NADPH Oxidases pp 429-446 | Cite as

High-Throughput Screening of NOX Inhibitors

  • Jacek ZielonkaEmail author
  • Monika Zielonka
  • Gang Cheng
  • Micael Hardy
  • Balaraman Kalyanaraman
Part of the Methods in Molecular Biology book series (MIMB, volume 1982)


Development of new, selective inhibitors of nicotinamide adenine dinucleotide phosphate oxidase (NOX) isoforms is important both for basic studies on the role of these enzymes in cellular redox signaling, cell physiology, and proliferation and for development of new drugs for diseases carrying a component of increased NOX activity, such as several types of cancer and cardiovascular and neurodegenerative diseases. High-throughput screening (HTS) of large libraries of compounds remains the major approach for development of new NOX inhibitors. Here, we describe the protocol for the HTS campaign for NOX inhibitors using rigorous assays for superoxide radical anion and hydrogen peroxide, based on oxidation of hydropropidine, coumarin boronic acid, and Amplex Red. We propose using these three probes to screen for and identify new inhibitors, by selecting positive hits that show inhibitory effects in all three assays. Protocols for the synthesis of hydropropidine and for confirmatory assays, including oxygen consumption measurements, electron paramagnetic resonance spin trapping of superoxide, and simultaneous monitoring of superoxide and hydrogen peroxide, are also provided.

Key words

High-throughput screening Superoxide radical anion Hydrogen peroxide Hydropropidine Coumarin boronic acid Amplex Red Fluorescence EPR spin trapping Seahorse extracellular flux analyzer 



This work was supported by NIH grants NCI U01 CA178960 and R01 AA022986 to B.K.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Jacek Zielonka
    • 1
    • 2
    • 3
    Email author
  • Monika Zielonka
    • 1
    • 2
  • Gang Cheng
    • 1
    • 2
  • Micael Hardy
    • 4
  • Balaraman Kalyanaraman
    • 1
    • 2
    • 3
  1. 1.Department of BiophysicsMedical College of WisconsinMilwaukeeUSA
  2. 2.Free Radical Research CenterMedical College of WisconsinMilwaukeeUSA
  3. 3.Cancer CenterMedical College of WisconsinMilwaukeeUSA
  4. 4.Aix Marseille Université, CNRS, ICR, UMR 7273MarseilleFrance

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