Chun-wa Chung

Suppression of inflammation by a synthetic histone mimic

Interaction of pathogens with cells of the immune system results in activation of inflammatory gene expression. This response, although vital for immune defence, is frequently deleterious to the host due to the exaggerated production of inflammatory proteins. The scope of inflammatory responses reflects the activation state of signalling proteins upstream of inflammatory genes as well as signal-induced assembly of nuclear chromatin complexes that support mRNA expression. Recognition of post-translationally modified histones by nuclear proteins that initiate mRNA transcription and support mRNA elongation is a critical step in the regulation of gene expression. Here we present a novel pharmacological approach that targets inflammatory gene expression by interfering with the recognition of acetylated histones by the bromodomain and extra terminal domain (BET) family of proteins. We describe a synthetic compound (I-BET) that by ‘mimicking’ acetylated histones disrupts chromatin complexes responsible for the expression of key inflammatory genes in activated macrophages, and confers protection against lipopolysaccharide-induced endotoxic shock and bacteria-induced sepsis. Our findings suggest that synthetic compounds specifically targeting proteins that recognize post-translationally modified histones can serve as a new generation of immunomodulatory drugs.

Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia

Recurrent chromosomal translocations involving the mixed lineage leukaemia (MLL) gene initiate aggressive forms of leukaemia, which are often refractory to conventional therapies. Many MLL-fusion partners are members of the super elongation complex (SEC), a critical regulator of transcriptional elongation, suggesting that aberrant control of this process has an important role in leukaemia induction. Here we use a global proteomic strategy to demonstrate that MLL fusions, as part of SEC and the polymerase-associated factor complex (PAFc), are associated with the BET family of acetyl-lysine recognizing, chromatin ‘adaptor’ proteins. These data provided the basis for therapeutic intervention in MLL-fusion leukaemia, via the displacement of the BET family of proteins from chromatin. We show that a novel small molecule inhibitor of the BET family, GSK1210151A (I-BET151), has profound efficacy against human and murine MLL-fusion leukaemic cell lines, through the induction of early cell cycle arrest and apoptosis. I-BET151 treatment in two human leukaemia cell lines with different MLL fusions alters the expression of a common set of genes whose function may account for these phenotypic changes. The mode of action of I-BET151 is, at least in part, due to the inhibition of transcription at key genes (BCL2, C-MYC and CDK6) through the displacement of BRD3/4, PAFc and SEC components from chromatin. In vivo studies indicate that I-BET151 has significant therapeutic value, providing survival benefit in two distinct mouse models of murine MLL–AF9 and human MLL–AF4 leukaemia. Finally, the efficacy of I-BET151 against human leukaemia stem cells is demonstrated, providing further evidence of its potent therapeutic potential. These findings establish the displacement of BET proteins from chromatin as a promising epigenetic therapy for these aggressive leukaemias.

A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response

The jumonji (JMJ) family of histone demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance, as well as in development, physiology and disease. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX). The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.

Discovery and Characterization of Small Molecule Inhibitors of the Bet Family Bromodomains.

Epigenetic mechanisms of gene regulation have a profound role in normal development and disease processes. An integral part of this mechanism occurs through lysine acetylation of histone tails which are recognized by bromodomains. While the biological and structural characterization of many bromodomain containing proteins has advanced considerably, the therapeutic tractability of this protein family is only now becoming understood. This paper describes the discovery and molecular characterization of potent (nM) small molecule inhibitors that disrupt the function of the BET family of bromodomains (Brd2, Brd3, and Brd4). By using a combination of phenotypic screening, chemoproteomics, and biophysical studies, we have discovered that the protein-protein interactions between bromodomains and acetylated histones can be antagonized by selective small molecules that bind at the acetylated lysine recognition pocket. X-ray crystal structures of compounds bound into bromodomains of Brd2 and Brd4 elucidate the molecular interactions of binding and explain the precisely defined stereochemistry required for activity.

Fragment-based discovery of bromodomain inhibitors part 1: inhibitor binding modes and implications for lead discovery.

Bromodomain-containing proteins are key epigenetic regulators of gene transcription and readers of the histone code. However, the therapeutic benefits of modulating this target class are largely unexplored due to the lack of suitable chemical probes. This article describes the generation of lead molecules for the BET bromodomains through screening a fragment set chosen using structural insights and computational approaches. Analysis of 40 BRD2/fragment X-ray complexes highlights both shared and disparate interaction features that may be exploited for affinity and selectivity. Six representative crystal structures are then exemplified in detail. Two of the fragments are completely new bromodomain chemotypes, and three have never before been crystallized in a bromodomain, so our results significantly extend the limited public knowledge-base of crystallographic small molecule/bromodomain interactions. Certain fragments (including paracetamol) bind in a consistent mode to different bromodomains such as CREBBP, suggesting their potential to act as generic bromodomain templates. An important implication is that the bromodomains are not only a phylogenetic family but also a system in which chemical and structural knowledge of one bromodomain gives insights transferrable to others.

