Figures S1 and S2; Table S1 from Inhibitors of both the <i>N</i>-methyl lysyl- and arginyl- demethylase activities of the JmjC oxygenases

Time course assays using LC-MS showing the extent of KDM (A) or RDM (B) demethylation.; Comparison of the KDM and RDM activities of KDM4E.; Kinetic parameters (KM, kcat and kcat/ KM)) for substrates and 2O

Tetracyanoresorcin[4]arene selectively recognises trimethyllysine and inhibits its enzyme-catalysed demethylation

<p><i>N</i>ε-methylation of lysine within proteins is a critical biological process that, among other roles, is involved in the control of gene expression. Compounds that recognise <i>N</i>ε-methylated lysine may therefore be useful probes for the study of the associated biological mechanisms and have therapeutic potential. Here, we show that tetracyanoresorcin[4]arene (<b><i>1</i></b>) selectively recognises <i>N</i>ε-trimethyllysine and binds to <i>N</i>ε-trimethyllysine within the context of a short peptide. Its binding properties compare favourably to a previously characterised <i>N</i>ε-trimethyllysine binder, <i>p</i>-sulfonatocalix[4]arene (<b><i>2</i></b>). We also show that both <b><i>1</i></b> and <b><i>2</i></b> inhibit the demethylation of <i>N</i>ε-trimethyllysine within a histone-derived peptide by the histone demethylase KDM4A.</p

The Activity of JmjC Histone Lysine Demethylase KDM4A is Highly Sensitive to Oxygen Concentrations

The JmjC histone lysine demethylases (KDMs) are epigenetic regulators involved in the removal of methyl groups from post-translationally modified lysyl residues within histone tails, modulating gene transcription. These enzymes require molecular oxygen for catalytic activity and, as 2-oxoglutarate (2OG)-dependent oxygenases, are related to the cellular oxygen sensing HIF hydroxylases PHD2 and FIH. Recent studies have indicated that the activity of some KDMs, including the pseudogene-encoded KDM4E, may be sensitive to changing oxygen concentrations. Here, we report detailed analysis of the effect of oxygen availability on the activity of the KDM4 subfamily member KDM4A, importantly demonstrating a high level of O₂ sensitivity both with isolated protein and in cells. Kinetic analysis of the recombinant enzyme revealed a high <i>K</i>_M^app(O₂) of 173 ± 23 μM, indicating that the activity of the enzyme is able to respond sensitively to a reduction in oxygen concentration. Furthermore, immunofluorescence experiments in U2OS cells conditionally overexpressing KDM4A showed that the cellular activity of KDM4A against its primary substrate, H3K9me3, displayed a graded response to depleting oxygen concentrations in line with the data obtained using isolated protein. These results suggest that KDM4A possesses the potential to act as an oxygen sensor in the context of chromatin modifications, with possible implications for epigenetic regulation in hypoxic disease states. Importantly, this correlation between the oxygen sensitivity of the catalytic activity of KDM4A in biochemical and cellular assays demonstrates the utility of biochemical studies in understanding the factors contributing to the diverse biological functions and varied activity of the 2OG oxygenases

Formaldehyde quantification using ampicillin is not selective

Formaldehyde (HCHO) is a simple and highly reactive human metabolite but its biochemistry is poorly defined. A limiting factor in HCHO research is lack of validated quantification methods for HCHO relevant to biological samples. We describe spectroscopic studies on a reported fluorescence-based HCHO detection method involving its reaction with ampicillin. The results validate the structure and fluorescence properties of the HCHO-ampicillin reaction product. However, the same adduct is observed after reaction of ampicillin with glyoxylate. Related fluorophores were formed with other biologically relevant carbonyl compounds. Overall, our studies suggest the ampicillin method is not reliable for selective detection and quantification of HCHO in biological samples

Arginine demethylation is catalysed by a subset of JmjC histone lysine demethylases.

While the oxygen-dependent reversal of lysine N(ɛ)-methylation is well established, the existence of bona fide N(ω)-methylarginine demethylases (RDMs) is controversial. Lysine demethylation, as catalysed by two families of lysine demethylases (the flavin-dependent KDM1 enzymes and the 2-oxoglutarate- and oxygen-dependent JmjC KDMs, respectively), proceeds via oxidation of the N-methyl group, resulting in the release of formaldehyde. Here we report detailed biochemical studies clearly demonstrating that, in purified form, a subset of JmjC KDMs can also act as RDMs, both on histone and non-histone fragments, resulting in formaldehyde release. RDM catalysis is studied using peptides of wild-type sequences known to be arginine-methylated and sequences in which the KDM's methylated target lysine is substituted for a methylated arginine. Notably, the preferred sequence requirements for KDM and RDM activity vary even with the same JmjC enzymes. The demonstration of RDM activity by isolated JmjC enzymes will stimulate efforts to detect biologically relevant RDM activity

