Mun Chiang Chan

Plant Growth Regulator Daminozide Is a Selective Inhibitor of Human KDM2/7 Histone Demethylases

The JmjC oxygenases catalyze the N-demethylation of N(ε)-methyl lysine residues in histones and are current therapeutic targets. A set of human 2-oxoglutarate analogues were screened using a unified assay platform for JmjC demethylases and related oxygenases. Results led to the finding that daminozide (N-(dimethylamino)succinamic acid, 160 Da), a plant growth regulator, selectively inhibits the KDM2/7 JmjC subfamily. Kinetic and crystallographic studies reveal that daminozide chelates the active site metal via its hydrazide carbonyl and dimethylamino groups.

Dynamic Combinatorial Chemistry Employing Boronic Acids/Boronate Esters Leads to Potent Oxygenase Inhibitors

The application of dynamic reactions is a promising approach for the discovery of small-molecule ligands for proteins. To date, however, this method is limited by the few appropriate reactions and the techniques used for the analysis of protein– ligand complexes. “Dynamic” functional group interconvertions that have been employed include the conversion of thiols to disulfides, the aldol reaction, and the addition of nucleophiles to ketones and aldehydes. The reaction of boronic acids with diols to form boronate esters is attractive for dynamic-library formation, because it is reversible in aqueous solution in a pH-dependent manner. The dynamic boronic acid/boronate ester system has been used to form supramolecular switches, some of which have been used for sugar detection. 5] However, this system has not been used for the identification of protein ligands. Proof of principle work with proteases, which react reversibly with boronic acids, suggests that boronic acid/boronate ester systems might be useful for the identification of enzyme inhibitors. One issue with the application of reversible reactions for ligand identification is the need to analyze labile complexes that are derived from mixtures. High-resolution techniques, such as NMR spectroscopy and X-ray crystallography, are applicable, but these are time-consuming. Our research group and that of Poulsen, have used non-denaturing protein mass spectrometry to identify protein–ligand complexes formed from equilibrating mixtures of thiols/disulfides and aldehydes/hydrazones. The dynamic-combinatorial mass spectrometry (DCMS) technique has the advantages of being efficient and providing information on mass shifts, which can be used for assigning structures to the ligands that bind preferentially. Herein we demonstrate that boronic acid/boronate ester dynamic systems coupled with protein mass spectrometry analysis are useful for the identification of protein inhibitors (Scheme 1). Our target model enzyme was prolyl hydroxylase domain isoform 2 (PHD2), which is a Fe and 2-oxoglutarate (2OG) oxygenase that regulates the human hypoxic response. PHD2 inhibition is of therapeutic interest for the treatment of anemia and ischemia-related diseases. DCMS experiments were carried out using “support ligands” 2 and 3 (Scheme 2), which were designed to participate in Fe chelation in the active site and, through the incorporation of a boronic acid moiety, participate in boronate ester exchange. We selected the 2-(picolinamido)acetic acid scaffold because, based on crystal structures of PHD2, it is predicted to fit into the active site through its chelation with Fe. The low potency of 2-(picolinamido)acetic acid (IC50> 1 mm) enabled the effect of boronate ester substitution to be monitored. Modeling studies suggested that whereas the boronic acid group in support ligand 2 would fit into the active-site subpocket, that of 3 would clash with the active-site wall. Hence, it was envisaged that the reactivity of 3 might serve as a control to investigate possible non-specific binding. The analysis of mixtures of 2 or 3 with PHD2·Fe through the use of non-denaturing ESI-MS led to the observation of a new peak at 27 887 Da (187 2 Da shift), corresponding to a small molecule/protein adduct, in which the OH groups of the boronic acids moiety are cleaved. We have previously observed, through the use of non-denaturing ESI-MS, analogous apparent fragmentation of boronic acids complexed with other enzymes. Notably, the mixture of boronate ester 4 and PHD2·Fe gave the same mass shift (187 2 Da) as that observed with 2 and 3 at a cone voltage of 80 V. However, when a lower cone voltage was used (30 V), the mass shift corresponding to an adduct of 4 with the protein, without fragmentation, was apparent (358 2 Da), demonstrating that boronate ester formation can be observed when sufficiently mild ionization is used. Both 2 and 3 compete with the 2OG analogue N-oxalylglycine (NOG) for the 2OG binding site of PHD2. To ensure that boronate ester formation involving 2 and 3 was favorable under the conditions used (NH4OAc [*] M. Demetriades, I. K. H. Leung, Dr. R. Chowdhury, M. C. Chan, Dr. M. A. McDonough, Dr. K. K. Yeoh, Dr. T. D. W. Claridge, Prof. C. J. Schofield Chemistry Research Laboratory, University of Oxford 12 Mansfield Road, Oxford, OX1 3TA (UK) E-mail: christopher.schofield@chem.ox.ac.uk

