Revisit of the admitted control RND efflux pumps’ inhibitor PAβN: use limitations, and structural revelation in complex with MexB

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.