Exploring allosteric efflux pump inhibitors and the role of bacterial membrane potential in modulating drug resistance

Multi-drug resistance (MDR) bacteria pose a significant global health challenge, primarily driven by the activity of multi-drug efflux pumps and their intimate coupling to bacterial membrane bioenergetics. Among these systems, proton-motive force (PMF) dependent Resistance, Nodulation–Division (RND) efflux pumps, such as AcrAB-TolC, play a central role in both intrinsic and acquired antibiotic resistance by expelling structurally diverse antimicrobial agents. Recent evidence indicates that efflux pumps are not merely drug extrusion devices but also key regulators of bacterial physiology, influencing membrane potential, redox balance, metabolic state, stress adaptation, and growth-phase transitions. Structural and mechanistic advances have uncovered conserved allosteric sites within RND pumps that are distinct from substrate-binding pockets, enabling the development of allosteric efflux pump inhibitors (EPIs) that disrupt conformational cycling and proton relay without competing with antibiotics. Pyridylpiperazine-based inhibitors, including BDM-series compounds, have demonstrated potent efflux inhibition, antibiotic potentiation, and in-vivo efficacy in preclinical models. This review integrates current knowledge on efflux pump architecture, PMF-driven transport mechanisms, membrane potential dynamics, and allosteric inhibition, emphasizing the therapeutic potential of dual-targeting strategies that combine efflux inhibition with bioenergetics disruption. This study also focused on translational challenges, including drug penetration, resistance evolution, and pharmacokinetic constraints, and future directions for incorporating efflux–bioenergetic targeting into next-generation antimicrobial discovery pipelines to restore antibiotic efficacy against MDR Gram-negative pathogens.

The escalating prevalence of multi-drug resistance (MDR) Gram-negative bacterial pathogens constitutes one of the most critical global health challenges of the 21st century. Clinically significant species, including Pseudomonas aeruginosa, Acinetobacter baumannii, and carbapenem-resistant Enterobacterales, have exhibited a remarkable capacity for the rapid acquisition and horizontal dissemination of antimicrobial resistance determinants, resulting in severely limited therapeutic options and substantially increased morbidity and mortality worldwide (Macesic et al., 2025). The intrinsic resilience of Gram-negative bacteria is largely attributable to their unique cell envelope architecture, particularly the outer membrane, which acts as a formidable permeability barrier to many antibiotics. This intrinsic resistance is further compounded by multiple acquired mechanisms, including target modification, enzymatic inactivation of antibiotics, and the overexpression of multidrug efflux systems (Gauba and Rahman, 2023). Reflecting this growing threat, the World Health Organization (WHO) has designated several Gram-negative pathogens as priority organisms due to their escalating resistance to last-resort antibiotics such as carbapenems and polymyxins (Gauba and Rahman, 2023). Efforts to combat MDR Gram-negative infections are severely constrained by the limited pipeline of novel antibiotics and the rapid evolution of resistance even against newly introduced agents. These limitations underscore the urgent need for alternative and adjunctive therapeutic strategies, including phage therapy, anti-virulence approaches, and rational antibiotic combination therapies (Mascellino et al., 2024; Macesic et al., 2025).

In parallel, robust infection control measures and antimicrobial stewardship programs remain essential to curtail the dissemination of resistant strains in both healthcare and community settings (Kavya et al., 2024). Among the diverse resistance mechanisms employed by Gram-negative bacteria, multidrug efflux pumps play a central and multifaceted role. These membrane-embedded transport systems actively expel a wide spectrum of structurally unrelated antimicrobial agents, thereby reducing intracellular drug concentrations to sub-inhibitory levels. Nodulation–Division (RND) family efflux pumps, such as AcrAB–TolC in Escherichia coli and Salmonella spp., which are powered by the proton motive force (PMF) across the inner membrane, are tripartite complexes that span the Gram-negative cell envelope to actively expel toxic compounds including antibiotics (Gaurav et al., 2023; Kumawat et al., 2023). The functional dependence of efflux activity on membrane energetics establishes a critical link between antimicrobial resistance and bacterial energy metabolism. Recent transcriptomic, biophysical, and physiological studies have revealed that perturbation or inhibition of major RND efflux pumps, including AcrB, results in altered proton flux across the inner membrane, leading to membrane hyperpolarization (Whittle et al., 2024). This energetic imbalance influences global regulatory networks, such as the ArcBA two-component system, thereby reshaping bacterial redox homeostasis, delaying entry into stationary phase, and modulating stress adaptation. Stationary-phase cells typically exhibit reduced antibiotic permeability and enhanced tolerance, these findings highlight an underappreciated role of efflux pumps in governing bacterial physiological states beyond drug extrusion (Whittle et al., 2024).

