title: Aminoacyl β-naphthylamides as substrates and modulators of AcrB multidrug efflux pump.
abstract: Efflux pumps of the resistance-nodulation division superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. Inhibitors of such pumps would improve the efficacy of antibiotics, and ameliorate the crisis in health care caused by the prevalence of multidrug resistant Gram-negative pathogens. Phenylalanyl-arginine β-naphthylamide (PAβN), is a well-known inhibitor of AcrB and its homologs. However, its mechanism of inhibition is not clear. Because the hydrolysis of PAβN in Escherichia coli was nearly entirely dependent on an aminopeptidase, PepN, expression of PepN in periplasm allowed us to carry out a quantitative determination of PAβN efflux kinetics through the determination of its periplasmic concentrations by quantitation of the first hydrolysis product, phenylalanine, after a short period of treatment. We found that PAβN is efficiently pumped out by AcrB, with a sigmoidal kinetics. We also examined the behavior of PAβN homologs, Ala β-naphthylamide, Arg β-naphthylamide, and Phe β-naphthylamide, as substrates of AcrB and as modulators of nitrocefin efflux through AcrB. Furthermore, molecular dynamics simulations indicated that the mode of binding of these compounds to AcrB affects the modulatory activity on the efflux of other substrates. These results, and the finding that PAβN changes the nitrocefin kinetics into a sigmoidal one, suggested that PAβN inhibited the efflux of other drugs by binding to the bottom of the distal binding pocket, the so-called hydrophobic trap, and also by interfering with the binding of other drug substrates to the upper part of the binding pocket.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202;
authors.forename: Alfred D
authors.initials: AD
authors.lastname: Kinana
authors.affiliation: Department of Physics, University of Cagliari, 09042 Monserrato, Italy.
authors.forename: Attilio V
authors.initials: AV
authors.lastname: Vargiu
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202;
authors.forename: Thithiwat
authors.initials: T
authors.lastname: May
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202; nhiroshi@berkeley.edu.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nikaido
mesh_terms: D029968:Escherichia coli Proteins / Q000378:metabolism*; D007700:Kinetics; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism*; D009281:Naphthalenes / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009281:Naphthalenes; C083496:AcrB protein, E coli; C072587:2-naphthylamide
keywords: Phe-Arg-β-naphthylamide; RND transporters; efflux inhibitors; molecular dynamics simulations