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

The efficiency of bacterial efflux pumps is closely linked to the energetic state of the cell, particularly the membrane potential (ΔΨ) component of the proton motive force (PMF), which drives proton-dependent transport systems, including the RND, MFS, and MATE pump families. Perturbation of ΔΨ reduces the energetic coupling required for substrate export, leading to impaired efflux and increased intracellular retention of antibiotics. For example, the artificial dissipation of ΔΨ using protonophores, such as CCCP, significantly increases intracellular drug accumulation in Escherichia coli and Salmonella enterica, demonstrating that efflux pump activity is directly dependent on maintaining membrane electrochemical gradients (Wang et al., 2021). Recent findings further reveal a bidirectional relationship in which loss or inhibition of major efflux systems, such as AcrB, alters membrane potential itself, indicating that ΔΨ not only drives but is also stabilized by efflux function (Whittle et al., 2024). Additionally, membrane potential influences the translocation of charged antibiotics across the membrane, affecting both uptake and export kinetics (Hillman, 2023). Computational analysis also supports the concept that PMF modulation can tune efflux specificity and turnover efficiency (Gerry et al., 2025). Together, these observations highlight ΔΨ as a critical regulatory axis in antimicrobial resistance, suggesting that targeted interference with membrane energetics may serve as a promising strategy to enhance intracellular drug accumulation and restore antibiotic susceptibility.

The activity and accessibility of allosteric efflux pump inhibitors (EPIs) are strongly influenced by the physiological microenvironment of bacterial cells, including variations in pH, nutrient availability, and metabolic state. Changes in environmental pH can significantly modulate the proton motive force (PMF), depends on the membrane potential (ΔΨ) and the transmembrane proton gradient (ΔpH), both of which drive the catalytic cycle of proton-dependent efflux pumps such as AcrAB–TolC. Changes in extracellular or cytoplasmic pH may therefore affect protonation states of both efflux pump residues and inhibitor molecules, potentially influencing the accessibility, binding affinity, and stability of allosteric inhibitor interactions within the transmembrane regulatory pocket of AcrB (Plé et al., 2022; Yang et al., 2023). Environmental pH changes can affect membrane permeability and the electrochemical gradient (membrane potential) across the bacterial membrane, affecting the diffusion or accumulation of EPIs in the periplasmic space, where many regulatory binding sites are located (Hillman, 2023; Wan et al., 2023). Under nutrient-rich conditions, metabolism sustains strong PMF generation through the electron transport chain, enabling robust efflux activity. In contrast, nutrient limitation or stationary-phase growth can lead to metabolic remodeling and adaptive regulation of PMF to maintain essential processes, including multidrug efflux, despite reduced energy availability (Wang et al., 2021; Wan et al., 2023). Such metabolic adaptations may influence the structural accessibility of allosteric binding pockets, thereby affecting the inhibitory efficiency of EPIs.

Many promising inhibitors exhibit potent in-vitro activity but face significant barriers related to absorption, distribution, metabolism, and excretion (ADME), as well as limited penetration across the Gram-negative outer membrane (Thakur et al., 2021; Duffey et al., 2024). Determining the dissociation constants (KD) and binding free energies of allosteric inhibitors across different environmental pH ranges would provide critical insights into how protonation states of efflux pump residues and inhibitor molecules influence binding affinity and residence time at the allosteric regulatory site. Acidic environmental conditions have been reported to stimulate transcriptional upregulation of multidrug efflux systems in several Gram-negative bacteria, which may further reduce intracellular accumulation of EPIs and compromise their inhibitory activity. Therefore, systematic biophysical and biochemical studies measuring pH-dependent KD values and inhibitor residence times will be essential to clarify how bacterial microenvironmental factors influence allosteric inhibitor binding and to guide the rational design of next-generation efflux pump inhibitors (Nikaido and Pagès, 2012; Li et al., 2015; Wang et al., 2017; Du et al., 2018).

