Ivanhoe K. H. Leung

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.

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

A Photoreactive Small-Molecule Probe for 2-Oxoglutarate Oxygenases

2-oxoglutarate (2-OG)-dependent oxygenases have diverse roles in human biology. The inhibition of several 2-OG oxygenases is being targeted for therapeutic intervention, including for cancer, anemia, and ischemic diseases. We report a small-molecule probe for 2-OG oxygenases that employs a hydroxyquinoline template coupled to a photoactivable crosslinking group and an affinity-purification tag. Following studies with recombinant proteins, the probe was shown to crosslink to 2-OG oxygenases in human crude cell extracts, including to proteins at endogenous levels. This approach is useful for inhibitor profiling, as demonstrated by crosslinking to the histone demethylase FBXL11 (KDM2A) in HEK293T nuclear extracts. The results also suggest that small-molecule probes may be suitable for substrate identification studies.

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.

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 K(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.

Diphenylacetylene‐Linked Peptide Strands Induce Bidirectional β‐Sheet Formation

In the search for synthetic mimics of protein secondary structures relevant to the mediation of protein-protein interactions, we have synthesized a series of tetrasubstituted diphenylacetylenes that display β-sheet structures in two directions. Extensive X-ray crystallographic and NMR solution phase studies are consistent with these proteomimetics adopting sheet structures, displaying both hydrophobic and hydrophilic amino acid side chains.

An approach to enzyme inhibition employing reversible boronate ester formation

Dynamic combinatorial chemistry (DCC) is a potentially useful method for the identification of biomacromolecule ligands; however, the number of reactions applicable to DCC in aqueous solution is limited. We report studies that investigate the reversible reaction of boronic acids with alcohols as an approach to enzyme inhibition, employing α-chymotrypsin as a model system. NMR techniques (11B NMR and 1H waterLOGSY) were used to observe ternary complexes of boronic acids, sugars and α-chymotrypsin, and were useful for distinguishing preferentially binding combinations of boronic acids and sugars. The results reveal that both the propensity of boronate ester formation in solution and affinity of the boronate ester for the target enzyme determine whether ternary complex formation is observed. The results also provide proof of principle for the boronate ester approach to DCC versusprotein targets.

Investigating the contribution of the active site environment to the slow reaction of hypoxia-inducible factor prolyl hydroxylase domain 2 with oxygen

The prolyl hydroxylase domain proteins (PHDs) catalyse the post-translational hydroxylation of the hypoxia-inducible factor (HIF), a modification that regulates the hypoxic response in humans. The PHDs are Fe(II)/2-oxoglutarate (2OG) oxygenases; their catalysis is proposed to provide a link between cellular HIF levels and changes in O2 availability. Transient kinetic studies have shown that purified PHD2 reacts slowly with O2 compared with some other studied 2OG oxygenases, a property which may be related to its hypoxia-sensing role. PHD2 forms a stable complex with Fe(II) and 2OG; crystallographic and kinetic analyses indicate that an Fe(II)-co-ordinated water molecule, which must be displaced before O2 binding, is relatively stable in the active site of PHD2. We used active site substitutions to investigate whether these properties are related to the slow reaction of PHD2 with O2. While disruption of 2OG binding in a R383K variant did not accelerate O2 activation, we found that substitution of the Fe(II)-binding aspartate for a glutamate residue (D315E) manifested significantly reduced Fe(II) binding, yet maintained catalytic activity with a 5-fold faster reaction with O2. The results inform on how the precise active site environment of oxygenases can affect rates of O2 activation and provide insights into limiting steps in PHD catalysis.

Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases.

The response to hypoxia in animals involves the expression of multiple genes regulated by the αβ-hypoxia-inducible transcription factors (HIFs). The hypoxia-sensing mechanism involves oxygen limited hydroxylation of prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of HIFα isoforms, as catalysed by prolyl hydroxylases (PHD 1–3). Prolyl hydroxylation promotes binding of HIFα to the von Hippel–Lindau protein (VHL)–elongin B/C complex, thus signalling for proteosomal degradation of HIFα. We reveal that certain PHD2 variants linked to familial erythrocytosis and cancer are highly selective for CODD or NODD. Crystalline and solution state studies coupled to kinetic and cellular analyses reveal how wild-type and variant PHDs achieve ODD selectivity via different dynamic interactions involving loop and C-terminal regions. The results inform on how HIF target gene selectivity is achieved and will be of use in developing selective PHD inhibitors.