Reversal of the Drug Binding Pocket Defects of the AcrB Multidrug Efflux Pump Protein of Escherichia coli

Soparkar, Ketaki; Kinana, Alfred; Weeks, Jon W.; Morrison, Keith; Nikaido, Hiroshi; Misra, Rajeev

doi:10.1128/jb.00547-15

Cited by 14 publications

(17 citation statements)

References 52 publications

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“…Finally, even nitrocefin may be pumped out with sigmoidal kinetics when its binding to the binding pocket becomes weaker, for example, by the simultaneous presence of inhibitor PAβN (Fig. 4) or by mutation within the binding site, such as F610A (31). These results further reinforce the idea that the loose binding of substrates is the main cause of the positive cooperativity and sigmoidal kinetics.…”

Section: (9) or D13-9001 (22)supporting

confidence: 63%

Aminoacyl β-naphthylamides as substrates and modulators of AcrB multidrug efflux pump

Kinana

Vargiu

May

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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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 wellknown 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.RND transporters | efflux inhibitors | Phe-Arg-β-naphthylamide | molecular dynamics simulations T he emergence and spread of antibiotic-resistant pathogenic bacteria, especially those of multidrug-resistant or even panresistant Gram-negative bacteria, is a major problem (1). Multidrug efflux pumps, especially those of the resistance-nodulation division (RND) family, contribute strongly to this type of resistance (2). AcrB of Escherichia coli has very wide substrate specificity (3) and its structure and mechanism have been studied extensively as a prototype RND efflux pump (4, 5). AcrB enhances the intrinsic resistance of E. coli, especially to large or lipophilic antibiotics (3), and makes the cell more resistant when overproduced (6). Because of this role, inhibitors of AcrB and its relatives are important not only in basic research but also possibly in clinical medicine, as they could reduce the resistance level of pathogens. The first such inhibitor was Phe-Arg β-naphthylamide (PAβN), reported by Lomovskaya et al. in 2001 (7). These researchers also showed that a PAβN analog, Ala β-naphthylamide, is a good substrate for the AcrB homolog MexB of Pseudomonas aeruginosa, by examining its intracellular accumulation followed by enzymatic hydrolysis that generated the fluorescent naphthylamine. The researchers suggested that PAβN is also a substrate of the AcrB/MexB pump, because pump overproducer...

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Section: (9) or D13-9001 (22)supporting

confidence: 63%

Aminoacyl β-naphthylamides as substrates and modulators of AcrB multidrug efflux pump

Kinana

Vargiu

May

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…The alteration of one residue, F610A, had a strong effect on the efflux of most substrates, as was shown by the MIC data by Bohnert and others [9] and by the nitrocefin efflux assay [11]. This residue appears to play a special role in the efflux process (see Discussion).…”

Section: Resultsmentioning

confidence: 57%

“…1A) in the upper “groove” subdomain, where most of the nitrocefin molecule binds. We have earlier reported [11] that F610A mutation produces a drastic change in the nitrocefin efflux kinetics, which now becomes clearly sigmoidal with a very large K 0.5 value (around 1 mM) and a large estimated V max (around 3 nmol/mg/s), with a Hill coefficient of about 1.5. Among other mutants in the cave region, F628A was less effective in nitrocefin efflux in 2 out of 3 assays (Fig.…”

Section: Resultsmentioning

confidence: 96%

“…Details on this plasmid can be found in our previous publication [11]. Mutations were introduced in the acrB gene by site-directed mutagenesis using a QuickChange Lightning Site Directed Mutagenesis kit (Agilent Technologies).…”

Section: Methodsmentioning

confidence: 99%

“…Although the binding of substrate drugs and inhibitors to the RND (Resistance-Nodulation-Division) transporter AcrB has been studied by crystallography [3-6], molecular docking [7], as well as molecular dynamics simulations [8], an early effort to use site-directed mutagenesis found that only F610A mutation, in the distal (or deep) binding pocket of AcrB, produced a change in the efflux of many drugs [9]. A ciprofloxacin-resistant clinical isolate of Salmonella Typhimurium was found to contain a G288D mutation in the upper part of the distal binding pocket of AcrB, which apparently improves the efficiency of ciprofloxacin efflux but adversely affects the efflux of minocycline and doxorubicin [10]; however an identical change in Escherichia coli AcrB was not found to alter the drug efflux phenotype [11]. …”

