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Development of a 384-well colorimetric assay to quantify hydrogen peroxide generated by the redox cycling of compounds in the presence of reducing agents

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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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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Item Type: Article
Status: Published
CreatorsEmailPitt UsernameORCID
Johnston, PApaj18@pitt.eduPAJ180000-0001-5815-3091
Soares, KM
Shinde, SN
Foster, CA
Shun, TY
Takyi, HK
Wipf, Ppwipf@pitt.eduPWIPF
Lazo, JS
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


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