Moreover, efflux inhibition–induced alterations in membrane potential may amplify antibiotic-mediated reactive oxygen species (ROS) production, further potentiating bactericidal activity. These observations position efflux pumps as integral hubs linking antimicrobial resistance, membrane energetics, metabolic regulation, and stress responses. Their dual function as both transporters and modulators of cellular bioenergetics suggests that targeting efflux systems could dismantle resistance at both physical and energetic levels.

Accordingly, the development of efflux pump inhibitors that simultaneously disrupt drug export and proton motive force utilization represents a promising strategy for restoring antibiotic efficacy against MDR Gram-negative pathogens (Gaurav et al., 2023; Kumawat et al., 2023). The objective of this review is to provide a comprehensive and integrative analysis of the roles of multi-drug efflux pumps and membrane energetics in shaping antimicrobial resistance in Gram-negative bacteria. Moving beyond conventional efflux-centered paradigms, this review integrates recent insights into efflux-mediated regulation of metabolism, stress responses, virulence, and resistance evolution. Furthermore, it critically evaluates current progress and challenges in efflux pump inhibitor development, emphasizing the necessity of targeting both efflux machinery and its energetic drivers (Thakur et al., 2021). This review provides a unified framework that redefines efflux pumps as central regulators of bacterial adaptation rather than passive determinants of multi-drug resistance.

Bacterial efflux pumps, critical contributors to multidrug resistance, are classified mainly into five major families based on their energy sources and structural features such as resistance-nodulation-cell division (RND), major facilitator superfamily (MFS), ATP-binding cassette (ABC) transporters, multidrug and toxic compound extrusion (MATE), and small multidrug resistance (SMR) families (Chetri, 2023; Gaurav et al., 2023). The RND family, primarily found in Gram-negative bacteria, includes tripartite assemblies such as AcrAB-TolC, which expel a wide range of antibiotics and toxic compounds using the proton motive force for energy (Chetri, 2023; Duffey et al., 2024). The MFS pumps are ubiquitous across bacterial species, functioning primarily as single-component transporters that harness proton gradients and are responsible for exporting structurally diverse antibiotic substrates, such as tetracyclines and chloramphenicol (Chetri, 2023; Gaurav et al., 2023). In contrast to other efflux pumps, ABC transporters are mechanistically distinct in their use of ATP hydrolysis to energize substrate transport. A prominent efflux system in Gram-negative bacteria such as MacAB-TolC system, which is known to act as an ABC-type tripartite efflux system, where an inner membrane ATP-binding cassette system, MacB, is assisted by a periplasmic adaptor protein, MacA, and an outer membrane channel (TolC), to export macrolide compounds and other substrates (Chetri, 2023; Duffey et al., 2024). The MATE family utilizes sodium or proton antiport to extrude cationic drugs, including fluoroquinolones and aminoglycosides, playing a crucial role in resistance in multiple bacterial genera (Chetri, 2023; Gaurav et al., 2023).

Finally, SMR pumps are typically small homodimers with narrow substrate recognition profiles, known for expelling antiseptics and disinfectants, contributing to resistance against quaternary ammonium compounds and some antibiotics (Chetri, 2023). Collectively, these families illustrate diverse structural and functional adaptations that enable bacterial survival under antimicrobial pressure. Understanding these distinct pump classes provides a framework for designing targeted inhibitors to mitigate efflux-mediated resistance, an essential strategy for restoring the clinical efficacy of existing antibiotics (Chetri, 2023; Duffey et al., 2024). Different efflux pumps with their other characteristics are given in the below Figure 1 and Table 1.