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Effect of site-directed mutations in multidrug efflux pump AcrB examined by quantitative efflux assays

Kinana

Vargiu

Nikaido

2016

Biochemical and Biophysical Research Communications

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Background The Resistance-Nodulation-Division (RND) family transporter AcrB plays a major role in the intrinsic and increased resistance of Escherichia coli to a large number of antibiotics. The distal binding pocket within this multidrug efflux transporter is very large, but the effort to define the roles of various residues facing this pocket through site-directed mutagenesis so far involved only the determination of minimal inhibitory concentrations of drugs in mutants. Methods We measured in intact E. coli cells the kinetics of efflux of two substrates, nitrocefin (a cephalosporin) that is predicted mainly to bind to the upper, “groove” domain of the pocket, and L-alanyl-β-naphthylamide (Ala-Naph) that is likely to bind to the lower, “cave” domain, in a number of site-directed mutants of AcrB, where a hydrophobic or aromatic residue was changed into alanine. Results The efflux of nitrocefin became attenuated by some mutations in the groove domain, such as I278A and F178A, but in some experiments a mutation in the cave domain, F628A produced a similar result. In some cases an increased value of KM was detected. The efflux of Ala-Naph was increased by mutations in the cave domain, such as F136A and I626A, but also by those in the groove domain (I277A, I278A, F178A). In most cases the increased Vmax values appeared to be responsible. F610A mutation had a profound effect on the efflux of both substrates, as reported earlier. Conclusions Our data show for the first time effects of various substrate-binding pocket mutations on the kinetics of efflux of two substrates by the AcrB pump. They also confirm interactions between substrates and drugs predicted by MD simulation studies, and also reveal areas that need future research.

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Using Chemical Probes to Assess the Feasibility of Targeting SecA for Developing Antimicrobial Agents against Gram‐Negative Bacteria

et al. 2016

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With the wide-spread emergence of drug resistance, there is an urgent need to search for new antimicrobials, especially those against Gram-negative bacteria. Along this line, the identification of viable targets is a critical first step. SecA is a protein translocase and is commonly believed to be an excellent target for the development of broad-spectrum antimicrobials. In recent years, we have developed three structural classes of SecA inhibitors, which have proven to be very effective against Gram-positive bacteria. However, we have not achieved the same level of success against Gram-negative bacteria, despite the potent inhibition of SecA in enzyme assays by the same inhibitors. In this study, we use representative inhibitors as chemical probes to gain understanding as to why these inhibitors were not effective against Gram-negative bacteria. The results validate our initial postulation that the major difference in effectiveness against Gram-positive and Gram-negative bacteria is in the additional permeability barrier posed by the outer membrane in Gram-negative bacteria. We have also found that expression of efflux pumps, which are responsible for multi-drug resistance, have no effect on the effectiveness of these SecA inhibitors. Identification of an inhibitor-resistant mutant and complementation tests of the plasmids containing secA in a secAts mutant showed that a single secA-azi-9 mutation increased the resistance, providing genetic evidence that SecA indeed is the target of these inhibitors in bacteria. Such results strongly suggest SecA as an excellent target for developing effective antimicrobials against Gram-negative bacteria with the intrinsic ability to overcome MDR. A key future research direction should be the optimization of membrane permeability.

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Reversal of the Drug Binding Pocket Defects of the AcrB Multidrug Efflux Pump Protein of Escherichia coli

Cited by 14 publications

References 52 publications

Aminoacyl β-naphthylamides as substrates and modulators of AcrB multidrug efflux pump

Aminoacyl β-naphthylamides as substrates and modulators of AcrB multidrug efflux pump

Effect of site-directed mutations in multidrug efflux pump AcrB examined by quantitative efflux assays

Using Chemical Probes to Assess the Feasibility of Targeting SecA for Developing Antimicrobial Agents against Gram‐Negative Bacteria

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