Additional file 1: Figure S1. of H3K27me3 demethylases regulate in vitro chondrogenesis and chondrocyte activity in osteoarthritis

The results of treatment with GSK-5, the less active enantiomer of GSK-4, on MSCs undergoing chondrogenesis. (A-E) Assessment of gene expression at days 7 and 14 of MSC chondrogenesis to articular cartilage discs revealed following GSK-J4 and GSK-J5 treatment. (F) NANOG expression in monolayer MSCs treated for 24 h with GSK-J4 and GSK-J5. (G) PAI1 and JMJD3 expression were decreased in monolayer MSCs treated for 1 h with GSK-J4 and GSK-J5. (H) H3K27Me3 staining (green) in MSCs cultured for 24 h in control, chondrongenic or chondrogenic medium plus GSK-J4 or GSK-J5. Cell cytoskeleton/actin (phalloidin, red), nuclear staining (DAPI, blue). (TIFF 2818 kb

Additional file 3: Figure S2. of H3K27me3 demethylases regulate in vitro chondrogenesis and chondrocyte activity in osteoarthritis

siRNA validation qPCR on MSCs and validation chondrogenic gene expression following treatment with an additional siRNA against JMJD3 and UTX. MSC were treated with non-targeting siRNA control and two siRNA against JMJD3 (A) and UTX (B) at 2.5, 5 and 10 nM for 48 h. MTC = mock transfection control (no siRNA), NTC = no transfection control. (C) Targeting JMJD3 and UTX with additional siRNA. MSCs were pre-treated with siRNA_2 against JMJD3, UTX or non-targeting siRNA control prior to chondrogenic induction in transwell culture. RNA was extracted and cDNA synthesized at day 7 of chondrogenesis and expression of SOX9, ACAN COL2A1, COL10A1 and COL1A1 assessed by RT-qPCR (n = 4 patients, n =2 technical replicates per patient). Dashed line represents expression level following MSC treatment with non-targeting siRNA control. (TIFF 372 kb

Quantitative Analysis of Histone Demethylase Probes Using Fluorescence Polarization

We previously reported methylstat as a selective inhibitor of jumonji C domain-containing histone demethylases (JHDMs). Herein, we describe the synthesis of a fluorescent analogue of methylstat and its application as a tracer in fluorescence polarization assays. Using this format, we have evaluated the binding affinities of several known JHDM probes, as well as the native cofactor and substrate of JHDM1A. This fluorophore allowed a highly robust and miniaturized competition assay sufficient for high-throughput screening

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

SALMON: Solvent Accessibility, Ligand binding, and Mapping of ligand Orientation by NMR Spectroscopy

Quinone oxidoreductase 2 (NQO2) binds the prodrug tretazicar (also known as CB1954, 5-(aziridin-1-yl)-2,4-dinitrobenzamide), which exhibits a profound antitumor effect in human cancers when administered together with caricotamide. X-ray structure determination allowed for two possible orientations of the ligand. Here we describe a new NMR method, SALMON (solvent accessibility, ligand binding, and mapping of ligand orientation by NMR spectroscopy), based on waterLOGSY to determine the orientation of a ligand bound to a protein by mapping its solvent accessibility, which was used to unambiguously determine the orientation of CB1954 in NQO2

Human Lysine Demethylase JMJD2D (KDM4D); A Target Enabling Package

<p>There are 4 members of the Lysine Demethylase JMJD2 (KDM4) family. SGC Oxford has expressed, purified and crystallized the catalytic domains of JMJD2A, JMJD2B, JMJD2C and JMJD2D as part of the probe programme. Fragment screening and X-ray crystallography identified a large number of binders, some of which were progressed into a medicinal chemistry programme. Despite significant effort molecules with probe properties were not obtained. Consequently it has been decided to put the information generated into the public domain.</p

Human Lysine Demethylase JMJD2D (KDM4D); A Target Enabling Package

<p>There are 4 members of the Lysine Demethylase JMJD2 (KDM4) family. SGC Oxford has expressed, purified and crystallized the catalytic domains of JMJD2A, JMJD2B, JMJD2C and JMJD2D as part of the probe programme. Fragment screening and X-ray crystallography identified a large number of binders, some of which were progressed into a medicinal chemistry programme. Despite significant effort molecules with probe properties were not obtained. Consequently it has been decided to put the information generated into the public domain.</p

Human Lysine Demethylase JMJD1B (KDM3B); A Target Enabling Package

<p>There are 3 members of the Lysine Demethylase JMJD1 (KDM3) family, JMJD1A-C. SGC Oxford has expressed, purified and crystallized the catalytic domains of JMJD1A, JMJD1B and JMJD1C as part of the probe programme. Fragment screening and X-ray crystallography identified a large number of binders, some of which were progressed into a medicinal chemistry programme. Despite significant effort molecules with probe properties were not obtained. Consequently it has been decided to put the information generated into the public domain.</p

Selective Inhibitors of the JMJD2 Histone Demethylases: Combined Nondenaturing Mass Spectrometric Screening and Crystallographic Approaches

Ferrous ion and 2-oxoglutarate (2OG) oxygenases catalyze the demethylation of <i>N</i>^ε-methylated lysine residues in histones. Here we report studies on the inhibition of the JMJD2 subfamily of histone demethylases, employing binding analyses by nondenaturing mass spectrometry (MS), dynamic combinatorial chemistry coupled to MS, turnover assays, and crystallography. The results of initial binding and inhibition assays directed the production and analysis of a set of <i>N</i>-oxalyl-d<i>-</i>tyrosine derivatives to explore the extent of a subpocket at the JMJD2 active site. Some of the inhibitors were shown to be selective for JMJD2 over the hypoxia-inducible factor prolyl hydroxylase PHD2. A crystal structure of JMJD2A in complex with one of the potent inhibitors was obtained; modeling other inhibitors based on this structure predicts interactions that enable improved inhibition for some compounds

Discovery of NCT-501, a Potent and Selective Theophylline-Based Inhibitor of Aldehyde Dehydrogenase 1A1 (ALDH1A1)

Aldehyde dehydrogenases (ALDHs) metabolize reactive aldehydes and possess important physiological and toxicological functions in areas such as CNS, metabolic disorders, and cancers. Increased ALDH (e.g., ALDH1A1) gene expression and catalytic activity are vital biomarkers in a number of malignancies and cancer stem cells, highlighting the need for the identification and development of small molecule ALDH inhibitors. A new series of theophylline-based analogs as potent ALDH1A1 inhibitors is described. The optimization of hits identified from a quantitative high throughput screening (qHTS) campaign led to analogs with improved potency and early ADME properties. This chemotype exhibits highly selective inhibition against ALDH1A1 over ALDH3A1, ALDH1B1, and ALDH2 isozymes as well as other dehydrogenases such as HPGD and HSD17β4. Moreover, the pharmacokinetic evaluation of selected analog <b>64</b> (NCT-501) is also highlighted