title: 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.
abstract: 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 colistrain 3-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 EPIs BM-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.
authors.affiliation: Institute of Medical Microbiology, University Hospital, Friedrich Schiller University, Jena, Germany juergen@bohnert.name.
authors.forename: Jürgen A
authors.initials: JA
authors.lastname: Bohnert
authors.affiliation: Center for Infectious Diseases and Travel Medicine, University Hospital, and Department of Medicine, Albert Ludwigs University, Freiburg, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Schuster
authors.affiliation: Center for Infectious Diseases and Travel Medicine, University Hospital, and Department of Medicine, Albert Ludwigs University, Freiburg, Germany.
authors.forename: Winfried V
authors.initials: WV
authors.lastname: Kern
authors.affiliation: Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Faculty of Pharmacy, Kraków, Poland.
authors.forename: Tadeusz
authors.initials: T
authors.lastname: Karcz
authors.affiliation: Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Faculty of Pharmacy, Kraków, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Olejarz
authors.affiliation: Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Faculty of Pharmacy, Kraków, Poland.
authors.forename: Aneta
authors.initials: A
authors.lastname: Kaczor
authors.affiliation: Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Faculty of Pharmacy, Kraków, Poland.
authors.forename: Jadwiga
authors.initials: J
authors.lastname: Handzlik
authors.affiliation: Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Faculty of Pharmacy, Kraków, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Kieć-Kononowicz
mesh_terms: D000900:Anti-Bacterial Agents / Q000138:chemical synthesis / Q000494:pharmacology*; D001425:Bacterial Outer Membrane Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D001681:Biological Assay; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002511:Cephalosporins / Q000737:chemistry; D017291:Clarithromycin / Q000494:pharmacology; D004357:Drug Synergism; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000494:pharmacology*; D015870:Gene Expression; D007093:Imidazoles / Q000138:chemical synthesis / Q000494:pharmacology*; D007202:Indicators and Reagents / Q000737:chemistry; D007700:Kinetics; D064704:Levofloxacin / Q000494:pharmacology; D000069349:Linezolid / Q000494:pharmacology; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D010068:Oxacillin / Q000494:pharmacology; D010879:Piperazines / Q000138:chemical synthesis / Q000494:pharmacology*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012293:Rifampin / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D002511:Cephalosporins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D007093:Imidazoles; D007202:Indicators and Reagents; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010879:Piperazines; D012097:Repressor Proteins; C041610:arcA protein, E coli; C073126:tolC protein, E coli; D064704:Levofloxacin; D011494:Protein Kinases; C065271:arcB protein, E coli; C021720:nitrocefin; D017291:Clarithromycin; D000069349:Linezolid; D010068:Oxacillin; D012293:Rifampin
references.citation: J Antimicrob Chemother. 2011 Sep;66(9):2057-60
references.pmid: 21700628
references.citation: Int J Antimicrob Agents. 2011 Feb;37(2):145-51
references.pmid: 21194895
references.citation: Biosci Biotechnol Biochem. 2010;74(11):2237-41
references.pmid: 21071837
references.citation: Antimicrob Agents Chemother. 2014 Nov;58(11):6870-8
references.pmid: 25182653
references.citation: Bioorg Med Chem Lett. 2001 Jul 23;11(14):1903-6
references.pmid: 11459657
references.citation: Antimicrob Agents Chemother. 2005 Feb;49(2):849-52
references.pmid: 15673787
references.citation: PLoS One. 2014;9(7):e101840
references.pmid: 25025665
references.citation: J Antimicrob Chemother. 2010 Jun;65(6):1215-23
references.pmid: 20304975
references.citation: Antimicrob Agents Chemother. 2000 Apr;44(4):814-20
references.pmid: 10722475
references.citation: Farmaco. 2002 Jan;57(1):39-44
references.pmid: 11902644
references.citation: Pharmazie. 1998 Oct;53(10):680-4
references.pmid: 9812333
references.citation: Farmaco. 2002 May;57(5):355-62
references.pmid: 12058809
references.citation: Acta Pol Pharm. 2012 Jan-Feb;69(1):149-56
references.pmid: 22574520
references.citation: J Nat Prod. 2015 Mar 27;78(3):402-12
references.pmid: 25646964
references.citation: Open Microbiol J. 2013;7:83-6
references.pmid: 23560030
references.citation: Antimicrob Agents Chemother. 2008 Oct;52(10):3604-11
references.pmid: 18694955
references.citation: PLoS One. 2011;6(6):e21196
references.pmid: 21698261
references.citation: Antimicrob Agents Chemother. 2010 May;54(5):1800-6
references.pmid: 20160052
references.citation: Antimicrob Agents Chemother. 2014;58(2):722-33
references.pmid: 24247144
references.citation: Antimicrob Agents Chemother. 2001 Jan;45(1):105-16
references.pmid: 11120952
references.citation: Antimicrob Agents Chemother. 2010 Sep;54(9):3770-5
references.pmid: 20606071
references.citation: FEMS Microbiol Rev. 2012 Mar;36(2):340-63
references.pmid: 21707670
references.citation: Curr Top Med Chem. 2013;13(24):3079-100
references.pmid: 24200360
references.citation: Bioorg Med Chem. 2015 May 1;23(9):2024-34
references.pmid: 25818767
references.citation: Antimicrob Agents Chemother. 1999 Feb;43(2):424-7
references.pmid: 9925552