title: Mitigation of NADPH Oxidase 2 Activity as a Strategy to Inhibit Peroxynitrite Formation.
abstract: Using high throughput screening-compatible assays for superoxide and hydrogen peroxide, we identified potential inhibitors of the NADPH oxidase (Nox2) isoform from a small library of bioactive compounds. By using multiple probes (hydroethidine, hydropropidine, Amplex Red, and coumarin boronate) with well defined redox chemistry that form highly diagnostic marker products upon reaction with superoxide (O2 (̇̄)), hydrogen peroxide (H2O2), and peroxynitrite (ONOO(-)), the number of false positives was greatly decreased. Selected hits for Nox2 were further screened for their ability to inhibit ONOO(-)formation in activated macrophages. A new diagnostic marker product for ONOO(-)is reported. We conclude that the newly developed high throughput screening/reactive oxygen species assays could also be used to identify potential inhibitors of ONOO(-)formed from Nox2-derived O2 (̇̄)and nitric oxide synthase-derived nitric oxide.
authors.affiliation: From the Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, jzielonk@mcw.edu.
authors.forename: Jacek
authors.initials: J
authors.lastname: Zielonka
authors.affiliation: From the Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.
authors.forename: Monika
authors.initials: M
authors.lastname: Zielonka
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142.
authors.forename: Lynn
authors.initials: L
authors.lastname: VerPlank
authors.affiliation: From the Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.
authors.forename: Gang
authors.initials: G
authors.lastname: Cheng
authors.affiliation: the Aix-Marseille Université, CNRS, ICR UMR 7273, 13397 Marseille, France.
authors.forename: Micael
authors.initials: M
authors.lastname: Hardy
authors.affiliation: the Aix-Marseille Université, CNRS, ICR UMR 7273, 13397 Marseille, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Ouari
authors.affiliation: the Aix-Marseille Université, CNRS, ICR UMR 7273, 13397 Marseille, France.
authors.forename: Mehmet Menaf
authors.initials: MM
authors.lastname: Ayhan
authors.affiliation: From the Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.
authors.forename: Radosław
authors.initials: R
authors.lastname: Podsiadły
authors.affiliation: the Institute of Applied Radiation Chemistry, Faculty of Chemistry, Lodz University of Technology, Żeromskiego 116, 90-924 Lodz, Poland, and.
authors.forename: Adam
authors.initials: A
authors.lastname: Sikora
authors.affiliation: the Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30322.
authors.forename: J David
authors.initials: JD
authors.lastname: Lambeth
authors.affiliation: From the Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, balarama@mcw.edu.
authors.forename: Balaraman
authors.initials: B
authors.lastname: Kalyanaraman
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003374:Coumarins / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005470:Fluorometry; D015870:Gene Expression; D018922:HL-60 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D006861:Hydrogen Peroxide / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D008262:Macrophage Activation / Q000187:drug effects; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015335:Molecular Probes / Q000737:chemistry*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010078:Oxazines / Q000737:chemistry; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry; D010617:Phenanthridines / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D013481:Superoxides / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D003374:Coumarins; D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D015335:Molecular Probes; D010078:Oxazines; D010617:Phenanthridines; D000644:Quaternary Ammonium Compounds; D054852:Small Molecule Libraries; C582101:hydropropidine; D013481:Superoxides; C470430:Amplex Red; D030421:Peroxynitrous Acid; C058428:hydroethidine; D006861:Hydrogen Peroxide; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D013755:Tetradecanoylphorbol Acetate
keywords: NADPH oxidase; NADPH oxidase inhibitors; fluorescence probes; high throughput screening (HTS); reactive nitrogen species (RNS); reactive oxygen species (ROS); superoxide ion
references.citation: J Med Chem. 2010 Apr 8;53(7):2719-40
references.pmid: 20131845
references.citation: Cell Mol Life Sci. 2012 Jul;69(14):2435-42
references.pmid: 22581366
references.citation: Cancer Res. 2010 Nov 15;70(22):9287-97
references.pmid: 20884632
references.citation: Cell Mol Life Sci. 2012 Jul;69(14):2365-71
references.pmid: 22618245
references.citation: Antioxid Redox Signal. 2014 Jun 10;20(17):2741-54
references.pmid: 24070014
references.citation: Br J Pharmacol. 2010 Oct;161(4):885-98
