TY  - JOUR
A1  - Hatch, Stephanie B.
A1  - Yapp, Clarence
A1  - Montenegro, Raquel Carvalho
A1  - Savitsky, Pavel
A1  - Gamble, Vicki
A1  - Tumber, Anthony
A1  - Ruda, Gian Filippo
A1  - Bavetsias, Vassilios
A1  - Fedorov, Oleg
A1  - Atrash, Butrus
A1  - Raynaud, Florence
A1  - Lanigan, Rachel
A1  - Carmichael, LeAnne
A1  - Tomlin, Kathy
A1  - Burke, Rosemary
A1  - Westaway, Susan M.
A1  - Brown, Jack A.
A1  - Prinjha, Rab K.
A1  - Martinez, Elisabeth D.
A1  - Oppermann, Udo
A1  - Schofield, Christopher J.
A1  - Bountra, Chas
A1  - Kawamura, Akane
A1  - Blagg, Julian
A1  - Brennan, Paul E.
A1  - Rossanese, Olivia
A1  - Müller, Susanne
T1  - Assessing histone demethylase inhibitors in cells : lessons learned
T2  - Epigenetics & chromatin
N2  - Background: Histone lysine demethylases (KDMs) are of interest as drug targets due to their regulatory roles in chromatin organization and their tight associations with diseases including cancer and mental disorders. The first KDM inhibitors for KDM1 have entered clinical trials, and efforts are ongoing to develop potent, selective and cell-active ‘probe’ molecules for this target class. Robust cellular assays to assess the specific engagement of KDM inhibitors in cells as well as their cellular selectivity are a prerequisite for the development of high-quality inhibitors. Here we describe the use of a high-content cellular immunofluorescence assay as a method for demonstrating target engagement in cells.
Results: A panel of assays for the Jumonji C subfamily of KDMs was developed to encompass all major branches of the JmjC phylogenetic tree. These assays compare compound activity against wild-type KDM proteins to a catalytically inactive version of the KDM, in which residues involved in the active-site iron coordination are mutated to inactivate the enzyme activity. These mutants are critical for assessing the specific effect of KDM inhibitors and for revealing indirect effects on histone methylation status. The reported assays make use of ectopically expressed demethylases, and we demonstrate their use to profile several recently identified classes of KDM inhibitors and their structurally matched inactive controls. The generated data correlate well with assay results assessing endogenous KDM inhibition and confirm the selectivity observed in biochemical assays with isolated enzymes. We find that both cellular permeability and competition with 2-oxoglutarate affect the translation of biochemical activity to cellular inhibition.
Conclusions: High-content-based immunofluorescence assays have been established for eight KDM members of the 2-oxoglutarate-dependent oxygenases covering all major branches of the JmjC-KDM phylogenetic tree. The usage of both full-length, wild-type and catalytically inactive mutant ectopically expressed protein, as well as structure-matched inactive control compounds, allowed for detection of nonspecific effects causing changes in histone methylation as a result of compound toxicity. The developed assays offer a histone lysine demethylase family-wide tool for assessing KDM inhibitors for cell activity and on-target efficacy. In addition, the presented data may inform further studies to assess the cell-based activity of histone lysine methylation inhibitors.
KW  - Histone lysine demethylase
KW  - Chromatin
KW  - Immunofluorescence
KW  - Cell proliferation
KW  - Apoptosis
KW  - Toxicity
KW  - Epigenetics
KW  - 2-Oxoglutarate oxygenases
Y1  - 2017
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/43005
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-430059
UR  - https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333395
SN  - 1756-8935
N1  - © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
VL  - 10
IS  - Art. 9
SP  - 1
EP  - 17
PB  - BioMed Central
CY  - London
ER  -