Issue 2, 2010
From the journal:

Molecular BioSystems

A miniaturized screen for inhibitors of Jumonji histone demethylases†

Masaaki Sakurai,‡§a   Nathan R. Rose,‡b   Lena Schultz,a   Amy M. Quinn,a   Ajit Jadhav,a   Stanley S. Ng,cd   Udo Oppermann,cd   Christopher J. Schofield*b  and  Anton Simeonov*a  

* Corresponding authors

a NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, USA
E-mail: asimeono@mail.nih.gov
Fax: +1-301-217-5736
Tel: +1-301-217-5721

b Department of Chemistry and the Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, UK
E-mail: christopher.schofield@chem.ox.ac.uk
Fax: +44 (0)1865 275674
Tel: +44 (0)1865 275625

c The Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Headington, UK

d Botnar Research Centre, Oxford Biomedical Research Unit, Oxford, UK

Abstract

2-Oxoglutarate- and Fe(II)-dependent oxygenases are a major class of Nε-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 μL volume in 1536-well format and was used to screen the library of pharmacologically active compounds (LOPAC1280). Inhibitors identified by the screen were further characterized in secondary assays including FDH counterscreen and demethylationassays 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.

Graphical abstract: A miniaturized screen for inhibitors of Jumonji histone demethylases

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Article information

DOI
https://doi.org/10.1039/B912993F
Article type
Paper
Submitted
06 Jul 2009
Accepted
25 Aug 2009
First published
08 Oct 2009

Mol. BioSyst., 2010,6, 357-364

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A miniaturized screen for inhibitors of Jumonji histone demethylases

M. Sakurai, N. R. Rose, L. Schultz, A. M. Quinn, A. Jadhav, S. S. Ng, U. Oppermann, C. J. Schofield and A. Simeonov, Mol. BioSyst., 2010, 6, 357 DOI: 10.1039/B912993F

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