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Corresponding Authors: Monika Zieonka: Dept. of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226., Tel.: 414-955-4000; Fax: 414- 955-6512; E-mail: jkielonk@mcw.edu

Corresponding Authors: Balaraman Kalyanaraman:Dept. of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226., Tel.: 414-955-4000; Fax: 414- 955-6512; E-mail: balarama@mcw.edu

B. K. conceived and coordinated the study and wrote the paper.

J. Z. designed, performed, analyzed, and interpreted the data, prepared the figures, and revised the paper critically.

M. Z. performed the experiments and provided the technical assistance.

G. C. designed and performed the Seahorse experiments and obtained cell proliferation data.

M. H. and M. M. A. synthesized hydropropidine and cyclo-o-MitoPh product, respectively.

O. O. provided assistance in these syntheses and structure analyses.

R. P. synthesized o-MitoPhB(OH)2, its oxidation/nitration products and corresponding deuterated analogs.

A. S. helped in characterization of minor products of o-MitoPhB(OH)2 oxidation.

J. D. L. revised the manuscript for intellectual content.

L. V. provided training, assisted in screening of the bioactive libraries at Broad Institute, and performed first pass analysis of the screening results.

All authors reviewed the results and approved the final version of the manuscript.

LC-MS/MS analyses were performed at the Cancer Center Bioenergetics Shared Resource at the Medical College of Wisconsin.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

The authors declare that they have no conflicts of interest with the contents of this article.

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Research Funding:

This work was supported by National Institutes of Health Grant R01 HL073056 (to B. K.) and in part by Molecular Libraries Probe Development Network Grant U54 HG005032.

Keywords:

  • Science & Technology
  • Life Sciences & Biomedicine
  • Biochemistry & Molecular Biology
  • high throughput screening (HTS)
  • NADPH oxidase
  • reactive nitrogen species (RNS)
  • reactive oxygen species (ROS)
  • superoxide ion
  • NADPH oxidase inhibitors
  • fluorescence probes
  • REACTIVE OXYGEN
  • NITRIC-OXIDE
  • EXTRACELLULAR-SUPEROXIDE
  • THERAPEUTIC TARGETS
  • NOX INHIBITORS
  • IN-VIVO
  • CELLS
  • DISEASE
  • BIOLOGY
  • CANCER

Mitigation of NADPH Oxidase 2 Activity as a Strategy to Inhibit Peroxynitrite Formation

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Journal Title:

Journal of Biological Chemistry

Volume:

Volume 291, Number 13

Publisher:

, Pages 7029-7044

Type of Work:

Article | Final Publisher PDF

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.

Copyright information:

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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