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. 2017 Dec;75(3-4):335-349.
doi: 10.1007/s12013-017-0813-6. Epub 2017 Jun 29.

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

Jacek Zielonka  1 , Micael Hardy  2 , Radosław Michalski  3 , Adam Sikora  3 , Monika Zielonka  4 , Gang Cheng  4 , Olivier Ouari  2 , Radosław Podsiadły  5 , Balaraman Kalyanaraman  4
Affiliations

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

Jacek Zielonka et al. Cell Biochem Biophys. 2017 Dec.

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.

Keywords: Fluorescent probes; HPLC; High-throughput screening; NADPH oxidase; Reactive oxygen species.

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Conflict of interest statement

Conflict of Interest: The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
The enzymatic function of NADPH oxidases.
Figure 2
Figure 2
Cascade of reactive oxygen and nitrogen species initiated by one- or two-electron reduction of molecular oxygen, leading to oxidation of biomolecules.
Figure 3
Figure 3
Involvement of HE•+ intermediate in the conversion of HE to 2-OH-E+.
Figure 4
Figure 4
Dependence of the products of HE oxidation on the identity of the oxidant.
Figure 5
Figure 5
Hydropropidine as a cell membrane-impermeable probe for superoxide. (A) Chemical structures of HPr+ and the superoxide-specific oxidation product, 2-OH-Pr2+. (B) Comparison of the cell-medium distribution of HE and HPr+ probes upon incubation with RAW 264.7 cells. (Reprinted from Free Radic. Biol. Med., vol. 54, Michalski, R., Zielonka, J., Hardy, M., Joseph, J., & Kalyanaraman, B., Hydropropidine: a novel, cell-impermeant fluorogenic probe for detecting extracellular superoxide, 135–147. Copyright 2013, with permission from Elsevier.) (72)
Figure 6
Figure 6
Spin trapping of O2•− by selected cyclic nitrones.
Figure 7
Figure 7
EPR spin trapping of superoxide generated by NADPH oxidase. DEPMPO spin trap was incubated with dHL60 cells in the absence (control) or presence of PMA. Where indicated, SOD or catalase was also present. (This research was originally published in Journal of Biological Chemistry. Zielonka, J., Cheng, G., Zielonka, M., Ganesh, T., Sun, A., Joseph, J., Michalski, R., O’Brien, W. J., Lambeth, J. D., & Kalyanaraman, B. High-throughput assays for superoxide and hydrogen peroxide: design of a screening workflow to identify inhibitors of NADPH oxidases. J. Biol. Chem. 2014; 289: 16176–16189. © the American Society for Biochemistry and Molecular Biology.) (79)
Figure 8
Figure 8
Involvement of Amplex Red-derived radical in the oxidative conversion of Amplex Red into resorufin.
Figure 9
Figure 9
Oxidation of aromatic boronic acids into phenolic products.
Figure 10
Figure 10
Principles of simultaneous detection of NADPH oxidase-derived O2•− and H2O2 by a mixture of HE and CBA probes.
Figure 11
Figure 11
Simultaneous monitoring of superoxide and H2O2 generated by different isoforms of NADPH oxidase. Cells were stimulated, where necessary, as indicated and incubated with HE and CBA probes. During the incubation the media were probed repeatedly at different time points, and analyzed by rapid HPLC for the formation of 2-OH-E+ and COH. (A) Nox2 model: dHL60 cells stimulated with PMA; (B) Nox4 model: HEK 293 cells with stably overexpressed Nox4; (C) Nox5 model: HEK 293 cells with stably overexpressed Nox5 and stimulated with ionomycin. (This research was originally published in Journal of Biological Chemistry. Zielonka, J., Cheng, G., Zielonka, M., Ganesh, T., Sun, A., Joseph, J., Michalski, R., O’Brien, W. J., Lambeth, J. D., & Kalyanaraman, B. High-throughput assays for superoxide and hydrogen peroxide: design of a screening workflow to identify inhibitors of NADPH oxidases. J. Biol. Chem. 2014; 289: 16176–16189. © the American Society for Biochemistry and Molecular Biology.) (79)
Figure 12
Figure 12
Measurement of NADPH oxidase activity by monitoring the rates of OCR. (A) Comparison of nondifferentiated and differentiated HL60 cells in their response to PMA stimulation. (B) Effect of mitochondrial inhibitors (1 μM rotenone, ROT, and 1 μM antimycin A, ANT) on basal and PMA-stimulated oxygen consumption by dHL60 cells. (C,D) Effect of diphenyleneiodonium (DPI, 10 μM) on basal (ΔOCRBasal) and PMA-stimulated (ΔOCRPMA) oxygen consumption by dHL60 cells. (This research was originally published in Journal of Biological Chemistry. Zielonka, J., Cheng, G., Zielonka, M., Ganesh, T., Sun, A., Joseph, J., Michalski, R., O’Brien, W. J., Lambeth, J. D., & Kalyanaraman, B. High-throughput assays for superoxide and hydrogen peroxide: design of a screening workflow to identify inhibitors of NADPH oxidases. J. Biol. Chem. 2014; 289:16176–16189, and Zielonka, J., Zielonka, M., VerPlank, L., Cheng, G., Hardy, M., Ouari, O., Ayhan, M. M., Podsiadly, R., Sikora, A., Lambeth, J. D., & Kalyanaraman, B. Mitigation of NADPH Oxidase 2 Activity as a Strategy to Inhibit Peroxynitrite Formation. J. Biol. Chem. 2016; 291:7029–7044. © the American Society for Biochemistry and Molecular Biology) (24, 79)