The AcrAB-TolC efflux pump is a prototypical and extensively studied tripartite resistance-nodulation-division (RND) family multidrug efflux system predominantly found in Gram-negative bacteria such as Escherichia coli and Salmonella sp. This pump significantly contributes to multidrug resistance by extruding a broad spectrum of antibiotics and toxic compounds, thereby decreasing intracellular drug accumulation and efficacy (Vargiu et al., 2022; Sharma et al., 2024). Structurally, AcrAB-TolC comprises three components: AcrB, an inner membrane RND transporter that forms a homotrimer; AcrA, a periplasmic membrane fusion protein (MFP) that bridges AcrB and TolC; and TolC, an outer membrane channel forming the exit duct (Vargiu et al., 2022; Sharma et al., 2024). Each AcrB protomer consists of three domains—the transmembrane domain (TMD), which harnesses the proton motive force to energize transport, the porter domain (PD) responsible for substrate recognition and binding, and the funnel domain (FD) that interfaces with AcrA (Vargiu et al., 2022). The pump operates through a functional rotation mechanism that cycles the protomers through distinct conformational states, known as access (loose), binding (tight), and extrusion (open), enabling substrate (antibiotic) capture, transport, and expulsion, respectively (Nishino et al., 2021; Vargiu et al., 2022). TolC forms a long α/β-barrel channel that undergoes conformational changes from closed to open states to release substrates into the extracellular space, coordinated by conformational shifts in AcrA and AcrB during the efflux cycle (Nishino et al., 2021; Sharma et al., 2024). This tripartite assembly enables the efflux of structurally diverse molecules, including antibiotics from multiple classes, detergents, dyes, and bile salts, contributing to broad substrate specificity (Vargiu et al., 2022; Ren et al., 2024).

The precise interaction of the AcrA hexamer with AcrB and TolC forms a continuous conduit spanning both membranes, preventing leakage and enhancing specificity (Vargiu et al., 2022). Recent structural studies, employing cryo-electron microscopy and X-ray crystallography, have elucidated the atomic-level details of the pump’s assembly and operational dynamics, laying the groundwork for the rational design of efflux pump inhibitors targeting key conformational states and substrate binding sites (Figure 2; Nishino et al., 2021; Kavanaugh et al., 2024).

Efflux pumps represent a fundamental mechanism of intrinsic and acquired resistance to a broad spectrum of antimicrobial agents. Intrinsic resistance arises from the basal or constitutive expression of efflux systems that actively extrude antibiotics and toxic compounds, thus lowering intracellular drug concentrations to sub-lethal levels (Soto, 2013; Sharma et al., 2024). For instance, the RND-family efflux pumps, such as AcrAB-TolC in Escherichia coli and Mex systems in Pseudomonas aeruginosa, contribute significantly to this innate defense by transporting diverse substrates, including β-lactams, fluoroquinolones, and tetracyclines (Dulanto Chiang and Dekker, 2024; Sharma et al., 2024). Acquired resistance mechanisms often involve the upregulation or mutation-driven overexpression of efflux pumps, which enhances substrate expulsion and enables bacteria to survive higher concentrations of antibiotics (Chetri, 2023; Li et al., 2025). Additionally, efflux pumps play a key role in the rising of resistance against the antibiotics within the bacterial cells. Such sub inhibitory exposure to antibiotics may promote accumulation of secondary mutations in the antibiotics targets including mutations in DNA gyrase or topoisomerase IV which confer resistance to fluoroquinolones and changes in the ribosomal proteins linked to macrolide resistance or changes in the penicillin binding proteins that prevent beta lactam binding. This process finally leads to the development of target mediated antibiotic resistance (Soto, 2013; Chetri, 2023). Beyond direct drug extrusion, efflux pumps also play roles in biofilm formation and the secretion of virulence factors, indirectly promoting resistance by creating protective microbial communities that are less susceptible to antibiotics (Kumawat et al., 2023; Sharma et al., 2024).

Efflux pumps proteins in the bacteria influence biofilm formation by expelling some QS molecules such as AHLs (acyl-homoserine lactones) and other signaling molecules which are considered as a virulence factors and those molecules also regulate concerned gene regulation involved in the biofilms maturation and EPS production which are key constituents of biofilm (Soto, 2013). These biofilms in the bacteria increase the tolerance to antibiotics by reducing the entry of antibiotics in the bacterial cell. Efflux pumps proteins also reported for the secretion of virulence factors, toxins, adhesin proteins and regulate global network of bacteria. These all activities of the bacteria contribute to the formation of microbial communities which help to exist in the presence of heavy attacks of antimicrobial compounds by enhancing collective resistance to different stresses and antimicrobial molecules (Du et al., 2018; Sharma et al., 2019). The dual role of these pumps in both intrinsic protection and the acquisition of enhanced resistance underscores the critical need for novel therapeutic strategies targeting efflux systems to restore antibiotic efficacy (Chetri, 2023; Dulanto Chiang and Dekker, 2024).

