A miniaturized screen for inhibitors of Jumonji histone demethylases.
2-Oxoglutarate- and Fe(ii)-dependent oxygenases are a major class of N(epsilon)-methyl lysine demethylases that are involved in epigenetic regulation. Assays suitable for implementation in a high-throughput manner have been lacking for these enzymes. Here, we describe the design and implementation o...
| Váldodahkkit: | , , , , , , , , |
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| Materiálatiipa: | Journal article |
| Giella: | English |
| Almmustuhtton: |
2010
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| Fáttát: |
| _version_ | 1826305915963310080 |
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| author | Sakurai, M Rose, N Schultz, L Quinn, A Jadhav, A Ng, S Oppermann, U Schofield, C Simeonov, A |
| author_facet | Sakurai, M Rose, N Schultz, L Quinn, A Jadhav, A Ng, S Oppermann, U Schofield, C Simeonov, A |
| author_sort | Sakurai, M |
| collection | OXFORD |
| description | 2-Oxoglutarate- and Fe(ii)-dependent oxygenases are a major class of N(epsilon)-methyl lysine demethylases that are involved in epigenetic regulation. Assays suitable for implementation in a high-throughput manner have been lacking for these enzymes. Here, we describe the design and implementation of a robust and miniaturized high-throughput kinetic assay for inhibitors of JMJD2E using a formaldehyde dehydrogenase-coupled reaction with real-time fluorescence detection. Reactant compatibility studies resulted in simplification of the assay scheme to the mixing of two reagent solutions, both of which were stable overnight. The assay was miniaturized to a 4 microL volume in 1536-well format and was used to screen the library of pharmacologically active compounds (LOPAC(1280)). Inhibitors identified by the screen were further characterized in secondary assays including FDH counterscreen and demethylation assays that monitored demethylation by MALDI-TOF MS. The assay developed here will enable the screening of large compound libraries against the Jumonji demethylases in a robust and automated fashion. |
| first_indexed | 2024-03-07T06:40:04Z |
| format | Journal article |
| id | oxford-uuid:f8f61f20-8ae3-46a6-b618-3c10a9472c9d |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T06:40:04Z |
| publishDate | 2010 |
| record_format | dspace |
| spelling | oxford-uuid:f8f61f20-8ae3-46a6-b618-3c10a9472c9d2022-03-27T12:54:28ZA miniaturized screen for inhibitors of Jumonji histone demethylases.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:f8f61f20-8ae3-46a6-b618-3c10a9472c9dKineticsmethodsSpectrometry, FluorescenceEnzyme InhibitorspharmacologyAldehyde OxidoreductasesSpectrometry, Mass, Matrix-Assisted Laser Desorption-Ionizationantagonists and inhibitorsJumonji Domain-Containing Histone DemethylasesmetabolismDrug DiscoveryHumansMiniaturizationEnglishStructural Genomics Consortium2010Sakurai, MRose, NSchultz, LQuinn, AJadhav, ANg, SOppermann, USchofield, CSimeonov, A2-Oxoglutarate- and Fe(ii)-dependent oxygenases are a major class of N(epsilon)-methyl lysine demethylases that are involved in epigenetic regulation. Assays suitable for implementation in a high-throughput manner have been lacking for these enzymes. Here, we describe the design and implementation of a robust and miniaturized high-throughput kinetic assay for inhibitors of JMJD2E using a formaldehyde dehydrogenase-coupled reaction with real-time fluorescence detection. Reactant compatibility studies resulted in simplification of the assay scheme to the mixing of two reagent solutions, both of which were stable overnight. The assay was miniaturized to a 4 microL volume in 1536-well format and was used to screen the library of pharmacologically active compounds (LOPAC(1280)). Inhibitors identified by the screen were further characterized in secondary assays including FDH counterscreen and demethylation assays that monitored demethylation by MALDI-TOF MS. The assay developed here will enable the screening of large compound libraries against the Jumonji demethylases in a robust and automated fashion. |
| spellingShingle | Kinetics methods Spectrometry, Fluorescence Enzyme Inhibitors pharmacology Aldehyde Oxidoreductases Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization antagonists and inhibitors Jumonji Domain-Containing Histone Demethylases metabolism Drug Discovery Humans Miniaturization Sakurai, M Rose, N Schultz, L Quinn, A Jadhav, A Ng, S Oppermann, U Schofield, C Simeonov, A A miniaturized screen for inhibitors of Jumonji histone demethylases. |
| title | A miniaturized screen for inhibitors of Jumonji histone demethylases. |
| title_full | A miniaturized screen for inhibitors of Jumonji histone demethylases. |
| title_fullStr | A miniaturized screen for inhibitors of Jumonji histone demethylases. |
| title_full_unstemmed | A miniaturized screen for inhibitors of Jumonji histone demethylases. |
| title_short | A miniaturized screen for inhibitors of Jumonji histone demethylases. |
| title_sort | miniaturized screen for inhibitors of jumonji histone demethylases |
| topic | Kinetics methods Spectrometry, Fluorescence Enzyme Inhibitors pharmacology Aldehyde Oxidoreductases Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization antagonists and inhibitors Jumonji Domain-Containing Histone Demethylases metabolism Drug Discovery Humans Miniaturization |
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