Johnston, PA and Soares, KM and Shinde, SN and Foster, CA and Shun, TY and Takyi, HK and Wipf, P and Lazo, JS
(2008)
Development of a 384-well colorimetric assay to quantify hydrogen peroxide generated by the redox cycling of compounds in the presence of reducing agents.
Assay and Drug Development Technologies, 6 (4).
505 - 518.
ISSN 1540-658X
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Abstract
We report here the development and optimization of a simple 384-well colorimetric assay to measure H2O2 generated by the redox cycling of compounds incubated with reducing agents in high-throughput screening (HTS) assay buffers. The phenol red-horseradish peroxidase (HRP) assay readily detected H2O2 either added exogenously or generated by the redox cycling of compounds in dithiothreitol (DTT). The generation of H 2O2 was dependent on the concentration of both the compound and DTT and was abolished by catalase. Although both DTT and tris(2-carboxyethyl) phosphine sustain the redox cycling generation of H 2O2 by a model quinolinedione, 6-chloro-7-(2-morpholin-4- yl-ethylamino)-quinoline-5,8-dione (NSC 663284; DA3003-1), other reducing agents such as β-mercaptoethanol, glutathione, and cysteine do not. The assay is compatible with HTS. Once terminated, the assay signal was stable for at least 5 h, allowing for a reasonable throughput. The assay tolerated up to 20% dimethyl sulfoxide, allowing a wide range of compound concentrations to be tested. The assay signal window was robust and reproducible with average Z-factors of ≥0.8, and the redox cycling generation of H2O2 by DA3003-1 in DTT exhibited an average 50% effective concentration of 0.830 ± 0.068 μM. Five of the mitogen-activated protein kinase phosphatase (MKP) 1 inhibitors identified in an HTS were shown to generate H 2O2 in the presence of DTT, and their inhibition of MKP-1 activity was shown to be time dependent and was abolished or significantly reduced by either 100 U of catalase or by higher DTT levels. A cross-target query of the PubChem database with three structurally related pyrimidotriazinediones revealed active flags in 36-39% of the primary screening assays. Activity was confirmed against a number of targets containing active site cysteines, including protein tyrosine phosphatases, cathepsins, and caspases, as well as a number of cellular cytotoxicity assays. Rather than utilize resources to conduct a hit characterization effort involving several secondary assays, the phenol red-HRP assay provides a simple, rapid, sensitive, and inexpensive method to identify compounds that redox cycle in DTT or tris(2-carboxyethyl)phosphine to produce H2O2 that may indirectly modulate target activity and represent promiscuous false-positives from a primary screen. © 2008 Mary Ann Liebert, Inc.
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Details
| Item Type: |
Article
|
| Status: |
Published |
| Creators/Authors: |
|
| Date: |
1 August 2008 |
| Date Type: |
Publication |
| Journal or Publication Title: |
Assay and Drug Development Technologies |
| Volume: |
6 |
| Number: |
4 |
| Page Range: |
505 - 518 |
| DOI or Unique Handle: |
10.1089/adt.2008.151 |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Chemistry |
| Refereed: |
Yes |
| ISSN: |
1540-658X |
| MeSH Headings: |
Catalase--pharmacology; Colorimetry--instrumentation; Colorimetry--methods; Coloring Agents; Drug Evaluation, Preclinical--instrumentation; Dual Specificity Phosphatase 1--analysis; Dual Specificity Phosphatase 1--antagonists & inhibitors; Dual Specificity Phosphatase 1--metabolism; Hydrogen Peroxide--analysis; Indicators and Reagents; Nanotechnology; Oxidation-Reduction; Phenolsulfonphthalein; Reducing Agents--chemistry |
| Other ID: |
NLM NIHMS124031, NLM PMC2752819 |
| PubMed Central ID: |
PMC2752819 |
| PubMed ID: |
18699726 |
| Date Deposited: |
15 Jul 2013 19:55 |
| Last Modified: |
22 Jun 2021 13:56 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/19275 |
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