Early efforts to combat multidrug resistance through classical efflux pump inhibitors (EPIs) were largely based on competitive inhibition of substrate-binding sites within the multidrug transporters. Despite promising in-vitro potentiation of antibiotics, most classical EPIs failed to progress into clinical use due to a combination of pharmacological, mechanistic, and toxicological limitations. One major obstacle was substrate competition, as competitive EPIs must directly contend with high intracellular or periplasmic concentrations of antibiotics and endogenous metabolites. In Gram-negative bacteria, the remarkable substrate polyspecificity and high turnover rate of RND pumps such as AcrAB-TolC render competitive inhibition inherently inefficient, particularly under clinical drug exposure conditions where substrate overload can easily outcompete inhibitors (Opperman and Nguyen, 2015; Rouvier et al., 2025). Another critical limitation was poor permeability across the Gram-negative outer membrane. Many early EPIs, including phenylalanine-arginine β-naphthylamide (PAβN), exhibited limited penetration and were often themselves substrates of efflux pumps, resulting in insufficient intracellular accumulation at target sites (Duffey et al., 2024; Rouvier et al., 2025). Furthermore, classical EPIs frequently displayed dose-limiting toxicity, arising from off-target interactions with mammalian membranes, ion channels, or transporters, thereby narrowing their therapeutic window and precluding systemic administration (Thakur et al., 2021; Duffey et al., 2024). These toxic effects were particularly pronounced for membrane-active or amphipathic compounds, which required reaching effective inhibitory concentrations.

From a resistance standpoint, classical EPIs were also vulnerable to rapid resistance development, as mutations in substrate-binding pockets or pump overexpression readily diminished inhibitor efficacy without substantially compromising pump function (Opperman and Nguyen, 2015; Thomson and Saadhali, 2025). Additionally, competitive EPIs typically failed to disrupt the energetic coupling between efflux activity and the proton motive force (PMF), allowing bacteria to maintain efflux competence through metabolic adaptation or compensatory upregulation of alternative transporters (Nishino et al., 2021; Rouvier et al., 2025). Collectively, these shortcomings highlighted the need for mechanistically distinct strategies—such as allosteric inhibition—that bypass substrate competition, exploit conserved conformational control points, and destabilize efflux–bioenergetic coupling more effectively in Gram-negative pathogens.

Allosteric inhibition of bacterial efflux pumps is characterized by the binding of an inhibitor molecule to a site distinct from the active or substrate-binding site on the pump protein, known as the allosteric site. This binding induces conformational changes in the pump structure, disrupting its functional cycle and thereby reducing or abolishing its ability to expel antimicrobial agents from the bacterial cell (Plé et al., 2022). Unlike competitive inhibitors, which directly compete for occupancy at the active site, allosteric inhibitors modulate pump activity indirectly by altering the topology or dynamics of the efflux machinery (Opperman and Nguyen, 2015; Rouvier et al., 2025). This mode of inhibition often leads to a non-competitive or uncompetitive phenotype whereby inhibition efficiency is independent of substrate concentration, providing a consistent suppression of efflux activity. One major advantage of allosteric inhibition over competitive inhibition is its potential to overcome the intrinsic limitation of substrate competition. Furthermore, allosteric inhibitors can target conserved allosteric pockets critical for the efflux pump’s conformational transitions during the efflux cycle, thereby potentially providing a broader spectrum of inhibition against multiple pump substrates (Plé et al., 2022; Rouvier et al., 2025). Additionally, the unique binding to allosteric sites reduces the chance of cross-resistance with substrates that share the active site, offering new avenues for circumventing multidrug resistance in Gram-negative bacteria.