Figure 13
Figure 13
Probe chemistry and assay design. (A) Probes and products formed in primary assays. (B) Probes and products formed in orthogonal assays. (C) The workflow scheme for screening of Nox inhibitors. (This research was originally published in Journal of Biological Chemistry. Zielonka, J., Cheng, G., Zielonka, M., Ganesh, T., Sun, A., Joseph, J., Michalski, R., O’Brien, W. J., Lambeth, J. D., & Kalyanaraman, B. High-throughput assays for superoxide and hydrogen peroxide: design of a screening workflow to identify inhibitors of NADPH oxidases. J. Biol. Chem. 2014; 289: 16176–16189. © the American Society for Biochemistry and Molecular Biology.) (79)
Figure 14
Figure 14
Monitoring NADPH oxidase-2 activity in dHL60 cells using primary assays. (A) Effect of SOD, catalase, DPI and VAS2870 on the time-dependent increase in fluorescence intensity due to oxidation of HPr+ probe, induced by PMA. (B) Same as in (A), but CBA probe was used. (This research was originally published in Journal of Biological Chemistry. Zielonka, J., Cheng, G., Zielonka, M., Ganesh, T., Sun, A., Joseph, J., Michalski, R., O’Brien, W. J., Lambeth, J. D., & Kalyanaraman, B. High-throughput assays for superoxide and hydrogen peroxide: design of a screening workflow to identify inhibitors of NADPH oxidases. J. Biol. Chem. 2014; 289: 16176–16189. © the American Society for Biochemistry and Molecular Biology.) (79)
Figure 15
Figure 15
Monitoring NADPH oxidase-2 activity in dHL60 cells using secondary assays. (A) Effect of SOD, catalase, DPI, and VAS2870 on the yield of 2-OH-E+ formed from HE probe by dHL60 cells stimulated with PMA, as measured by rapid HPLC analyses. (B) Effect of SOD, CAT, DPI, and VAS2870 on the time-dependent increase in fluorescence intensity due to oxidation of Amplex Red probe, induced by PMA. (This research was originally published in Journal of Biological Chemistry. Zielonka, J., Cheng, G., Zielonka, M., Ganesh, T., Sun, A., Joseph, J., Michalski, R., O’Brien, W. J., Lambeth, J. D., & Kalyanaraman, B. High-throughput assays for superoxide and hydrogen peroxide: design of a screening workflow to identify inhibitors of NADPH oxidases. J. Biol. Chem. 2014; 289: 16176–16189. © the American Society for Biochemistry and Molecular Biology.) (79)
Figure 16
Figure 16
Confirmatory assays used for further characterization of the positive hits from HTS campaign. (A) Structure of the identified hit (compound 43 from ref. (79)). (B-C) Effect of the identified hit on the PMA-stimulated oxygen consumption rates (B) and formation of DEPMPO superoxide spin adduct (C). (D) Concentration dependence of the compound 43 on PMA-stimulated probe oxidation by dHL60 cells in the HPLC-based assays for simultaneous monitoring of O2•− and H2O2. (This research was originally published in Journal of Biological Chemistry. Zielonka, J., Cheng, G., Zielonka, M., Ganesh, T., Sun, A., Joseph, J., Michalski, R., O’Brien, W. J., Lambeth, J. D., & Kalyanaraman, B. High-throughput assays for superoxide and hydrogen peroxide: design of a screening workflow to identify inhibitors of NADPH oxidases. J. Biol. Chem. 2014; 289: 16176–16189. © the American Society for Biochemistry and Molecular Biology.) (79)
Figure 17
Figure 17
Results of screening of a library of bioactive compounds (~ 2,000) using three probes: HPr+ in the presence of DNA as a probe for O2•−, and CBA or Amplex Red in the presence of HRP as probes for H2O2. (A) Correlation of the results of the three assays for Nox2 activity. (B) Results of screening as a percentage of positive hits in one, two or all three assays. (This research was originally published in Journal of Biological Chemistry. Zielonka, J., Zielonka, M., VerPlank, L., Cheng, G., Hardy, M., Ouari, O., Ayhan, M. M., Podsiadly, R., Sikora, A., Lambeth, J. D., & Kalyanaraman, B. Mitigation of NADPH Oxidase 2 Activity as a Strategy to Inhibit Peroxynitrite Formation. J. Biol. Chem. 2016; 291:7029–7044. © the American Society for Biochemistry and Molecular Biology) (24)
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