Kar Kheng Yeoh

The oncometabolite 2-hydroxyglutarate inhibits histone lysine demethylases.

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 Nε‐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.

Differential Sensitivity of Hypoxia Inducible Factor Hydroxylation Sites to Hypoxia and Hydroxylase Inhibitors

Hypoxia inducible factor (HIF) is regulated by dual pathways involving oxygen-dependent prolyl and asparaginyl hydroxylation of its α-subunits. Prolyl hydroxylation at two sites within a central degradation domain promotes association of HIF-α with the von Hippel-Lindau ubiquitin E3 ligase and destruction by the ubiquitin-proteasome pathways. Asparaginyl hydroxylation blocks the recruitment of p300/CBP co-activators to a C-terminal activation domain in HIF-α. These hydroxylations are catalyzed by members of the Fe(II) and 2-oxoglutarate (2-OG) oxygenase family. Activity of the enzymes is suppressed by hypoxia, increasing both the abundance and activity of the HIF transcriptional complex. We have used hydroxy residue-specific antibodies to compare and contrast the regulation of each site of prolyl hydroxylation (Pro402, Pro564) with that of asparaginyl hydroxylation (Asn803) in human HIF-1α. Our findings reveal striking differences in the sensitivity of these hydroxylations to hypoxia and to different inhibitor types of 2-OG oxygenases. Hydroxylation at the three sites in endogenous human HIF-1α proteins was suppressed by hypoxia in the order Pro402 > Pro564 > Asn803. In contrast to some predictions from in vitro studies, prolyl hydroxylation was substantially more sensitive than asparaginyl hydroxylation to inhibition by iron chelators and transition metal ions; studies of a range of different small molecule 2-OG analogues demonstrated the feasibility of selectively inhibiting either prolyl or asparaginyl hydroxylation within cells.

Investigating the Dependence of the Hypoxia-Inducible Factor Hydroxylases (Factor Inhibiting HIF and Prolyl Hydroxylase Domain 2) on Ascorbate and Other Reducing Agents

The HIF (hypoxia-inducible factor) hydroxylases [PHDs or EGLNs (prolyl hydroxylases), which in humans are PHD isoforms 1-3, and FIH (factor inhibiting HIF)] regulate HIF levels and activity. These enzymes are Fe(II)/2-oxoglutarate-dependent oxygenases, many of which are stimulated by ascorbate. We have investigated the ascorbate dependence of PHD2-catalysed hydroxylation of two prolyl hydroxylation sites in human HIF-1alpha, and of FIH-catalysed hydroxylation of asparaginyl hydroxylation sites in HIF-1alpha and in a consensus ankyrin repeat domain peptide. The initial rate and extent of hydroxylation was increased in the presence of ascorbate for each of these reactions. When ascorbate was replaced with structural analogues, the results revealed that the ascorbate side chain was not important in its contribution to HIF hydroxylase catalysis, whereas modifications to the ene-diol portion of the molecule negated the ability to promote hydroxylation. We investigated whether alternative reducing agents (glutathione and dithiothreitol) could be used to promote HIF hydroxylase activity, and found partial stimulation of hydroxylation in an apparently enzyme- and substrate-specific manner. The results raise the possibility of developing reducing agents targeted to specific HIF hydroxylase-catalysed reactions.

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

Structural basis for inhibition of the fat mass and obesity associated protein (FTO)

The fat mass and obesity associated protein (FTO) is a potential target for anti-obesity medicines. FTO is a 2-oxoglutarate (2OG)-dependent N-methyl nucleic acid demethylase that acts on substrates including 3-methylthymidine, 3-methyluracil, and 6-methyladenine. To identify FTO inhibitors, we screened a set of 2OG analogues and related compounds using differential scanning fluorometry- and liquid chromatography-based assays. The results revealed sets of both cyclic and acyclic 2OG analogues that are FTO inhibitors. Identified inhibitors include small molecules that have been used in clinical studies for the inhibition of other 2OG oxygenases. Crystallographic analyses reveal inhibition by 2OG cosubstrate or primary substrate competitors as well as compounds that bind across both cosubstrate and primary substrate binding sites. The results will aid the development of more potent and selective FTO inhibitors.

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.

Identification of valid housekeeping genes for quantitative RT-PCR analysis of cardiosphere-derived cells preconditioned under hypoxia or with prolyl-4-hydroxylase inhibitors

Infarction irreversibly damages the heart, with formation of an akinetic scar that may lead to heart failure. Endogenous cardiac stem cells (CSCs) are a promising candidate cell source for restoring lost tissue and thereby preventing heart failure. CSCs may be isolated in vitro, via the formation of cardiospheres, to give cardiosphere-derived cells (CDCs). Although qRT-PCR analyses of CDCs have been performed, no justification for the selection of the housekeeping gene has been published. Here, we evaluated the most suitable housekeeping gene for RNA expression analysis in CDCs cultured under normoxia, hypoxia or with prolyl-4-hydroxylase inhibitors (PHDIs), from both neonatal and adult rats, to determine the effects of ageing and different culture conditions on the stability of the housekeeping gene for CDCs. Six candidate housekeeping genes, [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (Actb), hypoxanthine phosphoribosyltransferase 1 (HPRT-1), beta-2-microtubulin (β2M), 60S acidic ribosomal protein large P1 (RPLP-1) and TATA box binding protein (Tbp)] were evaluated in this study. Analysis using geNorm and NormFinder revealed that GAPDH was the most constant housekeeping gene among all genes tested under normoxia for both neonatal and adult CDCs, whereas Actb was the most stable housekeeping gene under hypoxia. For the PHDI-treated CDCs, overall, GADPH, Actb and β2M were more consistently expressed, whereas HPRT-1, RPLP-1 and Tbp showed unstable expression. The ranking for β2M, HPRT-1 and RPLP-1 stability was different for neonatal and adult cells, indicating that expression of these genes was age-dependent. Lastly, independent of age or culture conditions, Tbp was the least stable housekeeping gene. In conclusion, a combination of Actb and GADPH gave the most reliable normalization for comparative analyses of gene transcription in neonatal and adult rat CDCs preconditioned by hypoxia or PHDIs.

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.

Application of a Proteolysis/Mass Spectrometry Method for Investigating the Effects of Inhibitors on Hydroxylase Structure

Limited proteolysis coupled to matrix-assisted laser desorption/ionization (MALDI) mass spectrometric analyses can be used to screen for compounds that alter protein structure by monitoring stabilizing/destabilizing effects with respect to the rate and nature of proteolysis. When applied to prolyl hydroxylase 2, a key enzyme involved in human oxygen sensing, the method efficiently revealed differential effects on proteolytic stability for structurally similar compounds and for different substrates.