Revisit of the admitted control RND efflux pumps’ inhibitor PAβN: use limitations, and structural revelation in complex with MexB
- 1Université Paris Cité, CNRS, CiTCoM
- 2Université Marie et Louis Pasteur, Chrono-environnement UMR CNRS 6249
Abstract
Introduction: To bypass the health problem of antibiotic resistance in Gram-negative bacteria, a tempting approach is to use efflux pump inhibitors (EPI) to block the transporters before they expel antibiotics from the cell. One well-known EPI is the phenylalanine-arginine beta-naphthylamide (PAβN). Although it is described as toxic and therefore unsuitable for clinical use, it is highly effective and is commonly used as EPI reference compound in drug development. Nevertheless, the lack of toxicity on several cellular strains in our hands required us to revisit this known RND inhibitor, to redefine its limits.
Methods: The rational design of new EPIs requires the integration of complementary information on their microbiological activity, toxicity toward eukaryotic cells, and description of molecular interaction with their target at atomic resolution. These parameters should also be considered in comparison with those of an established reference compound such as PAβN. Accordingly, the effect of PAβN on Pseudomonas aeruginosa was evaluated by determining the minimum inhibitory concentrations of three β-lactam antibiotics in the presence or absence of PAβN and by defining concentrations that do not perturb the bacterial envelope. Its toxicity was assessed using four human cell lines and multiple cellular endpoints. Finally, the interaction of PAβN with the RND transporter MexB was characterized by cryo-electron microscopy and crystallography.
Results and Discussion: Microbiological analyses defined concentration ranges in which PAβN can be used to investigate efflux inhibition without causing detectable perturbation of the bacterial envelope. Across the four human cell models, PAβN displayed limited cytotoxicity, whereas β-naphthylamine was markedly more toxic. This points to the β-naphthylamide moiety as a potential contributor to PAβN toxicity, possibly resulting from metabolic proteolysis processing. Structural analyses revealed PAβN bound to MexB in the Tight conformation in both cryo-EM and crystal structures, but at distinct positions within the binding cavity. Comparison with the previously reported structure of PAβN bound to the artificially symmetric trimer of AcrB provides new insights into PAβN recognition and handling by RND pumps. Together, these microbiological, toxicological, and structural data refine the use of PAβN as a reference EPI and provide new perspectives on substrate transport by RND efflux pumps.
Related
- Preclinical-Clinical Discrepancies in Immune Checkpoint Inhibitor Efficacy for Triple-Negative Breast Cancer: Disease Stage, Antibody Properties, Biomarker Alignment, and Model Limitations.
- Advancing T Cell Engagers in Multiple Myeloma: Structural Design and Clinical Optimization to Address Current Limitations.
- Factors Associated with Emergency Department Revisit and Urologic Intervention Following Discharge of Renal Colic Patients: A Retrospective Cohort Study from a Military Hospital in the Kingdom of Bahrain
- From therapeutic landscape perception to revisit intention: the mediating role of restorative experience in Palandöken Urban Forest
- Discovery of scutellarin as a MRGX2 agonist via MRGX2/CMC strategy and revelation of its pseudo-allergic mechanism
BibTeX
@article{wehbi2026revisit,
title = {{Revisit of the admitted control RND efflux pumps’ inhibitor PAβN: use limitations, and structural revelation in complex with MexB}},
author = {Hilal Wehbi and Elodie Olivier and Quentin Cece and Maxime Mégret and Mélanie Sébastien and Nesrine Aissaoui and Catherine Llanes and Gilles Phan and Isabelle Broutin},
journal = {Frontiers in Pharmacology},
year = {2026},
month = oct,
doi = {10.3389/fphar.2026.1906963},
url = {https://www.frontiersin.org/articles/10.3389/fphar.2026.1906963/full},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Revisit of the admitted control RND efflux pumps’ inhibitor PAβN: use limitations, and structural revelation in complex with MexB
AU - Hilal Wehbi
AU - Elodie Olivier
AU - Quentin Cece
AU - Maxime Mégret
AU - Mélanie Sébastien
AU - Nesrine Aissaoui
AU - Catherine Llanes
AU - Gilles Phan
AU - Isabelle Broutin
PY - 2026
DA - 2026/10/19
JO - Frontiers in Pharmacology
DO - 10.3389/fphar.2026.1906963
UR - https://www.frontiersin.org/articles/10.3389/fphar.2026.1906963/full
AB - Introduction: To bypass the health problem of antibiotic resistance in Gram-negative bacteria, a tempting approach is to use efflux pump inhibitors (EPI) to block the transporters before they expel antibiotics from the cell. One well-known EPI is the phenylalanine-arginine beta-naphthylamide (PAβN). Although it is described as toxic and therefore unsuitable for clinical use, it is highly effective and is commonly used as EPI reference compound in drug development. Nevertheless, the lack of toxicity on several cellular strains in our hands required us to revisit this known RND inhibitor, to redefine its limits. Methods: The rational design of new EPIs requires the integration of complementary information on their microbiological activity, toxicity toward eukaryotic cells, and description of molecular interaction with their target at atomic resolution. These parameters should also be considered in comparison with those of an established reference compound such as PAβN. Accordingly, the effect of PAβN on Pseudomonas aeruginosa was evaluated by determining the minimum inhibitory concentrations of three β-lactam antibiotics in the presence or absence of PAβN and by defining concentrations that do not perturb the bacterial envelope. Its toxicity was assessed using four human cell lines and multiple cellular endpoints. Finally, the interaction of PAβN with the RND transporter MexB was characterized by cryo-electron microscopy and crystallography. Results and Discussion: Microbiological analyses defined concentration ranges in which PAβN can be used to investigate efflux inhibition without causing detectable perturbation of the bacterial envelope. Across the four human cell models, PAβN displayed limited cytotoxicity, whereas β-naphthylamine was markedly more toxic. This points to the β-naphthylamide moiety as a potential contributor to PAβN toxicity, possibly resulting from metabolic proteolysis processing. Structural analyses revealed PAβN bound to MexB in the Tight conformation in both cryo-EM and crystal structures, but at distinct positions within the binding cavity. Comparison with the previously reported structure of PAβN bound to the artificially symmetric trimer of AcrB provides new insights into PAβN recognition and handling by RND pumps. Together, these microbiological, toxicological, and structural data refine the use of PAβN as a reference EPI and provide new perspectives on substrate transport by RND efflux pumps.
SN - 1663-9812
ER -