Anthony Tumber

A Series of Potent CREBBP Bromodomain Ligands Reveals an Induced-Fit Pocket Stabilized by a Cation–π Interaction

The benzoxazinone and dihydroquinoxalinone fragments were employed as novel acetyl lysine mimics in the development of CREBBP bromodomain ligands. While the benzoxazinone series showed low affinity for the CREBBP bromodomain, expansion of the dihydroquinoxalinone series resulted in the first potent inhibitors of a bromodomain outside the BET family. Structural and computational studies reveal that an internal hydrogen bond stabilizes the protein-bound conformation of the dihydroquinoxalinone series. The side chain of this series binds in an induced-fit pocket forming a cation–π interaction with R1173 of CREBBP. The most potent compound inhibits binding of CREBBP to chromatin in U2OS cells.

Inhibition of histone demethylases by 4-carboxy-2,2'-bipyridyl compounds

In eukaryotes, nuclear DNA is packaged into chromatin by binding to histones and associated factors. Covalent modifications to histone tails are associated with specific transcriptional states of the associated DNA. Acetylation of lysine side chains normally correlates with transcriptional activation, while deacetylation leads to transcriptional silencing. The regulatory roles of lysine and arginine methylation appear to be more complex. Methylation of certain lysine residues is associated with active transcription, while methylation of others is associated with silencing and heterochromatin formation. Each methylation marker is placed, removed and interpreted in a site-specific manner by histone methyltransferases, demethylases and methyl binding domains, respectively. The biological functions of the individual enzymes are largely undefined and are the focus of current investigations (for Reviews see References [1, 2]) The JmjC histone demethylases are 2-oxoglutarate (2OG)-dependent oxygenases that catalyse N-lysyl demethylation via hydroxylation of the methyl group in a 2OGand Fe-dependent manner (Scheme 1). Human 2OG oxygenases catalyse a range of reactions, including hydroxylation of amino acids, DNA, and small molecules, and demethylation of proteins and DNA. 2OG oxygenases show promise as therapeutic targets; an inhibitor of g-butyrobetaine hydroxylase (BBOX) is used for the treatment of cardiovascular disease, and inhibitors of the hypoxia inducible factor (HIF) prolyl hydroxylases are in clinical trials for the treatment of anaemia. Inhibitors of the collagen prolyl hydroxylases have also been evaluated as potential therapeutics for the treatment of liver fibrosis. 8] The discovery of the JmjC domain histone demethylases, and the suggestion that some of them are potential therapeutic targets for cancer treatment, has stimulated interest in their inhibition, but relatively few studies have been described. Reported inhibitors of the JmjC demethylases include N-oxalyl amino acids, 8-hydroxyquinolines, pyridine dicarboxylates, hydroxamic acids and catechol-type flavonoids (Figure 1). Compounds that catalyse the ejection of a structural Zn ion from the JMJD2 demethylases have also been reported (Figure 1).

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

Development of homogeneous luminescence assays for histone demethylase catalysis and binding.

Covalent modifications to histones play important roles in chromatin dynamics and the regulation of gene expression. The JumonjiC (JmjC)-containing histone demethylases (HDMs) catalyze the demethylation of methylated lysine residues on histone tails. Here we report the development of homogeneous luminescence-based assay methods for measuring the catalytic activity and the binding affinities of peptides to HDMs. The assays use amplified luminescent proximity homogeneous assay (ALPHA) technology, are sensitive and robust, and can be used for small molecule inhibitor screening of HDMs. We have profiled known inhibitors of JMJD2E and demonstrate a correlation between the inhibitor potencies determined by the ALPHA and other types of assays. Although this study focuses on the JMJD2E isoform, the catalytic turnover and binding assays described here can be used in studies on other HDMs. The assays should be useful for the development of small molecule inhibitors selective for HDM isoforms.

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.

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

The 2‐oxoglutarate (2OG)‐dependent Jumonjiu2005C 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 4u2005μM). 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.

Discovery of a Highly Selective Cell‐Active Inhibitor of the Histone Lysine Demethylases KDM2/7

Abstract Histone lysine demethylases (KDMs) are of critical importance in the epigenetic regulation of gene expression, yet there are few selective, cell‐permeable inhibitors or suitable tool compounds for these enzymes. We describe the discovery of a new class of inhibitor that is highly potent towards the histone lysine demethylases KDM2A/7A. A modular synthetic approach was used to explore the chemical space and accelerate the investigation of key structure–activity relationships, leading to the development of a small molecule with around 75‐fold selectivity towards KDM2A/7A versus other KDMs, as well as cellular activity at low micromolar concentrations.