5-Carboxy-8-hydroxyquinoline is a Broad Spectrum 2-Oxoglutarate Oxygenase Inhibitor which Causes Iron Translocation.

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.

Linking of 2‐Oxoglutarate and Substrate Binding Sites Enables Potent and Highly Selective Inhibition of JmjC Histone Demethylases

N-Methylation of histone lysine residues is an “epigenetic modification” that can be either transcriptionally activating or deactivating, depending on the position of the lysine, its methylation state and the presence of other modifications. The largest family of demethylases, the JmjC enzymes, employ 2-oxoglutarate (2OG) as a cosubstrate (Figure 1a). 3] Some JmjC demethylases are targeted for cancer treatment and inflammatory diseases. There are 5 JmjC demethylase subfamilies, targeting histone lysines (H3K = histone 3 lysine-residue) including at H3K4, H3K9, H3K27, and H3K36 (Figure 1b). The factors determining JmjC selectivities are emerging, and involve both catalytic and non-catalytic domains. Although there are reports of JmjC inhibitors, to date there are no reported compounds that are selective for subfamilies/isoforms. Here we report that a strategy involving binding to both the 2OG and substrate binding sites leads to selective and potent inhibitors of the JMJD2 subfamily. There are predicted to be four human JMJD2 enzymes (A to D) and a “pseudogene” product JMJD2E. JMJD2A–C accept both H3K9me3/me2 and H3K36me3/me2, whereas JMJD2D–E only accept H3K9me3/me2. Most, if not all, reported JmjC inhibitors are 2OG analogues with limited or undetermined selectivity, and with the exception of some peptide-based inhibitors, have not, at least rationally, exploited the histone binding pocket. 21] We reasoned that “two-component inhibitors” that bind to 2OG and histone

Selective small molecule probes for the hypoxia inducible factor (HIF) prolyl hydroxylases.

The hypoxia inducible factor (HIF) system is central to the signaling of low oxygen (hypoxia) in animals. The levels of HIF-α isoforms are regulated in an oxygen-dependent manner by the activity of the HIF prolyl-hydroxylases (PHD or EGLN enzymes), which are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases. Here, we describe biochemical, crystallographic, cellular profiling, and animal studies on PHD inhibitors including selectivity studies using a representative set of human 2OG oxygenases. We identify suitable probe compounds for use in studies on the functional effects of PHD inhibition in cells and in animals.

Non-enzymatic chemistry enables 2-hydroxyglutarate-mediated activation of 2-oxoglutarate oxygenases

Accumulation of (R)-2-hydroxyglutarate in cells results from mutations to isocitrate dehydrogenase that correlate with cancer. A recent study reports that (R)-, but not (S)-2-hydroxyglutarate, acts as a co-substrate for the hypoxia-inducible factor prolyl hydroxylases via enzyme-catalysed oxidation to 2-oxoglutarate. Here we investigate the mechanism of 2-hydroxyglutarate-enabled activation of 2-oxoglutarate oxygenases, including prolyl hydroxylase domain 2, the most important human prolyl hydroxylase isoform. We observe that 2-hydroxyglutarate-enabled catalysis by prolyl hydroxylase domain 2 is not enantiomer-specific and is stimulated by ferrous/ferric ion and reducing agents including L-ascorbate. The results reveal that 2-hydroxyglutarate is oxidized to 2-oxoglutarate non-enzymatically, likely via iron-mediated Fenton-chemistry, at levels supporting in vitro catalysis by 2-oxoglutarate oxygenases. Succinic semialdehyde and succinate are also identified as products of 2-hydroxyglutarate oxidation. Overall, the results rationalize the reported effects of 2-hydroxyglutarate on catalysis by prolyl hydroxylases in vitro and suggest that non-enzymatic 2-hydroxyglutarate oxidation may be of biological interest.