Studies on the catalytic domains of multiple JmjC oxygenases using peptide substrates

<div><p>The JmjC-domain-containing 2-oxoglutarate-dependent oxygenases catalyze protein hydroxylation and <i>N^ϵ</i>-methyllysine demethylation via hydroxylation. A subgroup of this family, the JmjC lysine demethylases (JmjC KDMs) are involved in histone modifications at multiple sites. There are conflicting reports as to the substrate selectivity of some JmjC oxygenases with respect to KDM activities. In this study, a panel of modified histone H3 peptides was tested for demethylation against 15 human JmjC-domain-containing proteins. The results largely confirmed known <i>N^ϵ</i>-methyllysine substrates. However, the purified KDM4 catalytic domains showed greater substrate promiscuity than previously reported (i.e., KDM4A was observed to catalyze demethylation at H3K27 as well as H3K9/K36). Crystallographic analyses revealed that the <i>N^ϵ-</i>methyllysine of an H3K27me3 peptide binds similarly to <i>N^ϵ-</i>methyllysines of H3K9me3/H3K36me3 with KDM4A. A subgroup of JmjC proteins known to catalyze hydroxylation did not display demethylation activity. Overall, the results reveal that the catalytic domains of the KDM4 enzymes may be less selective than previously identified. They also draw a distinction between the <i>N^ϵ</i>-methyllysine demethylation and hydroxylation activities within the JmjC subfamily. These results will be of use to those working on functional studies of the JmjC enzymes.</p></div

Investigations on Small Molecule Inhibitors Targeting the Histone H3K4 tri-methyllysine Binding PHD-finger of JmjC Histone Demethylases

Plant homeodomain (PHD) containing proteins are important epigenetic regulators and are of interest as potential drug targets. Inspired by the amiodarone derivatives reported to inhibit the PHD finger 3 of KDM5A (KDM5A(PHD3)), a set of compounds were synthesised. Amiodarone and its derivatives were observed to weakly disrupt the interactions of a histone H3K4me3 peptide with KDM5A(PHD3). Selected amiodarone derivatives inhibited catalysis of KDM5A, but in a PHD-finger independent manner. Amiodarone derivatives also bind to H3K4me3-binding PHD-fingers from the KDM7 subfamily. Further work is required to develop potent and selective PHD finger inhibitors

Reporter Ligand NMR Screening Method for 2‑Oxoglutarate Oxygenase Inhibitors

The human 2-oxoglutarate (2OG) dependent oxygenases belong to a family of structurally related enzymes that play important roles in many biological processes. We report that competition-based NMR methods, using 2OG as a reporter ligand, can be used for quantitative and site-specific screening of ligand binding to 2OG oxygenases. The method was demonstrated using hypoxia inducible factor hydroxylases and histone demethylases, and <i>K</i>_D values were determined for inhibitors that compete with 2OG at the metal center. This technique is also useful as a screening or validation tool for inhibitor discovery, as exemplified by work with protein-directed dynamic combinatorial chemistry

Human histone demethylase KDM6B can catalyse sequential oxidations

Jumonji domain‐containing demethylases (JmjC‐KDMs) catalyse demethylation of Nε ‐methylated lysines on histones and play important rolesin gene regulation. We report selectivity studies on KDM6B (JMJD3), a disease‐relevant JmjC‐KDM, using synthetic lysine analogues. The results unexpectedly reveal that KDM6B accepts multiple Nε ‐alkylated lysine analogues, forming alcohol, aldehyde and carboxylic acid products

The inhibitory activity of compound 1 and its analogues.^a

a<p>The NAT activity was measured by the NAT-inhibition assay using 150 µM HLZ and 120 µM Ac-CoA as substrates. The level of enzyme inhibition was measured in the presence of 50 µM inhibitor and compared to the un-inhibited control. The antimycobacterial activity against <i>M. bovis</i> BCG and <i>M. tuberculosis</i> were determined. Inhibition curves were obtained by non-linear fitting of the % inhibition and the inhibitor concentration (µM) using the Log(inhibitor) vs. response module of GraphPad Prism 5.0. The time-dependent assay <i>k_obs</i> values were obtained from the slope of the semilogarithmic plots of the residual activity vs incubation time at 11.9 µM, except for <b>3</b> (5.9 µM). The results are presented as the mean ± S.D. of triplicate measurements at 24°C. t_1/2 is the apparent inactivation half-life calculated from <i>k_obs</i> (t_1/2 = 0.693/k_obs). ND is not determined.</p>b<p>See Methods for further experimental details.</p