title: A mass spectrometry-based assay for improved quantitative measurements of efflux pump inhibition.
abstract: Bacterial efflux pumps are active transport proteins responsible for resistance to selected biocides and antibiotics. It has been shown that production of efflux pumps is up-regulated in a number of highly pathogenic bacteria, including methicillin resistant Staphylococcus aureus. Thus, the identification of new bacterial efflux pump inhibitors is a topic of great interest. Existing assays to evaluate efflux pump inhibitory activity rely on fluorescence by an efflux pump substrate. When employing these assays to evaluate efflux pump inhibitory activity of plant extracts and some purified compounds, we observed severe optical interference that gave rise to false negative results. To circumvent this problem, a new mass spectrometry-based method was developed for the quantitative measurement of bacterial efflux pump inhibition. The assay was employed to evaluate efflux pump inhibitory activity of a crude extract of the botanical Hydrastis Canadensis, and to compare the efflux pump inhibitory activity of several pure flavonoids. The flavonoid quercetin, which appeared to be completely inactive with a fluorescence-based method, showed an IC50 value of 75 μg/mL with the new method. The other flavonoids evaluated (apigenin, kaempferol, rhamnetin, luteolin, myricetin), were also active, with IC50 values ranging from 19 μg/mL to 75 μg/mL. The assay described herein could be useful in future screening efforts to identify efflux pump inhibitors, particularly in situations where optical interference precludes the application of methods that rely on fluorescence.
authors.affiliation: Department of Chemistry/Biochemistry, The University of North Carolina Greensboro, Greensboro, North Carolina, United States of America.
authors.forename: Adam R
authors.initials: AR
authors.lastname: Brown
authors.affiliation: Department of Chemistry/Biochemistry, The University of North Carolina Greensboro, Greensboro, North Carolina, United States of America.
authors.forename: Keivan A
authors.initials: KA
authors.lastname: Ettefagh
authors.affiliation: Department of Chemistry/Biochemistry, The University of North Carolina Greensboro, Greensboro, North Carolina, United States of America.
authors.forename: Daniel
authors.initials: D
authors.lastname: Todd
authors.affiliation: Department of Chemistry/Biochemistry, The University of North Carolina Greensboro, Greensboro, North Carolina, United States of America.
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Cole
authors.affiliation: Department of Chemistry/Biochemistry, The University of North Carolina Greensboro, Greensboro, North Carolina, United States of America.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Egan
authors.affiliation: Department of Chemistry/Biochemistry, The University of North Carolina Greensboro, Greensboro, North Carolina, United States of America.
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Foil
authors.affiliation: Department of Chemistry/Biochemistry, The University of North Carolina Greensboro, Greensboro, North Carolina, United States of America.
authors.forename: Tyler N
authors.initials: TN
authors.lastname: Graf
authors.affiliation: Division of Infectious Disease, John Dingell Department of Veteran Affairs Medical Center and Department of Internal Medicine, Wayne State University School of Medicine, Detroit, Michigan, United States of America.
authors.forename: Bryan D
authors.initials: BD
authors.lastname: Schindler
authors.affiliation: Division of Infectious Disease, John Dingell Department of Veteran Affairs Medical Center and Department of Internal Medicine, Wayne State University School of Medicine, Detroit, Michigan, United States of America.
authors.forename: Glenn W
authors.initials: GW
authors.lastname: Kaatz
authors.affiliation: Department of Chemistry/Biochemistry, The University of North Carolina Greensboro, Greensboro, North Carolina, United States of America.
authors.forename: Nadja B
authors.initials: NB
authors.lastname: Cech
mesh_terms: D001692:Biological Transport / Q000187:drug effects*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D002853:Chromatography, Liquid; D004305:Dose-Response Relationship, Drug; D004996:Ethidium / Q000378:metabolism; D020128:Inhibitory Concentration 50; D013058:Mass Spectrometry; D010936:Plant Extracts / Q000494:pharmacology; D013050:Spectrometry, Fluorescence / Q000379:methods; D013211:Staphylococcus aureus / Q000187:drug effects / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D010936:Plant Extracts; D004996:Ethidium