Drug combinations can amplify antibacterial activity by targeting complementary pathways, improving cell entry, or disabling resistance mechanisms, thereby potentiating “old” drugs and expanding therapeutic windows. Mechanistically informed pairings—such as membrane permeabilizers with hydrophilic antibiotics, β-lactams with β-lactamase inhibitors, or efflux pump inhibitors (EPIs) with pump substrates—have repeatedly demonstrated supra-additive killing in-vitro and translational promise in-vivo (Bognár et al., 2024; Garza-Cervantes and León-Buitimea, 2024). A central objective of combination therapy is resistance prevention—slowing de novo resistance and suppressing pre-existing resistant subpopulations. Beyond antibiotics, phage–antibiotic combinations can deliver potent synergism and reduced resistance emergence when partners are orthogonal in mechanism—e.g., cell-wall active antibiotics with lytic phages—expanding the design space for anti-resistance therapy (Manohar et al., 2022). Recent state-of-the-art surveys have cataloged successful AMP–antibiotic and antibiotic–adjuvant combinations that reduce bacterial loads and improve outcomes in-vivo, reinforcing the translational potential of synergy when guided by mechanism (Garza-Cervantes and León-Buitimea, 2024). Together, these advances support mechanism-driven, pharmacodynamically tuned combination regimens that potentiate index drugs through permeabilization, efflux inhibition, enzyme neutralization, and resistance prevention, and are increasingly reflected in contemporary drug development pipelines and clinical trial designs (Si et al., 2023; Bognár et al., 2024).

Recent advances in phenotypic diagnostics have enabled rapid quantification of efflux-pump activity and membrane potential in clinical bacterial isolates—tools that are critical for translating mechanistic resistance insights into actionable diagnostics. Fluorescent dye-based efflux assays, such as Nile Red or ethidium bromide accumulation, when coupled with flow cytometry or microplate fluorimetry, enable real-time assessment of efflux capacity in clinical strains of Enterobacterales and Staphylococcus aureus, thereby correlating efflux kinetics with antimicrobial susceptibility phenotypes (Whittle et al., 2024). Parallel advances in membrane potential probes [(e.g., DiOC2 (Mascellino et al., 2024), DiSC3 (Gaurav et al., 2023)] enable the quantification of ΔΨ in living bacteria, allowing for the stratification of isolates by energetic phenotype and linking membrane hyperpolarization or depolarization with drug tolerance or efflux competence (Zand et al., 2021). Microfluidic single-cell platforms further promise ultra-rapid (<1 h) readouts of membrane potential shifts under antibiotic challenge, offering potential for point-of-care diagnostics of efflux-mediated resistance (Zagajewski et al., 2023). Collectively, the convergence of efflux and membrane potential assays provides a translational pipeline through which laboratory mechanistic findings can inform clinical decisions—such as identifying high-efflux phenotypes that may benefit from efflux-pump inhibitors or membrane-disrupting adjuvants in real time.

Several recent investigations highlight the translational promise of both allosteric inhibitors and proton-motive-force (PMF)-targeting agents for combating antimicrobial-resistant infections in-vivo. These findings are primarily derived from well-established animal models, including murine systemic infection models, pulmonary infection models, and MRSA infection models, which enable assessment of bacterial burden, survival outcomes, and therapeutic efficacy (Hosfelt et al., 2022). On the bioenergetics front, detailed reviews show that disruption of bacterial PMF—encompassing ΔΨ and ΔpH—undermines key physiological processes including ATP synthesis and efflux pump activity, thereby sensitizing pathogens in animal infection models and suggesting a clinically relevant paradigm for adjuvant therapy (Yang et al., 2023). Moreover, experimental work that applied small-molecule PMF dissipators to methicillin-resistant Staphylococcus aureus demonstrated potent bactericidal activity in-vivo and synergistic combination with conventional antibiotics (Mohiuddin et al., 2022). Together, the in-vivo data for both modalities reinforce the concept that mechanistically orthogonal interventions—targeting bacterial energetics or non-active-site binding—can expand the therapeutic toolkit, reduce the probability of cross-resistance, and provide viable leads for clinical development of next-generation anti-infectives.

