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Second-generation bromodomain and extra terminal (BET) inhibitors, which selectively target one of the two bromodomains in the BET proteins, have begun to emerge in the literature. These inhibitors aim to help determine the roles and functions of each domain and assess whether they can demonstrate an improved safety profile in clinical settings compared to pan-BET inhibitors. Herein, we describe the discovery of a novel BET BD2-selective chemotype using a structure-based drug design from a hit identified by DNA-encoded library technologies, showing a structural differentiation from key previously reported greater than 100-fold BD2-selective chemotypes GSK620, GSK046, and ABBV-744. Following a structure-based hypothesis for the selectivity and optimization of the physicochemical properties of the series, we identified 60 (GSK040), an in vitro ready and in vivo capable BET BD2-inhibitor of unprecedented selectivity (5000-fold) against BET BD1, excellent selectivity against other bromodomains, and good physicochemical properties. This novel chemical probe can be added to the toolbox used in the advancement of epigenetics research.

Domain-specific BET bromodomain ligands represent an attractive target for drug discovery with the potential to unlock the therapeutic benefits of antagonizing these proteins without eliciting the toxicological aspects seen with pan-BET inhibitors. While we have reported several distinct classes of BD2 selective compounds, namely, GSK620, GSK549, and GSK046, only GSK046 shows high aqueous solubility. Herein, we describe the lead optimization of a further class of highly soluble compounds based upon a picolinamide chemotype. Focusing on achieving >1000-fold selectivity for BD2 over BD1 ,while retaining favorable physical chemical properties, compound 36 was identified as being 2000-fold selective for BD2 over BD1 (Brd4 data) with >1 mg/mL solubility in FaSSIF media. 36 represents a valuable new in vivo ready molecule for the exploration of the BD2 phenotype.

A number of reports have recently been published describing the discovery and optimization of bromo and extraterminal inhibitors which are selective for the second bromodomain (BD2); these include our own work toward GSK046 (3) and GSK620 (5). This paper describes our approach to mitigating the genotoxicity risk of GSK046 by replacement of the acetamide functionality with a heterocyclic ring. This was followed by a template-hopping and hybridization approach, guided by structure-based drug design, to incorporate learnings from other BD2-selective series, optimize the vector for the amide region, and explore the ZA cleft, leading to the identification of potent, selective, and bioavailable compounds 28 (GSK452), 39 (GSK737), and 36 (GSK217).

The profound efficacy, yet associated toxicity of pan-BET inhibitors is well documented. The possibility of an ameliorated safety profile driven by significantly selective (>100-fold) inhibition of a subset of the eight bromodomains is enticing, but challenging given the close homology. Herein, we describe the X-ray crystal structure-directed optimization of a novel weak fragment ligand with a pan-second bromodomain (BD2) bias, to potent and highly BD2 selective inhibitors. A template hopping approach, enabled by our parallel research into an orthogonal template (15, GSK046), was the basis for the high selectivity observed. This culminated in two tool molecules, 20 (GSK620) and 56 (GSK549), which showed an anti-inflammatory phenotype in human whole blood, confirming their cellular target engagement. Excellent broad selectivity, developability, and in vivo oral pharmacokinetics characterize these tools, which we hope will be of broad utility to the field of epigenetics research.

Pan-bromodomain and extra terminal domain (BET) inhibitors interact equipotently with the eight bromodomains of the BET family of proteins and have shown profound efficacy in a number of in vitro phenotypic assays and in vivo pre-clinical models in inflammation or oncology. A number of these inhibitors have progressed to the clinic where pharmacology-driven adverse events have been reported. To better understand the contribution of each domain to their efficacy and improve their safety profile, selective inhibitors are required. This article discloses the profile of GSK046, also known as iBET-BD2, a highly selective inhibitor of the second bromodomains of the BET proteins that has undergone extensive pre-clinical in vitro and in vivo characterization.

Pan-bromodomain and extra terminal (BET) inhibitors interact equipotently with all eight bromodomains of the BET family of proteins. They have shown profound efficacy in vitro and in vivo in oncology and immunomodulatory models, and a number of them are currently in clinical trials where significant safety signals have been reported. It is therefore important to understand the functional contribution of each bromodomain to assess the opportunity to tease apart efficacy and toxicity. This article discloses the in vitro and cellular activity profiles of GSK789, a potent, cell-permeable, and highly selective inhibitor of the first bromodomains of the BET family.

