Small-Molecule-Based Inhibition of Histone Demethylation in Cells Assessed by Quantitative Mass Spectrometry

Abstract

Post-translational modifications on histones are an important mechanism for the regulation of gene expression and are involved in all aspects of cell growth and differentiation as well as pathological processes including neurodegeneration, autoimmunity, and cancer. A major challenge within the chromatin field is to develop methods for the quantitative analysis of histone modifications. Here we report a mass spectrometry (MS) approach based on ultra-performance high/low collision switching (UPLC-MS E ) to monitor histone modifications in cells. This approach is exemplified by the analysis of trimethylated lysine-9 levels in histone H3 following a simple chemical derivatization procedure with deuterated acetic anhydride. This method was used to study the inhibition of histone demethylases with pyridine-2,4-dicarboxylic acid (PDCA) derivatives that inhibit histone demethylases in cells. Our results show that the PDCA-dimethyl ester inhibits JMJD2A catalyzed demethylation of lysine-9 on histone H3 in human HEK 293T cells. Demethylase inhibition, as observed by MS analyses, was supported by immunoblotting with modification-specific antibodies. The results demonstrate that PDCA derived small molecules are cell permeable demethylase inhibitors, and reveal that quantitative MS is a useful tool for measuring post-translational histone modifications in cells.

A novel effect of DMOG on cell metabolism: direct inhibition of mitochondrial function precedes HIF target gene expression

“…These results suggested that in vivo DMOG administration can increase O 2 availability in inflamed colon tissue, and improved tissue oxygenation may contribute to the therapeutic effects of DMOG shown previously in mice with DSS-induced colitis [20,63]. demethylases) point towards multifunctional properties of this compound [20][21][22][23][24][25][26][27]. Inhibition of O 2 consumption by cells treated with DMOG has been reported previously [37][38][39].…”

“…As a PHD inhibitor, DMOG activates HIF-1 and NF-κB, playing a protective role in experimental model of colitis and diarrhoea [20,21], suppresses induced TNF-α expression [22] and abrogates TNF-α-induced intestinal epithelial damage [23]. On the other hand, DMOG was also reported to inhibit JHDM histone demethylases in 293T cells [24], similar to NOG [24][25][26]. Recently, DMOG was shown to activate AMPK signalling [27], which orchestrates global metabolic adaptation of cells to energy crisis and oxidative stress [28].…”

Quantitative Proteomic Analysis of Histone Modifications

“…Deuterated acetic acid was one of the earliest reagents used to derivatize histones. 136 The derivatization adds a deuterated acetyl group to originally unmodified lysine residues. Because the acetyl groups contain deuteron instead of hydrogen, which causes a 3-Da mass shift, they therefore can be distinguished from endogenous acetylated lysine residue.…”

KDM4B as a Target for Prostate Cancer: Structural Analysis and Selective Inhibition by a Novel Inhibitor

“…9 Yet, as a prodrug, the cytotoxic IC 50 of compound 2 (PD2) is in the millimolar range in cultured cells because of its poor cell-penetrating ability, 10 whereas 5-carboxy-8HQ, with a potent inhibitory effect on KDM4E in vitro (IC 50 = 0.2 μM), exhibits a relatively high cytotoxic IC 50 in HeLa cells (86.5 μM). 9 In this investigation, we determined the KDM4B· 2 ·H3K9me3 ternary complex structure and applied a structure-guided strategy to identify a selective inhibitor, 1,5-bis[( E )-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene (NSC636819), toward KDM4A and KDM4B.…”