Recent structural insights into Unlike competitive inhibitor RND family efflux pumps, such as AcrAB-TolC, have revealed novel allosteric pockets in the transmembrane domain and periplasmic regions, which, when occupied by selective inhibitors, prevent essential conformational cycling necessary for substrate extrusion (Figure 3; Plé et al., 2022). Antibiotic accumulation inside the bacterial cells can disrupt the Proton Motive Force (PMF) by increasing proton concentration inside the cell. After the accumulation of antibiotics, protons started to accumulate in the inner membrane of the bacteria, leads to directly damage the cytoplasmic membrane or indirectly affect components of the Electron Transport Chain, which lead to dissipation of the transmembrane proton gradient (ΔpH) and membrane potential (Δψ). Further transmembrane proton gradient (ΔpH) and membrane potential (Δψ) collapse and energy dependent defenses fail which lead to bacterial death (Plé et al., 2022). Since PMF is essential for ATP synthesis, nutrient transport, and the activity of energy-dependent systems such as RND efflux pumps, excessive intracellular accumulation of antibiotics ultimately collapses the electrochemical gradient and compromises cellular viability (Chetri, 2023).

Recent advances in identifying allosteric binding sites on bacterial efflux pumps have significantly enhanced the understanding of their inhibition mechanisms, particularly within the resistance-nodulation-division (RND) family of efflux pumps, such as AcrB. High-resolution cryo-electron microscopy (cryo-EM) and X-ray crystallography studies have unveiled novel allosteric sites distinct from the canonical substrate-binding pockets, predominantly located in the transmembrane domain, porter domain, and at subunit interfaces of the tripartite efflux machinery (Wang et al., 2017; Plé et al., 2022). These structural insights have established that binding of allosteric inhibitors at these sites modulates the conformational dynamics essential for pump function, disrupting proton motive force coupling and substrate transport without directly competing with antibiotics at the active site (Plé et al., 2022; Roy et al., 2024). Plé et al., identified a unique allosteric binding pocket within the transmembrane region of AcrB targeted by pyridylpiperazine-based inhibitors (Plé et al., 2022). This binding site includes residues critical for proton relay, and binding to this pocket effectively halts the efflux pump’s catalytic cycle, demonstrating a potent mechanism of inhibition that bypasses the limitations of competitive inhibition (Plé et al., 2022). Complementing these structural discoveries, molecular dynamics simulations and computational network analyses have mapped allosteric communication pathways linking the transmembrane proton-translocation domain with the periplasmic substrate-binding porter domain.

Classical efflux pump inhibitors (EPIs) are compounds that inhibit bacterial multidrug efflux pumps. Classical inhibitors generally act by blocking the substrate-binding site on efflux pumps and interfering with pump assembly and function (Sharma et al., 2019). The combined use of classical efflux pump inhibitors (EPIs) with pump substrates is under exploration to overcome efflux-mediated multidrug resistance. Phenylalanine-arginine β-naphthylamide (PAβN) is a well-studied classical EPI that is routinely combined with fluoroquinolone antibiotics, but few studies have assessed its utility in combination with β-lactam antibiotics (Compagne et al., 2023). PAβN inhibits the three RND pumps of P. aeruginosa (MexAB-OprM, MexCD-OprJ, and MexEF-OprN) and is also a substrate of these pumps, indicating it acts as a competitive inhibitor. This classical inhibitor, β-naphthylamide (PAβN), binds competitively and prevents drug extrusion. Reserpine is also a classical efflux pump inhibitor, which also blocks substrate binding sites of efflux pumps (Shaheen et al., 2019).

Several chemical classes of allosteric efflux pump inhibitors (EPIs) have emerged as promising agents to combat multidrug resistance mediated by RND-type efflux pumps, particularly AcrAB-TolC in Gram-negative bacteria. Among these, the pyridylpiperazines represent a pioneering family that binds to a novel allosteric site in the transmembrane domain of the AcrB L protomer, distinct from the classical drug-binding pockets (Table 2).

