa showing conservation of the residues that comprise the hydrophobic trap. Residues that interact with MBX-3132 and D13-9001 are indicated above the alignment with green and blue arrows to indicate hydrophobic and polar residues, respectively. Hydrophobic trap residues are underlined. The residues are numbered according to the E. coli AcrB sequence. Significant differences between the conserved residues of AcrB/MexB and AcrD and MexY are indicated by red and yellow circles, respectively. Residue F178 is highlighted by two asterisks (**) because the F178W substitution in MexY has been shown to prevent binding of D13-9001 [29]. The secondary structure of AcrB is shown below the alignment using green arrows to indicate beta strands and red cylinders to indicate alpha helices. The alignment was generated using Clustal X2 [52].

Fig. 5

The MBX compound binding site overlaps with substrate binding sites. Superimposition of MBX-3132 (cyan, carbon; red, oxygen; blue, nitrogen; yellow, sulfur) bound to AcrB with substrate compounds. A) MIN (green; PDB entry 4DX5 [43]), B) R6G (magenta; PDB entry 5ENS [30]) and C) doxorubicin (orange; PDB entry 4DX7 [43]). MBX-3132 bound to the hydrophobic trap sterically hinders substrate binding to the AcrB deep binding pocket. AcrB side-chains involved in the binding of substrates or EPI are indicated and shown as sticks (carbon = yellow). Reprinted with permission from Sjuts et al. [30].