| 18699726 |
Johnston PA, Soares KM, Shinde SN, Foster CA, Shun TY, Takyi HK, Wipf P, Lazo JS: 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 Drug Dev Technol. 2008 Aug;6(4):505-18.
We report here the development and optimization of a simple 384-well colorimetric assay to measure H (2) O (2) 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 H (2) O (2) either added exogenously or generated by the redox cycling of compounds in dithiothreitol (DTT). The generation of H (2) O (2) 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 (2) O (2) 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 beta-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 > or =0.8, and the redox cycling generation of H (2) O (2) by DA3003-1 in DTT exhibited an average 50% effective concentration of 0.830 +/- 0.068 microM. Five of the mitogen-activated protein kinase phosphatase (MKP) 1 inhibitors identified in an HTS were shown to generate H (2) O (2) 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 H (2) O (2) that may indirectly modulate target activity and represent promiscuous false-positives from a primary screen. |
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