eprintid: 19275
rev_number: 28
userid: 1419
dir: disk0/00/01/92/75
datestamp: 2013-07-15 19:55:58
lastmod: 2021-06-13 00:55:50
status_changed: 2013-07-15 19:55:58
type: article
metadata_visibility: show
item_issues_count: 0
eprint_status: archive
creators_name: Johnston, PA
creators_name: Soares, KM
creators_name: Shinde, SN
creators_name: Foster, CA
creators_name: Shun, TY
creators_name: Takyi, HK
creators_name: Wipf, P
creators_name: Lazo, JS
creators_email: paj18@pitt.edu
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creators_email: pwipf@pitt.edu
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creators_id: PAJ18
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creators_id: PWIPF
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title: Development of a 384-well colorimetric assay to quantify hydrogen peroxide generated by the redox cycling of compounds in the presence of reducing agents
ispublished: pub
divisions: sch_as_chemistry
full_text_status: public
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.
date: 2008-08-01
date_type: published
publication: Assay and Drug Development Technologies
volume: 6
number: 4
pagerange: 505 - 518
refereed: TRUE
issn: 1540-658X
id_number: 10.1089/adt.2008.151
other_id: NLM NIHMS124031
other_id: NLM PMC2752819
pmcid: PMC2752819
pmid: 18699726
mesh_headings: Catalase--pharmacology
mesh_headings: Colorimetry--instrumentation
mesh_headings: Colorimetry--methods
mesh_headings: Coloring Agents
mesh_headings: Drug Evaluation, Preclinical--instrumentation
mesh_headings: Dual Specificity Phosphatase 1--analysis
mesh_headings: Dual Specificity Phosphatase 1--antagonists & inhibitors
mesh_headings: Dual Specificity Phosphatase 1--metabolism
mesh_headings: Hydrogen Peroxide--analysis
mesh_headings: Indicators and Reagents
mesh_headings: Nanotechnology
mesh_headings: Oxidation-Reduction
mesh_headings: Phenolsulfonphthalein
mesh_headings: Reducing Agents--chemistry
chemical_names: Coloring Agents
chemical_names: Indicators and Reagents
chemical_names: Reducing Agents
chemical_names: Phenolsulfonphthalein
chemical_names: Hydrogen Peroxide
chemical_names: Catalase
chemical_names: DUSP1 protein, human
chemical_names: Dual Specificity Phosphatase 1
citation:   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     
document_url: http://d-scholarship-dev.library.pitt.edu/19275/1/licence.txt