The Art of War with Pseudomonas aeruginosa: Targeting Mex Efflux Pumps Directly to Strategically Enhance Antipseudomonal Drug Efficacy

Abstract

Pseudomonas aeruginosa (P. aeruginosa) poses a grave clinical challenge due to its multidrug resistance (MDR) phenotype, leading to severe and life-threatening infections. This bacterium exhibits both intrinsic resistance to various antipseudomonal agents and acquired resistance against nearly all available antibiotics, contributing to its MDR phenotype. Multiple mechanisms, including enzyme production, loss of outer membrane proteins, target mutations, and multidrug efflux systems, contribute to its antimicrobial resistance. The clinical importance of addressing MDR in P. aeruginosa is paramount, and one pivotal determinant is the resistance-nodulation-division (RND) family of drug/proton antiporters, notably the Mex efflux pumps. These pumps function as crucial defenders, reinforcing the emergence of extensively drug-resistant (XDR) and pandrug-resistant (PDR) strains, which underscores the urgency of the situation. Overcoming this challenge necessitates the exploration and development of potent efflux pump inhibitors (EPIs) to restore the efficacy of existing antipseudomonal drugs. By effectively countering or bypassing efflux activities, EPIs hold tremendous potential for restoring the antibacterial activity against P. aeruginosa and other Gram-negative pathogens. This review focuses on concurrent MDR, highlighting the clinical significance of efflux pumps, particularly the Mex efflux pumps, in driving MDR. It explores promising EPIs and delves into the structural characteristics of the MexB subunit and its substrate binding sites.

Keywords: MexAB-OprM; MexXY-OprM; Pseudomonas aeruginosa antimicrobial resistance; RND multidrug efflux pump; efflux pump inhibitor.

Figure 3

Regulation systems controlling the Mex efflux pumps in P. aeruginosa. Genes are represented by arrows, while proteins are depicted as oval shapes. The membrane fusion proteins (MFPs) are shown in green, the RND proteins or IMP in yellow, and the outer membrane factors (OMFs) in pale cyan. Repression is indicated by a “-” sign, and activation is represented by a “+” sign. Adapted with permission from ref. [168], 2018, Issa et al. Licensed under Creative Commons Attribution 4.0 International License. This figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution license (CC-BY).

Figure 1

Schematic overview of the major families of MDR efflux pumps in P. aeruginosa. This figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license.

Figure 2

Multidrug efflux pumps in P. aeruginosa. (a) Schematic structures of the four major efflux pumps implicated in antibiotic resistance in P. aeruginosa, presenting the resistance-nodulation-cell division transporters (MexB, MexY, MexD, and MexF) on IM; the periplasmic membrane fusion proteins (MexA, MexX, MexC, and MexE) on the periplasm; and the channel-forming OM factors (OprM, OprJ, and OprN) on the OM. Protein descriptions are based on a protein databank (PDB) MexAB-OprM structure; PDB id; 6TA6 [137]. (b) Schematic overview of RND efflux pumps in P. aeruginosa. This figure was created using PDB data and was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license.

Figure 4

Structure of PAβN. This figure was prepared with BIOVIA Draw 2022.

Figure 5

Structure of MP-601,205. This figure was prepared with BIOVIA Draw 2022.

Figure 6

Chemical structures of potent plant-derived EPIs targeting Mex efflux pumps in P. aeruginosa. This figure was prepared with BIOVIA Draw 2022.

Figure 7

Structure of PAβN and its two major derivatives. aa: amino acid. This figure was prepared with BIOVIA Draw 2022. Adapted with permission from [136], 2023, Compagne et al. Licensed under Creative Commons Attribution 4.0 International License (CC-BY 4.0).

Figure 8

PAβN structure and SAR overview for enhancing its EPI activity. This figure was prepared with BIOVIA Draw 2022. Adapted with permission from [136], 2023, Compagne et al. Licensed under Creative Commons Attribution 4.0 International License (CC-BY 4.0).

