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Recent Developments in the Probes and Assays for Measurement of the Activity of NADPH Oxidases

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Abstract

NADPH oxidases are a family of enzymes capable of transferring electrons from NADPH to molecular oxygen. A major function of NADPH oxidases is the activation of molecular oxygen into reactive oxygen species. Increased activity of NADPH oxidases has been implicated in various pathologies, including cardiovascular disease, neurological dysfunction, and cancer. Thus, NADPH oxidases have been identified as a viable target for the development of novel therapeutics exhibiting inhibitory effects on NADPH oxidases. Here, we describe the development of new assays for measuring the activity of NADPH oxidases enabling the high-throughput screening for NADPH oxidase inhibitors.

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Abbreviations

2-OH-E+ :

2-hydroxyethidium

2-OH-Pr2+ :

2-hydroxypropidium

BMPO:

5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide

CAT:

catalase

CBA:

coumarin-7-boronic acod

CD:

cyclodextrin

COH:

7-hydroxycoumarin

cyt c 3+ :

ferricytochrome c

DEPMPO:

5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide

DIPPMPO:

5-(diisopropoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide

DMPO:

5,5-dimethyl-1-pyrroline-N-oxide

DPI:

diphenyleneiodonium

E+ :

ethidium

E+-E+ :

diethidium

EPR:

electron paramagnetic resonance

GSH:

glutathione

HE:

hydroethidine (or dihydroethidium)

HE•+ :

HE radical cation

HPLC:

high-performance liquid chromatography

HPr+ :

hydropropidine

HRP:

horseradish peroxidase

HTS:

high-throughput screening

L-012:

8-amino-5-chloro-2,3-dihydro-7-phenyl-pyrido[3,4-d]pyridazine

MPO:

myeloperoxidase

NADPH:

nicotinamide adenine dinucleotide phosphate, reduced form

NBT:

nitroblue tetrazolium

Nox2:

NADPH oxidase-2

Nox4:

NADPH oxidase-4

Nox5:

NADPH oxidase-5

OCR:

oxygen consumption rate

PMA:

phorbol 12-myristate 13-acetate

SOD:

superoxide dismutase

VAS2870:

1,3-benzoxazol-2-yl-3-benzyl-3H-[1–3]triazolo[4,5-d]pyrimidin-7-yl sulfide

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Funding

This work was supported by a grant from NIH (R01 AA022986) to B.K. and from the French National Research Agency (ANR-16-CE07-0023-01) to O.O. and M.H. A.S. and R.M. were supported by a grant from Polish National Science Centre, No. 2015/18/E/ST4/00235.

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Authors and Affiliations

  1. Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA

    Jacek Zielonka, Monika Zielonka, Gang Cheng & Balaraman Kalyanaraman

  2. Aix Marseille Univ, CNRS, ICR, 13013, Marseille, France

    Micael Hardy & Olivier Ouari

  3. Institute of Applied Radiation Chemistry, Faculty of Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924, Lodz, Poland

    Radosław Michalski & Adam Sikora

  4. Institute of Polymer and Dye Technology, Faculty of Chemistry, Lodz University of Technology, Stefanowskiego 12/16, 90-924, Lodz, Poland

    Radosław Podsiadły

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  1. Jacek Zielonka
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  2. Micael Hardy
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  9. Balaraman Kalyanaraman
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Zielonka, J., Hardy, M., Michalski, R. et al. Recent Developments in the Probes and Assays for Measurement of the Activity of NADPH Oxidases. Cell Biochem Biophys 75, 335–349 (2017). https://doi.org/10.1007/s12013-017-0813-6

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