[Image: see text] Pan-BET inhibitors have shown profound efficacy in a number of in vivo preclinical models and have entered the clinic in oncology trials where adverse events have been reported. These inhibitors interact equipotently with the eight bromodomains of the BET family of proteins. To better understand the contribution of each domain to their efficacy and to improve from their safety profile, selective inhibitors are required. This Letter discloses the profile of GSK973, a highly selective inhibitor of the second bromodomains of the BET proteins that has undergone extensive preclinical in vitro and in vivo characterization.

This Perspective discusses the published data and recent developments in the research area of bromodomains in parasitic protozoa. Further work is needed to evaluate the tractability of this target class in the context of infectious diseases and launch drug discovery campaigns to identify and develop antiparasite drugs that can offer differentiated mechanisms of action.

Leishmania are unicellular parasites that cause human and animal disease. Alongside other organisms in kinetoplastida, they have evolved an unusual genome architecture that requires all RNA polymerase II transcribed genes to be expressed constitutively, with transcriptional start regions denoted by histone variants and histone lysine acetylation. However, the way these chromatin marks are interpreted by the cell is not understood. Seven predicted bromodomain factors (BDF1-7), the reader modules for acetyl-lysine, were identified across Leishmania genomes. Using L. mexicana as a model, Cas9-driven gene deletions indicate that BDF1-5 are essential for promastigote survival, whilst DiCre inducible gene deletion of the dual bromodomain factor BDF5 identified it to be essential for both promastigotes and amastigotes. ChIP-seq assessment of BDF5s genomic distribution revealed it as highly enriched at transcriptional start sites. Using an optimised proximity proteomic and phosphoproteomic technique, XL-BioID, we defined the BDF5-proximal environment to be enriched for other bromodomain factors, histone acetyltransferase 2, and proteins essential for transcriptional activity and RNA processing. Inducible deletion of BDF5, led to a disruption of pol II transcriptional activity and global defects in gene expression. Our results indicate the requirement of Leishmania to interpret histone acetylation marks for normal levels of gene expression and thus cellular viability.

Herein, a series of 2,3-dihydrobenzofurans have been developed as highly potent bromo and extra-terminal domain (BET) inhibitors with 1000-fold selectivity for the second bromodomain (BD2) over the first bromodomain (BD1). Investment in the development of two orthogonal synthetic routes delivered inhibitors that were potent and selective but had raised in vitro clearance and suboptimal solubility. Insertion of a quaternary center into the 2,3-dihydrobenzofuran core blocked a key site of metabolism and improved the solubility. This led to the development of inhibitor 71 (GSK852): a potent, 1000-fold-selective, highly soluble compound with good in vivo rat and dog pharmacokinetics.

The profound efficacy of pan-BET inhibitors is well documented, but these epigenetic agents have shown pharmacology-driven toxicity in oncology clinical trials. The opportunity to identify inhibitors with an improved safety profile by selective targeting of a subset of the eight bromodomains of the BET family has triggered extensive medicinal chemistry efforts. In this article, we disclose the identification of potent and selective drug-like pan-BD2 inhibitors such as pyrazole 23 (GSK809) and furan 24 (GSK743) that were derived from the pyrrole fragment 6. We transpose the key learnings from a previous pyridone series (GSK620 2 as a representative example) to this novel class of inhibitors, which are characterized by significantly improved solubility relative to our previous research.

The bromodomain and extraterminal domain (BET) family of epigenetic regulators comprises four proteins (BRD2, BRD3, BRD4, BRDT), each containing tandem bromodomains. To date, small molecule inhibitors of these proteins typically bind all eight bromodomains of the family with similar affinity, resulting in a diverse range of biological effects. To enable further understanding of the broad phenotype characteristic of pan-BET inhibition, the development of inhibitors selective for individual, or sets of, bromodomains within the family is required. In this regard, we report the discovery of a potent probe molecule possessing up to 150-fold selectivity for the N-terminal bromodomains (BD1s) over the C-terminal bromodomains (BD2s) of the BETs. Guided by structural information, a specific amino acid difference between BD1 and BD2 domains was targeted for selective interaction with chemical functionality appended to the previously developed I-BET151 scaffold. Data presented herein demonstrate that selective inhibition of BD1 domains is sufficient to drive anti-inflammatory and antiproliferative effects.

