Novel Piperazine Arylideneimidazolones Inhibit the AcrAB-TolC Pump in Escherichia coli and Simultaneously Act as Fluorescent Membrane Probes in a Combined Real-Time Influx and Efflux Assay.
Jürgen A Bohnert, Sabine Schuster, Winfried V Kern, Tadeusz Karcz, Agnieszka Olejarz, Aneta Kaczor, Jadwiga Handzlik, Katarzyna Kieć-Kononowicz
Author Information
Jürgen A Bohnert: Institute of Medical Microbiology, University Hospital, Friedrich Schiller University, Jena, Germany juergen@bohnert.name.
Sabine Schuster: Center for Infectious Diseases and Travel Medicine, University Hospital, and Department of Medicine, Albert Ludwigs University, Freiburg, Germany.
Winfried V Kern: Center for Infectious Diseases and Travel Medicine, University Hospital, and Department of Medicine, Albert Ludwigs University, Freiburg, Germany.
Tadeusz Karcz: Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Faculty of Pharmacy, Kraków, Poland.
Agnieszka Olejarz: Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Faculty of Pharmacy, Kraków, Poland.
Aneta Kaczor: Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Faculty of Pharmacy, Kraków, Poland.
Jadwiga Handzlik: Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Faculty of Pharmacy, Kraków, Poland.
Katarzyna Kieć-Kononowicz: Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Faculty of Pharmacy, Kraków, Poland.
In this study, we tested five compounds belonging to a novel series of Piperazine Arylideneimidazolones for the ability to inhibit the AcrAB-TolC efflux pump. The biphenylmethylene derivative (BM-19) and the fluorenylmethylene derivative (BM-38) were found to possess the strongest efflux pump inhibitor (EPI) activities in the AcrAB-TolC-overproducingEscherichia colistrain3-AG100, whereas BM-9, BM-27, and BM-36 had no activity at concentrations of up to 50 μM in a Nile red efflux assay. MIC microdilution assays demonstrated that BM-19 at 1/4 MIC (intrinsic MIC, 200 μM) was able to reduce the MICs of levofloxacin, oxacillin, linezolid, and clarithromycin 8-fold. BM-38 at 1/4 MIC (intrinsic MIC, 100 μM) was able to reduce only the MICs of oxacillin and linezolid (2-fold). Both compounds markedly reduced the MIC of rifampin (BM-19, 32-fold; and BM-38, 4-fold), which is suggestive of permeabilization of the outer membrane as an additional mechanism of action. Nitrocefin hydrolysis assays demonstrated that in addition to their EPI activity, both compounds were in fact weak permeabilizers of the outer membrane. Moreover, it was found that BM-19, BM-27, BM-36, and BM-38 acted as near-infrared-emitting fluorescent membrane probes, which allowed for their use in a combined influx and efflux assay and thus for tracking of the transport of an EPI across the outer membrane by an efflux pump in real time. The EPIsBM-38 and BM-19 displayed the most rapid influx of all compounds, whereas BM-27, which did not act as an EPI, showed the slowest influx.
