{'fraction_non_alphanumeric': 0.022248708780294002, 'fraction_numerical': 0.0, 'mean_word_length': 5.358585858585859, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Aminoacyl β-naphthylamides as substrates and modulators of AcrB multidrug efflux pump

Significance Resistance-nodulation division family efflux pumps, exemplified by AcrB of Escherichia coli, play an important role in multidrug resistance of Gram-negative bacteria by actively pumping out a very wide range of antimicrobial agents. Yet it is unclear with how much affinity and with how much rate this pumping process works, except for our earlier studies on cephalosporins and penicillins. Here we succeeded in determining these parameters for the earliest-discovered inhibitor Phenylalanyl-argenine β-naphthylamide and its relatives, and examined the binding of these compounds to AcrB binding sites by docking and molecular dynamics simulations. The results suggest how an inhibitor, even when it is itself pumped out, can reduce the pumping of other substrates. 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.