These inhibitors, exemplified by compounds like BDM73185 and its optimized analog BDM88855, act by sterically preventing crucial conformational changes in the pump’s catalytic cycle, thereby disabling the proton relay essential for drug efflux (Plé et al., 2022; Vieira Da Cruz et al., 2024). BDM88855, a lead compound in the BDM series, exhibits potent activity by directly binding the transmembrane allosteric pocket, stabilizing an inactive pump conformation, and significantly boosting antibiotic potency against AcrAB-TolC substrates such as chloramphenicol, tetracycline, and ciprofloxacin (Plé et al., 2022; Jiménez-Castellanos et al., 2023). The protonated piperazine moiety of BDM88855 forms critical interactions with catalytic residues within the pump, blocking the proton motive force essential for transport and disrupting the functional rotation mechanism (Table 2; Plé et al., 2022). Besides pyridylpiperazines, other allosteric EPIs have been identified, including arylpiperazines like NMP (1-(1-naphthylmethyl)-piperazine) and members of the MBX series, which target hydrophobic or transmembrane pockets proximal to drug-binding sites, thereby halting substrate extrusion through allosteric inhibition (Jang, 2023; Duffey et al., 2024). These inhibitors offer advantages over competitive EPIs by binding to less conserved allosteric sites, thereby reducing the development of resistance and allowing for the simultaneous inhibition of multiple efflux pump conformations (Plé et al., 2022; Jiménez-Castellanos et al., 2023).

Efflux pumps transport proteins of bacteria which participate in extrusion of substrates (generally antibiotics) from the cellular interior to the external environment (Sharma et al., 2019). The proton gradient or the ATPase that supplies energy to these pumps has been tried as targets of various EPIs (Sharma et al., 2019). Carbonyl cyanide-m-chlorophenylhydrazone (CCCP) is an EPI ionophore that disrupts the proton motive force (PMF) by affecting both its components, Δψ and ΔpH (Bhattacharyya et al., 2017). Verapamil is a small molecule that acts as an ion channel blocker and is used in the treatment of hypertension. Studies in Mycobacterium tuberculosis have shown that verapamil potentiates the activity of bedaquiline and ofloxacin (Singh et al., 2011; Gupta et al., 2014).

Allosteric efflux pump inhibitors (EPIs) exert their effects by binding to regulatory sites distinct from the substrate-binding pockets, inducing conformational changes that disrupt the functional cycle of multidrug efflux pumps, particularly those of the RND family, such as AcrAB-TolC (Sjuts et al., 2016; Plé et al., 2022). At the molecular level, many EPIs target the transmembrane domain (TMD; made of 12 transmembrane helices) of the inner membrane transporter AcrB, especially the L protomer’s allosteric pocket, where critical proton relay residues reside (Plé et al., 2022). The binding of inhibitors, such as pyridylpiperazines or BDM88855 changes the conformation of pumps in non-functional form that prevents the necessary conformational transitions between the access (L), binding (T), and extrusion (O) states for substrate transport (Sjuts et al., 2016; Plé et al., 2022; Jiménez-Castellanos et al., 2023). This blockage abrogates the Proton Motive Force (PMF)-driven functional rotation that effectively “freezing” the pump and halting drug extrusion (Wang et al., 2017; Plé et al., 2022). Molecular dynamics simulations also focus on inhibitors-based restriction of transmembrane helices flexibility, preventing the remodeling of the drug-binding pocket necessary for substrate access and export (Sjuts et al., 2016; Jamshidi et al., 2018).

Evidence from site-directed mutagenesis and high-resolution crystallography has been pivotal in substantiating the mechanistic basis of allosteric inhibition of multidrug efflux pumps, particularly AcrAB-TolC and related RND transporters. Structural analyses employing co-crystal structures of pyridylpiperazine-based inhibitors such as BDM88855 bound to AcrB reveal a unique allosteric pocket located within the transmembrane domain of the L protomer, spatially distinct from classical substrate-binding sites (Plé et al., 2022; Jiménez-Castellanos et al., 2023). This crystallographic insight demonstrated specific interactions between the inhibitor and critical proton-relaying residues, including aspartates essential for harnessing the proton motive force, thereby impeding the energy transduction necessary for pump operation (Plé et al., 2022).

