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

Jacek Zielonka (1) , Monika Zielonka (1) , Lynn Verplank (2) , Gang Cheng (1) , Micael Hardy (3) , Olivier Ouari (3) , Mehmet Menaf Ayhan (3) , Radosław Podsiadły (4, 1) , Adam Sikora (5) , J David Lambeth (6) , Balaraman Kalyanaraman (1)

1 Medical College of Wisconsin
2 BROAD INSTITUTE - Broad Institute of MIT and Harvard
3 ICR - Institut de Chimie Radicalaire
4 Institute of Polymer and Dye Technology [Łódź University of Technology]
5 Institute of Applied Radiation Chemistry, Technical University of Lodz
6 Department of Pathology & Laboratory Medicine

Using a high-throughput screening (HTS)-compatible assays for superoxide and hydrogen peroxide, we identified potential inhibitors of 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 (O 2 • –), hydrogen peroxide (H 2 O 2), 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. New diagnostic marker product for ONOO – is reported. We conclude that the newly developed HTS/ROS assays could also be used to identify potential inhibitors of ONOO – formed from Nox2-derived O 2 • – and nitric oxide synthase (NOS)-derived nitric oxide.

Keywords: hydrogen peroxide; superoxide radical anion; reactive nitrogen species; reactive oxygen species; fluorescence probes; NADPH oxidase inhibitors; high-throughput screening (HTS); peroxynitrite

Domains: Chemical Sciences; Medicinal Chemistry; Life Sciences [q-bio]; Biochemistry, Molecular Biology; Biochemistry [q-bio.BM]

Jacek Zielonka, Monika Zielonka, Lynn Verplank, Gang Cheng, Micael Hardy, et al.. Mitigation of NADPH Oxidase 2 Activity as a Strategy to Inhibit Peroxynitrite Formation. Journal of Biological Chemistry, 2016, 291, pp.7029-7044. ⟨10.1074/jbc.M115.702787⟩. ⟨hal-01400640⟩