Mutations in isocitrate dehydrogenases (IDHs) have a gain‐of‐function effect leading to R (−)‐2‐hydroxyglutarate ( R‐ 2HG) accumulation. By using biochemical, structural and cellular assays, we show that either or both R ‐ and S ‐2HG inhibit 2‐oxoglutarate (2OG)‐dependent oxygenases with varying potencies. Half‐maximal inhibitory concentration (IC50) values for the R ‐form of 2HG varied from approximately 25 μM for the histone N ε‐lysine demethylase JMJD2A to more than 5 mM for the hypoxia‐inducible factor (HIF) prolyl hydroxylase. The results indicate that candidate oncogenic pathways in IDH‐associated malignancy should include those that are regulated by other 2OG oxygenases than HIF hydroxylases, in particular those involving the regulation of histone methylation.

Protein tyrosine phosphatases (PTPs) play a critical role in regulating cellular functions by selectively dephosphorylating their substrates. Here we present 22 human PTP crystal structures that, together with prior structural knowledge, enable a comprehensive analysis of the classical PTP family. Despite their largely conserved fold, surface properties of PTPs are strikingly diverse. A potential secondary substrate-binding pocket is frequently found in phosphatases, and this has implications for both substrate recognition and development of selective inhibitors. Structural comparison identified four diverse catalytic loop (WPD) conformations and suggested a mechanism for loop closure. Enzymatic assays revealed vast differences in PTP catalytic activity and identified PTPD1, PTPD2, and HDPTP as catalytically inert protein phosphatases. We propose a "head-to-toe" dimerization model for RPTPgamma/zeta that is distinct from the "inhibitory wedge" model and that provides a molecular basis for inhibitory regulation. This phosphatome resource gives an expanded insight into intrafamily PTP diversity, catalytic activity, substrate recognition, and autoregulatory self-association.

2-Oxoglutarate (2OG) dependent oxygenases are ubiquitous iron enzymes that couple substrate oxidation to the conversion of 2OG to succinate and carbon dioxide. In humans their roles include collagen biosynthesis, fatty acid metabolism, DNA repair, RNA and chromatin modifications, and hypoxic sensing. Commercial applications of 2OG oxygenase inhibitors began with plant growth retardants, and now extend to a clinically used pharmaceutical compound for cardioprotection. Several 2OG oxygenases are now being targeted for therapeutic intervention for diseases including anaemia, inflammation and cancer. In this critical review, we describe studies on the inhibition of 2OG oxygenases, focusing on small molecules, and discuss the potential of 2OG oxygenases as therapeutic targets (295 references).

There is a growing recognition of the importance of protein kinases in the control of alternative splicing. To define the underlying regulatory mechanisms, highly selective inhibitors are needed. Here, we report the discovery and characterization of the dichloroindolyl enaminonitrile KH-CB19, a potent and highly specific inhibitor of the CDC2-like kinase isoforms 1 and 4 (CLK1/CLK4). Cocrystal structures of KH-CB19 with CLK1 and CLK3 revealed a non-ATP mimetic binding mode, conformational changes in helix αC and the phosphate binding loop and halogen bonding to the kinase hinge region. KH-CB19 effectively suppressed phosphorylation of SR (serine/arginine) proteins in cells, consistent with its expected mechanism of action. Chemical inhibition of CLK1/CLK4 generated a unique pattern of splicing factor dephosphorylation and had at low nM concentration a profound effect on splicing of the two tissue factor isoforms flTF (full-length TF) and asHTF (alternatively spliced human TF).

Small molecule modulators of epigenetic processes are currently sought as basic probes for biochemical mechanisms, and as starting points for development of therapeutic agents. N^ε-Methylation of lysine residues on histone tails is one of a number of post-translational modifications that together enable transcriptional regulation. Histone lysine demethylases antagonize the action of histone methyltransferases in a site- and methylation state-specific manner. N^ε-Methyllysine demethylases that use 2-oxoglutarate as co-factor are associated with diverse human diseases, including cancer, inflammation and X-linked mental retardation; they are proposed as targets for the therapeutic modulation of transcription. There are few reports on the identification of templates that are amenable to development as potent inhibitors in vivo and large diverse collections have yet to be exploited for the discovery of demethylase inhibitors. High-throughput screening of a ∼236,000-member collection of diverse molecules arrayed as dilution series was used to identify inhibitors of the JMJD2 (KDM4) family of 2-oxoglutarate-dependent histone demethylases. Initial screening hits were prioritized by a combination of cheminformatics, counterscreening using a coupled assay enzyme, and orthogonal confirmatory detection of inhibition by mass spectrometric assays. Follow-up studies were carried out on one of the series identified, 8-hydroxyquinolines, which were shown by crystallographic analyses to inhibit by binding to the active site Fe(II) and to modulate demethylation at the H3K9 locus in a cell-based assay. These studies demonstrate that diverse compound screening can yield novel inhibitors of 2OG dependent histone demethylases and provide starting points for the development of potent and selective agents to interrogate epigenetic regulation.

2-Oxoglutarate and iron dependent oxygenases are therapeutic targets for human diseases. Using a representative 2OG oxygenase panel, we compare the inhibitory activities of 5-carboxy-8-hydroxyquinoline (IOX1) and 4-carboxy-8-hydroxyquinoline (4C8HQ) with that of two other commonly used 2OG oxygenase inhibitors, N-oxalylglycine (NOG) and 2,4-pyridinedicarboxylic acid (2,4-PDCA). The results reveal that IOX1 has a broad spectrum of activity, as demonstrated by the inhibition of transcription factor hydroxylases, representatives of all 2OG dependent histone demethylase subfamilies, nucleic acid demethylases and γ-butyrobetaine hydroxylase. Cellular assays show that, unlike NOG and 2,4-PDCA, IOX1 is active against both cytosolic and nuclear 2OG oxygenases without ester derivatisation. Unexpectedly, crystallographic studies on these oxygenases demonstrate that IOX1, but not 4C8HQ, can cause translocation of the active site metal, revealing a rare example of protein ligand-induced metal movement.