Figure 9

NMP structure and SAR overview for enhancing its EPI activity. This figure was prepared with BIOVIA Draw. Adapted with permission from [136], 2023, Compagne et al. Licensed under Creative Commons Attribution 4.0 International License (CC-BY 4.0).

Figure 10

D13-9001 structure and SAR overview for enhancing its EPI activity. This figure was prepared with BIOVIA Draw. Adapted with permission from [136], 2023, Compagne et al. Licensed under Creative Commons Attribution 4.0 International License (CC-BY 4.0).

Figure 11

MBX2319 structure and SAR overview for enhancing its EPI activity. This figure was prepared with BIOVIA Draw. Adapted with permission from [136], 2023, Compagne et al. Licensed under Creative Commons Attribution 4.0 International License (CC-BY 4.0).

Figure 12

Structure of TXA09155, TXA01182, and aryl-alkyl diaminopentanamide analogue. This figure was prepared with BIOVIA Draw. The arrows in the figure show the lead optimization processes, highlighting the modifications made to the chemical structure to improve its drug-like properties, leading to the introduction of TXA09155 Adapted with permission from [136], 2023, Compagne et al. Licensed under Creative Commons Attribution 4.0 International License (CC-BY 4.0).

Figure 13

The EPIs EA-371α and EA-371δ, derived from a novel Streptomyces strain closely related to Streptomyces vellosus, appear to be potent EPIs against P. aeruginosa. This figure was prepared with BIOVIA Draw.

Figure 14

Structure of RP1, derived from the soil bacterium Streptomyces Sp., functions as an EPI targeting P. aeruginosa. This figure was prepared with BIOVIA Draw.

Figure 15

Schematic representation of the phage-based EPI approach using the outer efflux protein (OprM) as a receptor to block efflux. This leads to the accumulation of antibiotics in P. aeruginosa, offering a promising strategy to enhance antibiotic efficacy. This figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license.

Figure 16

MexB’s structure and architecture are represented by a ribbon diagram of the trimer, showcasing its arrangement in the membrane plane. It highlights the three major domains and provides an approximate location of the IM. A monomer is color-coded to delineate its subdomains, with cyan representing one subdomain and navy blue and green indicating the others. The subdomains are labeled, and the α-helices are numbered from 1 to 12. Reprinted with permission from ref. [307], 2009, Sennhauser et al. This article is licensed under the Creative Commons Attribution license (CC-BY).

Figure 17

Alignment of MexB and AcrB sequences. Secondary structure elements are shown above the MexB sequence based on subunit C of the MexB crystal structure. Different subdomain colors align with those presented in Figure 16. Reprinted with permission from ref. [307], 2009, Sennhauser et al. This article is licensed under the Creative Commons Attribution license (CC-BY).

Figure 18

MexB structure: the left panel highlights L, T, and O monomers of MexB colored in green, cyan, and magenta, respectively (PDB Id: 3W9J). The detailed visualization on the right shows the different DP_T sub-regions of MexB: the interface is colored in gray (residues S79, T91, K134, F573, F617, M662, and E673), the cave is colored in red (residues Q46, T89, T130, N135, F136, V139, Q176, K292, Y327, V571, R620, and F628), and the groove is colored in blue (residues K151, F178, G179, R180, D274, S276, I277, A279, S287, P326, F610, V612, F615, and V47; S48, Q125, G126, R128, Q163, D174, F175, and Q273 that are located near the exit gate, colored in orange). The switch loop is represented as a yellow cartoon. Reprinted with permission from ref. [315], 2022, Gervasoni et al. This article is licensed under a Creative Commons Attribution-Noncommercial 3.0 unported license (CC BY-NC 3.0).