Allosteric inhibitors have emerged as a promising class of therapeutics capable of modulating protein function by binding to regulatory sites distinct from the orthosteric (active) site. Their unique mechanisms provide advantages in overcoming resistance mutations that typically affect orthosteric inhibitors. However, resistance to allosteric inhibitors themselves presents a critical challenge to their long-term clinical efficacy. One significant challenge is the emergence of resistance mutations within the allosteric binding pockets or along the allosteric communication pathways. Such mutations can reduce the inhibitor’s binding affinity or impair the allosteric modulation of the target, thereby diminishing the drug’s potency (Lu et al., 2020). In addition, up-regulation of efflux transporters can lower intracellular concentrations of allosteric inhibitors, thereby compromising efficacy. Future strategies to overcome these barriers involve the rational design of next-generation allosteric inhibitors with enhanced binding specificity and resistance-resilient mechanisms. Combination therapies employing both orthosteric and allosteric inhibitors—either as separate agents or linked “bitopic” molecules—offer a promising avenue for reducing resistance development by simultaneously targeting multiple regulatory sites (Zhang et al., 2022). Advances in computational modeling and structural biology enable the prediction and optimization of allosteric sites and their dynamic properties, facilitating the design of inhibitors less prone to resistance mutations (Ni et al., 2020; Outhwaite et al., 2025).

Despite significant advances, critical research gaps and methodological challenges persist that hamper progress in many biomedical fields, including antibiotic discovery, drug delivery, and resistance mitigation. One major gap is the insufficient integration of interdisciplinary approaches, which limits a holistic understanding of complex physiological processes and bacterial resistance mechanisms. Future research must prioritize comprehensive frameworks that encompass molecular, cellular, and systemic levels to overcome these limitations (Hamers et al., 2023). Methodological deficiencies also impede the reproducibility and robustness of findings. Outdated or inadequate experimental models, lack of standardized protocols, and insufficiently representative preclinical systems constrain translation. Sophisticated, physiologically relevant models—such as organoids, microfluidic systems, and advanced in-vivo imaging—are urgently needed to better simulate human pathophysiology and drug interactions (Jansson-Löfmark et al., 2025). Data-related challenges include heterogeneity, poor integration across datasets, and unresolved ethical concerns regarding data privacy, particularly in clinical research. Developing standardized, interoperable data platforms and applying advanced analytics like artificial intelligence will enhance data interpretability and accelerate discovery. However, these tools require careful validation and transparency to avoid bias and ensure reliability (Nyanchoka et al., 2019). There remains a pressing need for methodological innovation in clinical trial designs, particularly in adaptive and pragmatic trials that better reflect real-world settings and facilitate the rapid and efficient evaluation of novel interventions.

Addressing gaps in patient diversity, including underrepresented populations and variable disease phenotypes, will further strengthen evidence and equity in healthcare outcomes (Hamers et al., 2023).

Next-generation antimicrobial therapies have witnessed significant advances through the discovery of novel antimicrobial compounds, innovative drug delivery platforms, and alternative therapeutic modalities, including bacteriophage therapy, antimicrobial peptides, and efflux pump inhibitors. These developments hold promise for overcoming the current limitations posed by multidrug-resistant pathogens and reinvigorating the antimicrobial pipeline. Comprehensive surveillance data emphasize the critical need for integrating these novel approaches with global stewardship and rapid diagnostics to enhance clinical impact (Naghavi et al., 2024). Looking forward, next-generation antimicrobial strategies must embrace precision medicine frameworks, leveraging omics technologies and artificial intelligence to tailor therapies to pathogen and patient-specific profiles. Enhanced global cooperation for data sharing, harmonized surveillance, and sustainable financing models will be essential to accelerate innovation and equitable deployment. Ultimately, a holistic and coordinated effort that bridges scientific innovation with public health imperatives will be crucial for mitigating the threat of antimicrobial resistance and ensuring effective infection management in the decades to come (Huang et al., 2025).