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High-throughput Assays for Superoxide and Hydrogen Peroxide: DESIGN OF A SCREENING WORKFLOW TO IDENTIFY INHIBITORS OF NADPH OXIDASES*
Authors:Jacek Zielonka  Gang Cheng  Monika Zielonka  Thota Ganesh  Aiming Sun  Joy Joseph  Rados?aw Michalski  William J O'Brien  J David Lambeth  Balaraman Kalyanaraman
Institution:From the Department of Biophysics and Free Radical Research Center and ;the Department of Ophthalmology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.;the Departments of §Pharmacology and ;**Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30322, and ;the Emory Institute for Drug Development, Yerkes National Primate Research Center, Atlanta, Georgia 30322
Abstract: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.
Keywords:Enzyme Inhibitor  Fluorescence  High-performance Liquid Chromatography (HPLC)  High-throughput Screening (HTS)  Hydrogen Peroxide  NADPH Oxidase  Reactive Oxygen Species (ROS)  Superoxide Ion
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