references.pmid: 20860666
references.citation: Chem Res Toxicol. 2012 Sep 17;25(9):1793-9
references.pmid: 22731669
references.citation: Proc Natl Acad Sci U S A. 1990 Feb;87(4):1620-4
references.pmid: 2154753
references.citation: Biochemistry. 2014 Aug 12;53(31):5111-20
references.pmid: 25062272
references.citation: Antioxid Redox Signal. 2009 Oct;11(10):2535-52
references.pmid: 19309261
references.citation: Nature. 2014 Sep 25;513(7519):481-3
references.pmid: 25254460
references.citation: Free Radic Biol Med. 2012 Jan 15;52(2):497-506
references.pmid: 22120494
references.citation: J Biol Chem. 2010 May 7;285(19):14210-6
references.pmid: 20194496
references.citation: Cardiovasc Ther. 2013 Jun;31(3):125-37
references.pmid: 22280098
references.citation: Free Radic Biol Med. 2012 Jan 1;52(1):1-6
references.pmid: 22027063
references.citation: Toxicol Sci. 2003 Dec;76(2):376-83
references.pmid: 14514966
references.citation: Free Radic Biol Med. 2013 Dec;65:1310-4
references.pmid: 24080119
references.citation: J Biomol Screen. 1999;4(2):67-73
references.pmid: 10838414
references.citation: J Vis Exp. 2010;(46). pii: 2511. doi: 10.3791/2511
references.pmid: 21189469
references.citation: Hypertension. 2008 Feb;51(2):211-7
references.pmid: 18086956
references.citation: Circulation. 2002 Jan 22;105(3):293-6
references.pmid: 11804982
references.citation: J Biol Chem. 2007 Mar 2;282(9):6324-37
references.pmid: 17200124
references.citation: Free Radic Biol Med. 2004 Jan 1;36(1):101-11
references.pmid: 14732294
references.citation: Antioxid Redox Signal. 2009 Oct;11(10):2607-19
references.pmid: 19438290
references.citation: J Biol Chem. 1991 Mar 5;266(7):4244-50
references.pmid: 1847917
references.citation: Free Radic Biol Med. 2015 Sep;86:239-49
references.pmid: 26013584
references.citation: Physiol Rev. 2007 Jan;87(1):315-424
references.pmid: 17237348
references.citation: Exp Neurol. 2007 May;205(1):154-65
references.pmid: 17349624
references.citation: Proc Natl Acad Sci U S A. 2010 Dec 14;107(50):21316-21
references.pmid: 21115844
references.citation: Blood. 1980 Jul;56(1):23-9
references.pmid: 6248151
references.citation: Free Radic Biol Med. 2011 Oct 1;51(7):1271-88
references.pmid: 21722728
references.citation: Brain Res. 2016 Jan 1;1630:159-70
references.pmid: 26596859
references.citation: J Biol Chem. 2014 Jun 6;289(23):16176-89
references.pmid: 24764302
references.citation: ACS Chem Biol. 2010 Oct 15;5(10):981-93
references.pmid: 20715845
references.citation: Nat Chem Biol. 2008 May;4(5):278-86
references.pmid: 18421291
references.citation: Arch Biochem Biophys. 2009 Apr 15;484(2):114-6
references.pmid: 19393377
references.citation: Proc Natl Acad Sci U S A. 2005 Apr 19;102(16):5727-32
references.pmid: 15824309
references.citation: Methods Mol Biol. 2015;1264:171-81
references.pmid: 25631013
references.citation: Drug Discov Today. 2006 Jun;11(11-12):524-33
references.pmid: 16713904
references.citation: Am J Physiol Heart Circ Physiol. 2009 May;296(5):H1466-83
references.pmid: 19286953
references.citation: J Immunol. 2009 May 1;182(9):5693-701
references.pmid: 19380816
references.citation: Antioxid Redox Signal. 2015 Aug 10;23(5):358-74
references.pmid: 26135714
references.citation: J Med Chem. 2010 Sep 23;53(18):6758-62
references.pmid: 20731357
references.citation: J Inorg Biochem. 2015 May;146:61-8
references.pmid: 25771434
references.citation: Nat Rev Immunol. 2004 Mar;4(3):181-9
references.pmid: 15039755
references.citation: ACS Med Chem Lett. 2015 Aug 31;6(10 ):1047-52
references.pmid: 26487910
references.citation: Free Radic Biol Med. 2013 Jan;54:135-47
references.pmid: 23051008
references.citation: Cell Metab. 2011 Apr 6;13(4):361-6
references.pmid: 21459321
references.citation: Free Radic Biol Med. 2009 Nov 15;47(10):1401-7
references.pmid: 19686842
references.citation: Redox Biol. 2013;1:244-57
references.pmid: 24024158
references.citation: Mol Cancer Res. 2013 Aug;11(8):901-11
references.pmid: 23696131
references.citation: J Biomol Screen. 2010 Apr;15(4):388-97
references.pmid: 20228280
references.citation: Nat Rev Drug Discov. 2011 Jun;10(6):453-71
references.pmid: 21629295
references.citation: J Biol Chem. 2012 Jan 27;287(5):2984-95
references.pmid: 22139901
references.citation: Nat Rev Mol Cell Biol. 2014 Jun;15(6):411-21
references.pmid: 24854789
references.citation: Chem Res Toxicol. 2011 May 16;24(5):687-97
references.pmid: 21434648
references.citation: Antioxid Redox Signal. 2015 Aug 10;23(5):406-27
references.pmid: 24383718
references.citation: Chem Res Toxicol. 2013 Jun 17;26(6):856-67
references.pmid: 23611338
references.citation: Nat Protoc. 2008;3(1):8-21
references.pmid: 18193017
references.citation: J Exp Med. 2011 Mar 14;208(3):519-33
references.pmid: 21282379