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High-Throughput Screening of NOX Inhibitors

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NADPH Oxidases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1982))

Abstract

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.

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Acknowledgment

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

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Authors and Affiliations

  1. Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI, USA

    Jacek Zielonka, Monika Zielonka, Gang Cheng & Balaraman Kalyanaraman

  2. Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI, USA

    Jacek Zielonka, Monika Zielonka, Gang Cheng & Balaraman Kalyanaraman

  3. Cancer Center, Medical College of Wisconsin, Milwaukee, WI, USA

    Jacek Zielonka & Balaraman Kalyanaraman

  4. Aix Marseille Université, CNRS, ICR, UMR 7273, Marseille, France

    Micael Hardy

Authors
  1. Jacek Zielonka
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  2. Monika Zielonka
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  3. Gang Cheng
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  4. Micael Hardy
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  5. Balaraman Kalyanaraman
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Corresponding author

Correspondence to Jacek Zielonka .

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Editors and Affiliations

  1. Conway Institute, School of Medicine, University College Dublin, Dublin, Ireland

    Ulla G. Knaus

  2. Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Thomas L. Leto

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Zielonka, J., Zielonka, M., Cheng, G., Hardy, M., Kalyanaraman, B. (2019). High-Throughput Screening of NOX Inhibitors. In: Knaus, U., Leto, T. (eds) NADPH Oxidases. Methods in Molecular Biology, vol 1982. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9424-3_25

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  • DOI: https://doi.org/10.1007/978-1-4939-9424-3_25

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9423-6

  • Online ISBN: 978-1-4939-9424-3

  • eBook Packages: Springer Protocols

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