The mimicry of host proteins by viruses contributes to their ability to suppress antiviral immunity and hijack host biosynthetic machinery1. Host adaptation to evade this exploitation depends on host protein functional redundancy2. Non-redundant, essential host proteins have limited potential to adapt without severe consequences3. Histones, which are essential for genome architecture and control of gene expression, are among the most evolutionary conserved proteins4. Here we show that the capsid protein of the flavivirus yellow fever virus (YFV), mimics histone H4 and interferes with chromatin gene regulation by BRD4, a bromodomain and extraterminal domain (BET) protein. Two acetyl-lysine residues of YFV capsid are embedded in a histone-like motif that interacts with the BRD4 bromodomain, affecting gene expression and influencing YFV replication. These findings reveal histone mimicry as a strategy employed by an RNA virus that replicates in the cytosol5 and define convergent and distinct molecular determinants for motif recognition of the viral mimic versus histone H4.

Bromodomain inhibitors revisited Bromodomain and extraterminal domain (BET) proteins contribute to the pathogenesis of cancer and immune diseases through their effects on transcriptional regulation. BET proteins contain two nearly identical bromodomains, BD1 and BD2, structural modules that have attracted great interest as targets for drug development. First-generation drugs that inhibited both BD1 and BD2 showed promising therapeutic activity in preclinical models but proved to be less efficacious in clinical trials. Gilan et al. took a different approach and designed drugs that selectively inhibited BD1 or BD2 (see the Perspective by Filippakopoulos and Knapp). They found that BD1 and BD2 inhibitors altered gene expression in different ways and that BD2 inhibitors had greater therapeutic activity than BD1 inhibitors in preclinical models of inflammation and autoimmune disease. Science , this issue p. 387 ; see also p. 367

Transcription of inflammatory genes is tightly regulated by acetylation and deacetylation of histone tails. An inhibitor of the acetylated-lysine reader bromodomain and extra-terminal domain (BET) proteins, I-BET151, is known to counteract the induction of expression of inflammatory genes in macrophages. We have investigated the effects of I-BET151 on dendritic cell function, including expression of co-stimulatory molecules and cytokines, and capacity for T cell activation. Treatment of mouse bone marrow derived dendritic cells (BMDC) and human monocyte derived DCs (mdDC) with I-BET151 reduced LPS-induced expression of co-stimulatory molecules, as well as the production of multiple cyokines and chemokines. Most strikingly, secretion of IL-6, IL-12 and IL-10 was significantly reduced to 89.7%, 99.9% and 98.6% respectively of that produced by control cells. I-BET151-treated mdDC showed a reduced ability to stimulate proliferation of autologous Revaxis-specific T cells. Moreover, while I-BET151 treatment of BMDC did not affect their ability to polarise ovalbumin specific CD4(+) CD62L(+) naive T cells towards Th1, Th2, or Th17 phenotypes, an increase in Foxp3 expressing Tregs secreting higher IL-10 levels was observed. Suppression assays demonstrated that Tregs generated in response to I-BET151-treated BMDC displayed anti-proliferative capacity. Finally, evidence that I-BET151 treatment can ameliorate inflammation in vivo in a T cell dependent colitis model is shown. Overall, these results demonstrate marked effects of BET inhibition on DC maturation, reducing their capacity for pro-inflammatory cytokine secretion and T cell activation and enhancing the potential of DC to induce Foxp3 expressing Treg with suppressive properties

Non-alcoholic fatty liver disease (NAFLD) is a leading form of chronic liver disease with large unmet need. Non-alcoholic steatohepatitis (NASH), a progressive variant of NAFLD, can lead to fibrosis, cirrhosis, and hepatocellular carcinoma. To identify potential new therapeutics for NASH, we used a computational approach based on Connectivity Map (CMAP) analysis, which pointed us to bromodomain and extra-terminal motif (BET) inhibitors for treating NASH. To experimentally validate this hypothesis, we tested a small-molecule inhibitor of the BET family of proteins, GSK1210151A (I-BET151), in the STAM mouse NASH model at two different dosing timepoints (onset of NASH and progression to fibrosis). I-BET151 decreased the non-alcoholic fatty liver disease activity score (NAS), a clinical endpoint for assessing the severity of NASH, as well as progression of liver fibrosis and interferon-γ expression. Transcriptional characterization of these mice through RNA-sequencing was consistent with predictions from the CMAP analysis of a human NASH signature and pointed to alterations in molecular mechanisms related to interferon signaling and cholesterol biosynthesis, as well as reversal of gene expression patterns linked to fibrotic markers. Altogether, these results suggest that inhibition of BET proteins may present a novel therapeutic opportunity in the treatment of NASH and liver fibrosis.