Fragment-Based Discovery of Bromodomain Inhibitors Part 2: Optimization of Phenylisoxazole Sulfonamides.

Bromodomains are epigenetic reader modules that regulate gene transcription through their recognition of acetyl-lysine modified histone tails. Inhibitors of this protein-protein interaction have the potential to modulate multiple diseases as demonstrated by the profound anti-inflammatory and antiproliferative effects of a recently disclosed class of BET compounds. While these compounds were discovered using phenotypic assays, here we present a highly efficient alternative approach to find new chemical templates, exploiting the abundant structural knowledge that exists for this target class. A phenyl dimethyl isoxazole chemotype resulting from a focused fragment screen has been rapidly optimized through structure-based design, leading to a sulfonamide series showing anti-inflammatory activity in cellular assays. This proof-of-principle experiment demonstrates the tractability of the BET family and bromodomain target class to fragment-based hit discovery and structure-based lead optimization.

Discovery of Epigenetic Regulator I-Bet762: Lead Optimization to Afford a Clinical Candidate Inhibitor of the Bet Bromodomains.

The bromo and extra C-terminal domain (BET) family of bromodomains are involved in binding epigenetic marks on histone proteins, more specifically acetylated lysine residues. This paper describes the discovery and structure-activity relationships (SAR) of potent benzodiazepine inhibitors that disrupt the function of the BET family of bromodomains (BRD2, BRD3, and BRD4). This work has yielded a potent, selective compound I-BET762 that is now under evaluation in a phase I/II clinical trial for nuclear protein in testis (NUT) midline carcinoma and other cancers.

Identification of a novel series of BET family bromodomain inhibitors: binding mode and profile of I-BET151 (GSK1210151A).

A novel series of quinoline isoxazole BET family bromodomain inhibitors are discussed. Crystallography is used to illustrate binding modes and rationalize their SAR. One member, I-BET151 (GSK1210151A), shows good oral bioavailability in both the rat and minipig as well as demonstrating efficient suppression of bacterial induced inflammation and sepsis in a murine in vivo endotoxaemia model.

BET Inhibition Silences Expression of MYCN and BCL2 and Induces Cytotoxicity in Neuroblastoma Tumor Models

BET family proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Selective inhibitors of BET proteins exhibit potent anti-proliferative activity in a number of hematologic cancer models, in part through suppression of the MYC oncogene and downstream Myc-driven pathways. However, little is currently known about the activity of BET inhibitors in solid tumor models, and whether down-regulation of MYC family genes contributes to sensitivity. Here we provide evidence for potent BET inhibitor activity in neuroblastoma, a pediatric solid tumor associated with a high frequency of MYCN amplifications. We treated a panel of neuroblastoma cell lines with a novel small molecule inhibitor of BET proteins, GSK1324726A (I-BET726), and observed potent growth inhibition and cytotoxicity in most cell lines irrespective of MYCN copy number or expression level. Gene expression analyses in neuroblastoma cell lines suggest a role of BET inhibition in apoptosis, signaling, and N-Myc-driven pathways, including the direct suppression of BCL2 and MYCN. Reversal of MYCN or BCL2 suppression reduces the potency of I-BET726-induced cytotoxicity in a cell line-specific manner; however, neither factor fully accounts for I-BET726 sensitivity. Oral administration of I-BET726 to mouse xenograft models of human neuroblastoma results in tumor growth inhibition and down-regulation MYCN and BCL2 expression, suggesting a potential role for these genes in tumor growth. Taken together, our data highlight the potential of BET inhibitors as novel therapeutics for neuroblastoma, and suggest that sensitivity is driven by pleiotropic effects on cell growth and apoptotic pathways in a context-specific manner.

The Discovery of I-Bet726 (Gsk1324726A), a Potent Tetrahydroquinoline Apoa1 Up-Regulator and Selective Bet Bromodomain Inhibitor.

Through their function as epigenetic readers of the histone code, the BET family of bromodomain-containing proteins regulate expression of multiple genes of therapeutic relevance, including those involved in tumor cell growth and inflammation. BET bromodomain inhibitors have profound antiproliferative and anti-inflammatory effects which translate into efficacy in oncology and inflammation models, and the first compounds have now progressed into clinical trials. The exciting biology of the BETs has led to great interest in the discovery of novel inhibitor classes. Here we describe the identification of a novel tetrahydroquinoline series through up-regulation of apolipoprotein A1 and the optimization into potent compounds active in murine models of septic shock and neuroblastoma. At the molecular level, these effects are produced by inhibition of BET bromodomains. X-ray crystallography reveals the interactions explaining the structure-activity relationships of binding. The resulting lead molecule, I-BET726, represents a new, potent, and selective class of tetrahydroquinoline-based BET inhibitors.