Dynamic combinatorial mass spectrometry leads to inhibitors of a 2-oxoglutarate-dependent nucleic acid demethylase.

2-Oxoglutarate-dependent nucleic acid demethylases are of biological interest because of their roles in nucleic acid repair and modification. Although some of these enzymes are linked to physiology, their regulatory roles are unclear. Hence, there is a desire to develop selective inhibitors for them; we report studies on AlkB, which reveal it as being amenable to selective inhibition by small molecules. Dynamic combinatorial chemistry linked to mass spectrometric analyses (DCMS) led to the identification of lead compounds, one of which was analyzed by crystallography. Subsequent structure-guided studies led to the identification of inhibitors of improved potency, some of which were shown to be selective over two other 2OG oxygenases. The work further validates the use of the DCMS method and will help to enable the development of inhibitors of nucleic acid modifying 2OG oxygenases both for use as functional probes and, in the longer term, for potential therapeutic use.

A Cell‐Permeable Ester Derivative of the JmjC Histone Demethylase Inhibitor IOX1

The 2‐oxoglutarate (2OG)‐dependent Jumonji C domain (JmjC) family is the largest family of histone lysine demethylases. There is interest in developing small‐molecule probes that modulate JmjC activity to investigate their biological roles. 5‐Carboxy‐8‐hydroxyquinoline (IOX1) is the most potent broad‐spectrum inhibitor of 2OG oxygenases, including the JmjC demethylases, reported to date; however, it suffers from low cell permeability. Here, we describe structure–activity relationship studies leading to the discovery of an n‐octyl ester form of IOX1 with improved cellular potency (EC50 value of 100 to 4 μM). These findings are supported by in vitro inhibition and selectivity studies, docking studies, activity versus toxicity analysis in cell cultures, and intracellular uptake measurements. The n‐octyl ester was found to have improved cell permeability; it was found to inhibit some JmjC demethylases in its intact ester form and to be more selective than IOX1. The n‐octyl ester of IOX1 should find utility as a starting point for the development of JmjC inhibitors and as a use as a cell‐permeable tool compound for studies investigating the roles of 2OG oxygenases in epigenetic regulation.

An experimentally validated network of nine haematopoietic transcription factors reveals mechanisms of cell state stability

Transcription factor (TF) networks determine cell-type identity by establishing and maintaining lineage-specific expression profiles, yet reconstruction of mammalian regulatory network models has been hampered by a lack of comprehensive functional validation of regulatory interactions. Here, we report comprehensive ChIP-Seq, transgenic and reporter gene experimental data that have allowed us to construct an experimentally validated regulatory network model for haematopoietic stem/progenitor cells (HSPCs). Model simulation coupled with subsequent experimental validation using single cell expression profiling revealed potential mechanisms for cell state stabilisation, and also how a leukaemogenic TF fusion protein perturbs key HSPC regulators. The approach presented here should help to improve our understanding of both normal physiological and disease processes. DOI: http://dx.doi.org/10.7554/eLife.11469.001

Betti reaction enables efficient synthesis of 8-hydroxyquinoline inhibitors of 2-oxoglutarate oxygenases.

There is interest in developing potent, selective, and cell-permeable inhibitors of human ferrous iron and 2-oxoglutarate (2OG) oxygenases for use in functional and target validation studies. The 3-component Betti reaction enables efficient one-step C-7 functionalisation of modified 8-hydroxyquinolines (8HQs) to produce cell-active inhibitors of KDM4 histone demethylases and other 2OG oxygenases; the work exemplifies how a template-based metallo-enzyme inhibitor approach can be used to give biologically active compounds.