A Mass Spectrometry-Based Assay for Improved Quantitative Measurements of Efflux Pump Inhibition

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Nadja B. Cech, Patricia A. Sullivan Distinguished Professor of Chemistry (Creator)
Tyler Graf, Research Scientist (Creator)
The University of North Carolina at Greensboro (UNCG )
Web Site: http://library.uncg.edu/

Abstract: Bacterial efflux pumps are active transport proteins responsible for resistance to selectedbiocides and antibiotics. It has been shown that production of efflux pumps is up-regulatedin a number of highly pathogenic bacteria, including methicillin resistant Staphylococcusaureus. 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 anefflux 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 massspectrometry-based method was developed for the quantitative measurement of bacterialefflux pump inhibition. The assay was employed to evaluate efflux pump inhibitory activity ofa 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 becompletely inactive with a fluorescence-based method, showed an IC50 value of 75 µg/mLwith 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 effluxpump inhibitors, particularly in situations where optical interference precludes the application of methods that rely on fluorescence.

Additional Information

Language: English
Date: 2015
Efflux pump, Efflux pump inhibitors

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