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1.  Profiling the NIH Small Molecule Repository for Compounds That Generate H2O2 by Redox Cycling in Reducing Environments 
We have screened the Library of Pharmacologically Active Compounds (LOPAC) and the National Institutes of Health (NIH) Small Molecule Repository (SMR) libraries in a horseradish peroxidase–phenol red (HRP-PR) H2O2 detection assay to identify redox cycling compounds (RCCs) capable of generating H2O2 in buffers containing dithiothreitol (DTT). Two RCCs were identified in the LOPAC set, the ortho-naphthoquinone β-lapachone and the para-naphthoquinone NSC 95397. Thirty-seven (0.02%) concentration-dependent RCCs were identified from 195,826 compounds in the NIH SMR library; 3 singleton structures, 9 ortho-quinones, 2 para-quinones, 4 pyrimidotriazinediones, 15 arylsulfonamides, 2 nitrothiophene-2-carboxylates, and 2 tolyl hydrazides. Sixty percent of the ortho-quinones and 80% of the pyrimidotriazinediones in the library were confirmed as RCCs. In contrast, only 3.9% of the para-quinones were confirmed as RCCs. Fifteen of the 251 arylsulfonamides in the library were confirmed as RCCs, and since we screened 17,868 compounds with a sulfonamide functional group we conclude that the redox cycling activity of the arylsulfonamide RCCs is due to peripheral reactive enone, aromatic, or heterocyclic functions. Cross-target queries of the University of Pittsburgh Drug Discovery Institute (UPDDI) and PubChem databases revealed that the RCCs exhibited promiscuous bioactivity profiles and have populated both screening databases with significantly higher numbers of active flags than non-RCCs. RCCs were promiscuously active against protein targets known to be susceptible to oxidation, but were also active in cell growth inhibition assays, and against other targets thought to be insensitive to oxidation. Profiling compound libraries or the hits from screening campaigns in the HRP-PR H2O2 detection assay significantly reduce the timelines and resources required to identify and eliminate promiscuous nuisance RCCs from the candidates for lead optimization.
doi:10.1089/adt.2009.0247
PMCID: PMC3098569  PMID: 20070233
2.  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 H2O2 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 H2O2 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 H2O2 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.
doi:10.1089/adt.2008.151
PMCID: PMC2752819  PMID: 18699726
3.  Development of a 384-Well Colorimetric Assay to Quantify Hydrogen Peroxide Generated by the Redox Cycling of Compounds in the Presence of Reducing Agents 
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 H2O2 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 H2O2 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 μ 0.068 μM. Five of the mitogen-activated protein kinase phosphatase (MKP) 1 inhibitors identified in an HTS were shown to generate H2O2 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.
doi:10.1089/adt.2008.151
PMCID: PMC2752819  PMID: 18699726

Results 1-3 (3)