Mutagenesis studies have further corroborated the allosteric mechanism by identifying pump variants with point mutations in transmembrane helices that abolish inhibitor binding and confer resistance to allosteric EPIs (Plé et al., 2022; Jiménez-Castellanos et al., 2023). For example, substitutions such as S450P and A446P in TM5 of AcrB were discovered in resistant Escherichia coli mutants exposed to pyridylpiperazine inhibitors, highlighting the functional importance of these residues in the allosteric binding pocket for inhibitor efficacy (Plé et al., 2022). These mutations, located distant from the substrate-binding pockets, support a mechanism in which allosteric inhibitors disrupt conformational cycling through steric hindrance. Complementary mutagenesis mapping, combined with thermodynamic stability assays, demonstrated that allosteric inhibitors stabilize pump conformations unfavorable for substrate transport. This stabilization was manifested as increased thermal shift values for wild-type complexes but not for resistant mutants, confirming direct inhibitor engagement at the allosteric site (Plé et al., 2022; Jiménez-Castellanos et al., 2023).

Leading allosteric efflux pump inhibitors (EPIs), particularly those based on the pyridylpiperazine (PyrPip) scaffold, such as BDM88855 and its analogs, exhibit potent inhibition of RND-type pumps like AcrAB-TolC, with broad-spectrum activity against clinically relevant Gram-negative pathogens, including Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii (Plé et al., 2022; Jiménez-Castellanos et al., 2023; Vieira Da Cruz et al., 2024). These PyrPip EPIs increase the intracellular accumulation of a wide range of antibiotics, leading to 2–8-fold enhancement in antibiotic efficacy in-vitro, particularly with substrates such as chloramphenicol, tetracycline, erythromycin, and ciprofloxacin (Plé et al., 2022). BDM88855, a lead compound, exhibits micromolar potency with half-maximal effective concentration (EC50) values in the low micromolar range (∼1–5 μM) across multiple efflux pump assays (Plé et al., 2022; Jiménez-Castellanos et al., 2023). In-vitro studies elucidate its selectivity for RND efflux pumps without affecting non-substrate antibiotics, underscoring its specificity (Plé et al., 2022). The spectrum of these inhibitors encompasses multiple multidrug-resistant strains, making them promising candidates for restoring antibiotic susceptibility where efflux-mediated resistance predominates (Jiménez-Castellanos et al., 2023; Vieira Da Cruz et al., 2024). Critically, proof-of-concept in-vivo studies validate the therapeutic potential of PyrPip EPIs. For example, BDM91288, an optimized analog of BDM88855, significantly potentiated the efficacy of levofloxacin in a murine model of K. pneumoniae pulmonary infection, reducing bacterial load and increasing survival rates compared to antibiotic treatment alone (Plé et al., 2022; Vieira Da Cruz et al., 2024).

Pharmacokinetic analyses highlight favorable metabolic stability and target tissue distribution, although further optimization is underway to enhance organ penetration and plasma retention (Vieira Da Cruz et al., 2024). Other allosteric EPIs, such as the MBX series, also show promising spectrum and in-vivo efficacy, with compounds like MBX3796 exhibiting improved ADME properties and activity against resistant Pseudomonas aeruginosa strains (Duffey et al., 2024). Collectively, these data underscore the progress toward clinically viable allosteric efflux pump inhibitors that can combat multidrug resistance in Gram-negative pathogens.

Efflux pumps of the RND family, among others, harness the energy from PMF to actively extrude a broad spectrum of antibiotics and harmful metabolites out of bacterial cells, thereby contributing critically to intrinsic and acquired antimicrobial resistance (Wan et al., 2023; Yang et al., 2023). The magnitude and maintenance of the membrane potential and proton gradient directly influence efflux efficiency, impacting intracellular drug accumulation and bacterial susceptibility (Wan et al., 2023). Recent investigations show bacteria dynamically regulate PMF under stress conditions, such as nutrient starvation or antibiotic exposure, to sustain efflux pump function, thus facilitating phenotypic tolerance and survival (Wan et al., 2023; Biquet-Bisquert et al., 2024). Moreover, the membrane potential itself contributes to the electrochemical driving force needed for proton translocation in pumps like AcrB, which couples proton influx to substrate expulsion, underpinning the proton relay mechanism critical for efflux pump catalytic cycles (Wan et al., 2023; Yang et al., 2023). Perturbation of PMF, for example, by protonophores or environmental stresses, leads to compromised efflux pump activity, higher intracellular antibiotic concentrations, and increased bacterial susceptibility, highlighting the essential role of membrane energetics in antimicrobial resistance (Yang et al., 2023; Biquet-Bisquert et al., 2024). Collectively, this bioenergetic foundation underscores the importance of targeting membrane potential and PMF in strategies aiming to overcome efflux-mediated drug resistance in bacterial pathogens (Yang et al., 2023).

