High-throughput assays for superoxide and hydrogen peroxide: design of a screening workflow to identify inhibitors of NADPH oxidases.
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.
From the Department of Biophysics and Free Radical Research Center and jzielonk@mcw.edu.
Jacek
J
Zielonka
From the Department of Biophysics and Free Radical Research Center and.
Gang
G
Cheng
From the Department of Biophysics and Free Radical Research Center and.
Monika
M
Zielonka
the Departments of Pharmacology and.
Thota
T
Ganesh
the Emory Institute for Drug Development, Yerkes National Primate Research Center, Atlanta, Georgia 30322.
Aiming
A
Sun
From the Department of Biophysics and Free Radical Research Center and.
Joy
J
Joseph
From the Department of Biophysics and Free Radical Research Center and.
Radosław
R
Michalski
the Department of Ophthalmology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.
William J
WJ
O'Brien
Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30322, and.
J David
JD
Lambeth
From the Department of Biophysics and Free Radical Research Center and balarama@mcw.edu.
Balaraman
B
Kalyanaraman
D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000378:metabolism / Q000494:pharmacology; D057809:HEK293 Cells; D018922:HL-60 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis* / Q000378:metabolism / Q000494:pharmacology; D013058:Mass Spectrometry; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism; D013113:Spin Labels; D013481:Superoxides / Q000032:analysis* / Q000378:metabolism / Q000494:pharmacology
D004791:Enzyme Inhibitors; D017382:Reactive Oxygen Species; D013113:Spin Labels; D013481:Superoxides; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases
Enzyme Inhibitor; Fluorescence; High-performance Liquid Chromatography (HPLC); High-throughput Screening (HTS); Hydrogen Peroxide; NADPH Oxidase; Reactive Oxygen Species (ROS); Superoxide Ion