High-throughput assays for superoxide and hydrogen peroxide: design of a screening workflow to identify inhibitors of NADPH oxidases. J Biol Chem 2014 Jun 06;289(23):16176-89
Date
04/26/2014Pubmed ID
24764302Pubmed Central ID
PMC4047388DOI
10.1074/jbc.M114.548693Scopus ID
2-s2.0-84902206908 (requires institutional sign-in at Scopus site) 64 CitationsAbstract
Recent progress characterizing the reaction mechanism(s) of fluorescent probes with reactive oxygen species has made it possible to rigorously analyze these reactive species in biological systems. We have developed rapid high throughput-compatible assays for monitoring cellular production of superoxide radical anion and hydrogen peroxide using hydropropidine and coumarin boronic acid probes, respectively. Coupling plate reader-based fluorescence measurements with HPLC-based simultaneous monitoring of superoxide radical anion and hydrogen peroxide provides the basis for the screening protocol for NADPH oxidase (Nox) inhibitors. Using this newly developed approach along with the medium-throughput plate reader-based oximetry and EPR spin trapping as confirmatory assays, it is now eminently feasible to rapidly and reliably identify Nox enzyme inhibitors with a markedly lower rate of false positives. These methodological advances provide an opportunity to discover selective inhibitors of Nox isozymes, through enhanced conceptual understanding of their basic mechanisms of action.
Author List
Zielonka J, Cheng G, Zielonka M, Ganesh T, Sun A, Joseph J, Michalski R, O'Brien WJ, Lambeth JD, Kalyanaraman BAuthors
Gang Cheng PhD Assistant Professor in the Biophysics department at Medical College of WisconsinBalaraman Kalyanaraman PhD Professor in the Biophysics department at Medical College of Wisconsin
Jacek M. Zielonka PhD Assistant Professor in the Biophysics department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
Chromatography, High Pressure LiquidEnzyme Inhibitors
HEK293 Cells
HL-60 Cells
High-Throughput Screening Assays
Humans
Hydrogen Peroxide
Mass Spectrometry
NADPH Oxidases
Oxidation-Reduction
Reactive Oxygen Species
Spin Labels
Superoxides
