<> "The repository administrator has not yet configured an RDF license."^^ . <> . . . "Development of a 384-well colorimetric assay to quantify hydrogen peroxide generated by the redox cycling of compounds in the presence of reducing agents"^^ . "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."^^ . "2008-08-01" . . "6" . "4" . . "Assay and Drug Development Technologies"^^ . . . "1540658X" . . . . . . . . . . . . . . . . . . . . . . . . . . . . "CA"^^ . "Foster"^^ . "CA Foster"^^ . . "PA"^^ . "Johnston"^^ . "PA Johnston"^^ . . "KM"^^ . "Soares"^^ . "KM Soares"^^ . . "P"^^ . "Wipf"^^ . "P Wipf"^^ . . "HK"^^ . "Takyi"^^ . "HK Takyi"^^ . . "SN"^^ . "Shinde"^^ . "SN Shinde"^^ . . "TY"^^ . "Shun"^^ . "TY Shun"^^ . . "JS"^^ . "Lazo"^^ . "JS Lazo"^^ . . . . . . "Development of a 384-well colorimetric assay to quantify hydrogen peroxide generated by the redox cycling of compounds in the presence of reducing agents (Plain Text)"^^ . . . "licence.txt"^^ . . . "Development of a 384-well colorimetric assay to quantify hydrogen peroxide generated by the redox cycling of compounds in the presence of reducing agents (Other)"^^ . . . . . . "indexcodes.txt"^^ . . "HTML Summary of #19275 \n\nDevelopment of a 384-well colorimetric assay to quantify hydrogen peroxide generated by the redox cycling of compounds in the presence of reducing agents\n\n" . "text/html" . .