Assessing histone demethylase inhibitors in cells: lessons learned

Abstract

BackgroundHistone 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.ResultsA 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.ConclusionsHigh-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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-017-0116-6) contains supplementary material, which is available to authorized users.

The histone demethylase JMJD2B regulates endothelial-to-mesenchymal transition

“…2 E and F). Furthermore, siJMJD2B-treated ECs showed preserved CDH5 protein expression and the typical CDH5 organization at adherens junctions after EndMT induction (SI Appendix, To address whether inhibition of the enzymatic activity of JMJD2B exhibits similar effects, we used the previously described JMJD2B inhibitor CCT365599 (39). Treatment with CCT365599 reduced TGF-β2-stimulated SM22 expression in a dose-dependent manner (SI Appendix, Fig.…”

Synthesis and Biological Evaluation of Tripartin, a Putative KDM4 Natural Product Inhibitor, and 1‐Dichloromethylinden‐1‐ol Analogues

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Design, Synthesis and Characterization of Covalent KDM5 Inhibitors

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