Figure 19

Horizontal cut view of the transmembrane region of the asymmetric AcrB trimer. Asp407, 408, and Lys940, which form ion pairs in the transmembrane core region, are depicted as ball-and-stick models. The inset shows the electron density observed in the transmembrane hole at the center of the MexB transmembrane trimers, indicating that the hole is filled with a phospholipid bilayer. The protein structures are depicted using ribbon models, with green, blue, and pink representing the access, binding, and extrusion monomers, respectively. Bound drugs are illustrated as stick models. Reprinted with permission from [61], 2015, Yamaguchi et al. This article is licensed under the Creative Commons Attribution license (CC-BY).

Figure 20

Intramolecular water-accessible channels in the AcrB trimer. The proximal pocket, distal pocket, entrances, and funnel-like exit are depicted in green, pink, gray, and yellow, respectively. The channel apertures at the entrance and exit are depicted in black. (1) IM entrance, (2) periplasmic entrance, (3) central cavity entrance. (A) Side view. The central cavity and central hole are depicted as dotted lines. (B) Horizontal cut view of the porter domain. The yellow circle indicates the closed pore-like structure comprising three central α-helices (depicted as a ribbon model with dense color), which was postulated to be a part of the putative substrate translocation channel during the early stages. Bound minocycline (cyan), doxorubicin (orange), rifampicin (magenta), and erythromycin (yellow) overlap in the space-filling model.

Figure 21

Chemical structure of lauryl maltose neopentyl glycol (LMNG). This figure was prepared with BIOVIA Draw.

Figure 22

Examples of fluoroquinolone (FQ) structures: (A) levofloxacin, (B) ofloxacin, (C) nadifloxacin, (D) pazufloxacin, (E) flumequine, and (F) orbifloxacin. The molecular scaffold shared by all quinolones is highlighted in blue, while the presence of fluorine atoms, characteristic of FQs, is indicated in green. Adapted with permission from ref. [315], 2022, Gervasoni et al. This article is licensed under a Creative Commons Attribution-Noncommercial 3.0 unported license (CC BY-NC 3.0).

Figure 23

Comparison between the crystal structure (colored in green) and the docking poses of the substrate localizations trapped inside MexB: (A) zwitterionic levofloxacin in violet, (B) non-zwitterionic levofloxacin (net charge-1) in magenta, (C) minocycline in yellow, and (D) D13-9001 in orange. Levofloxacin and minocycline are in a complex with AcrB (PDB Ids: 7B8T32 and 4DX5,45, respectively), while D13-9001 is in a complex with MexB (PDB Id 3W9J29). Protein–ligand interactions are represented as dotted lines. Reprinted with permission from ref. [315], 2022, Gervasoni et al. This article is licensed under a Creative Commons Attribution-Noncommercial 3.0 unported license (CC BY-NC 3.0).

Figure 24

Crystal structure of the MexB trimer complexed with the EPI D13-9001. This figure provides several key views, including the overall structure of the D13-9001-bound MexB trimer (a), a top-down cutaway view of the head-piece region in the D13-9001-bound MexB structure (b), a close-up view highlights the precise binding site of D13-9001 within MexB (c), and cutaway views of the distal drug-binding pocket from different angles: towards the exit ((d); green oval), looking down the hydrophobic trap from the substrate-translocation channel ((e); white oval), and towards the entrance ((f); green oval). Reprinted with permission from ref. [318], 2013, Nakashima et al. This article is licensed under the Creative Commons Attribution license (CC-BY).

Figure 25

A magnified view of the D13-9001 binding site is depicted as a surface model. D13-9001 is shown in stick form, F178 and W177 in white space-filling models, and V139, I138, and mutated Ala in magenta space-filling models. The symbols + and - indicate inhibition or the lack of inhibition by D13-9001. Crystal structures (A,B,E,F) and homology models (C,D,G,H) illustrate the binding of D13-9001 to AcrB, MexB, MexY, MexY W177F, AcrB F178W, ABI-PP-binding MexAB F178W, AcrB F178W V139A, and MexY I138A. Reprinted with permission from [61], 2015, Yamaguchi et al. This article is licensed under the Creative Commons Attribution license (CC-BY).