The catalytic cycles of multidrug efflux pumps, especially those in the RND and MFS families, are tightly coupled to the proton motive force (PMF) across the bacterial cytoplasmic membrane (Chetri, 2023; Li et al., 2024). This energy is transduced by dedicated proton relay networks within the inner membrane transporter (e.g., AcrB for RND pumps), where conserved charged residues (such as D407, D408, K940 in AcrB) cycle through protonation and deprotonation events to propel substrate extrusion and drive conformational transitions within the pump protomers (Plé et al., 2022; Li et al., 2024). Recent cryo-EM and mutagenesis studies highlight that substrate binding and release are coordinated with the passage of protons through the transmembrane domain, linking drug export directly to PMF-driven conformational cycling (Plé et al., 2022; Li et al., 2024). For example, each AcrB protomer transitions through loose (L), tight (T), and open (O) states, each of which is associated with protonation steps and accompanied by substrate recognition, binding, and expulsion, thereby ensure the vectorial transport of antibiotics out of the cell (Plé et al., 2022). Blockage or dissipation of the PMF—whether by membrane-acting antibiotics, metabolic inhibitors, or targeted small-molecule EPIs—interrupts proton translocation, “freezing” the pump in inactive conformations and leading to elevated intracellular drug concentrations (Gerry et al., 2025). Critically, membrane potential and ΔpH do not always act as a single unit; recent models show that they can individually fine-tune the overall pump throughput and substrate specificity, underlining the non-equilibrium and adaptable nature of energy transduction in efflux-mediated resistance (Li et al., 2024; Gerry et al., 2025).

Importantly, these mechanistic insights reveal that PMF perturbation can selectively reduce efflux efficiency and sensitize bacteria to antibiotics, highlighting PMF-coupled transport as a compelling target for adjuvant therapeutic strategies (Chetri, 2023).

Experimental and computational studies have increasingly elucidated how membrane potential dynamics regulate bacterial efflux pumps, particularly those of the resistance-nodulation-division (RND) family, such as AcrB. Efflux pumps harness the proton motive force (PMF), an electrochemical gradient across the inner membrane, as the primary energy source for substrate extrusion. Disruption or alteration of this membrane potential directly affects pump activity and efficiency (Whittle et al., 2024). Genetic knockout or functional inactivation of efflux pumps leads to increased bacterial membrane potential (hyperpolarization) due to decreased proton influx in bacterial cells. In the previous literature it has been demonstrated that Salmonella strains deficient in the AcrB pump or carrying mutations that block proton translocation exhibit significantly elevated membrane potential relative to wild-type cells (Whittle et al., 2024). This hyperpolarization affects global cellular physiology, including shifts in anaerobic energy metabolism, redox balance, and delayed entry into the stationary phase, establishing a direct link between efflux function and membrane energetics (Whittle et al., 2024). The protonophore CCCP collapse of PMF is routinely used experimentally to confirm the dependence of efflux on membrane potential (López et al., 2017). At the computational level, molecular dynamics (MD) simulations of intact tripartite pumps embedded in Gram-negative inner and outer membrane models reveal how pump conformations and inter-protein interfaces are energetically coupled to the surrounding membrane environment and proton gradients (López et al., 2017).

MD studies of MexAB-OprM from Pseudomonas aeruginosa reveal that the conformational dynamics responsible for opening the outer membrane channel and substrate extrusion are intimately regulated by proton flux-driven changes in the transmembrane regions, which are sensitive to variations in membrane potential (López et al., 2017). Simulations also clarify how allosteric inhibitor binding within transmembrane pockets interfere with proton relay and membrane-driven conformational cycling, offering molecular insight into efflux modulation (Plé et al., 2022; Roy et al., 2024). MD simulations predicted pyridylpiperazine inhibitors to preferentially interact with the cytosolic surface of the L protomer. Specifically, simulations indicated that compounds enter AcrB in the L protomer state via a cytoplasmic-open access channel leading to the inhibitor binding pocket (Figure 4; Plé et al., 2022). As the L protomer TM domain is in an inward open conformation, this entry pathway is more accessible than in the T or the O protomer, which are in an outward open (inward closed) or occluded conformation, respectively (Eicher et al., 2014).