Herein we describe the design and synthesis of a dual active histone deacetylase (HDAC)/bromodomain and extra terminal (BET) small molecule tool inhibitor, DUAL946 (1). Exploiting our extensive epigenetic toolbox, we achieved the functionalisation of a BET active tetrahydroquinoline (THQ) core, with a hydroxamic acid HDAC inhibitor (HDACi) motif. Dual inhibition of BET and HDAC proteins was confirmed by in vitro biochemical and biophysical testing and through chemoproteomic competition experiments in cell lysates. This activity was translated into potent cellular activity in both immune and cancer cells.

Small molecule inhibition of the BET family of proteins, which bind acetylated lysines within histones, has been shown to have a marked therapeutic benefit in pre-clinical models of mixed lineage leukemia (MLL) fusion protein-driven leukemias. Here, we report that I-BET151, a highly specific BET family bromodomain inhibitor, leads to growth inhibition in a human erythroleukemic (HEL) cell line as well as in erythroid precursors isolated from polycythemia vera patients. One of the genes most highly downregulated by I-BET151 was LMO2, an important oncogenic regulator of hematopoietic stem cell development and erythropoiesis. We previously reported that LMO2 transcription is dependent upon Janus kinase 2 (JAK2) kinase activity in HEL cells. Here, we show that the transcriptional changes induced by a JAK2 inhibitor (TG101209) and I-BET151 in HEL cells are significantly over-lapping, suggesting a common pathway of action. We generated JAK2 inhibitor resistant HEL cells and showed that these retain sensitivity to I-BET151. These data highlight I-BET151 as a potential alternative treatment against myeloproliferative neoplasms driven by constitutively active JAK2 kinase.

Trypanosoma brucei, the causative agent of African sleeping sickness, is transmitted to its mammalian host by the tsetse. In the fly, the parasite’s surface is covered with invariant procyclin, while in the mammal it resides extracellularly in its bloodstream form (BF) and is densely covered with highly immunogenic Variant Surface Glycoprotein (VSG). In the BF, the parasite varies this highly immunogenic surface VSG using a repertoire of ~2500 distinct VSG genes. Recent reports in mammalian systems point to a role for histone acetyl-lysine recognizing bromodomain proteins in the maintenance of stem cell fate, leading us to hypothesize that bromodomain proteins may maintain the BF cell fate in trypanosomes. Using small-molecule inhibitors and genetic mutants for individual bromodomain proteins, we performed RNA-seq experiments that revealed changes in the transcriptome similar to those seen in cells differentiating from the BF to the insect stage. This was recapitulated at the protein level by the appearance of insect-stage proteins on the cell surface. Furthermore, bromodomain inhibition disrupts two major BF-specific immune evasion mechanisms that trypanosomes harness to evade mammalian host antibody responses. First, monoallelic expression of the antigenically varied VSG is disrupted. Second, rapid internalization of antibodies bound to VSG on the surface of the trypanosome is blocked. Thus, our studies reveal a role for trypanosome bromodomain proteins in maintaining bloodstream stage identity and immune evasion. Importantly, bromodomain inhibition leads to a decrease in virulence in a mouse model of infection, establishing these proteins as potential therapeutic drug targets for trypanosomiasis. Our 1.25Å resolution crystal structure of a trypanosome bromodomain in complex with I-BET151 reveals a novel binding mode of the inhibitor, which serves as a promising starting point for rational drug design., The use of specific inhibitors reveals a role for bromodomain proteins in epigenetically switching from the tsetse fly form of the sleeping sickness parasite Trypanosoma brucei to the mammalian bloodstream form., Author Summary Many parasites undergo major changes in lifestyle as they cycle between their vectors and their hosts. We use Trypanosoma brucei, the causative agent of human and animal African Trypanosomiasis, as a model to study how these changes are regulated and maintained at the level of gene expression. T. brucei lives in the bloodstream of the mammalian host before migrating to the insect through the bite of its insect vector, the tsetse fly. It escapes immune detection by varying the proteins on its surface and by rapidly internalizing host antibodies. We report here that a class of proteins, called bromodomain proteins, help maintain the identity of the parasite in its bloodstream form. When these proteins are inhibited, the parasites express an unvarying epitope that is usually expressed only at the insect stage and are compromised in their ability to internalize host antibodies. Bromodomain proteins bind to DNA that is wrapped around histone proteins, acting as mediators that interact with the transcription machinery to determine which genes are turned on and which are kept repressed. Our crystal structure of a bromodomain inhibitor in complex with a trypanosome bromodomain reveals a novel binding mode and demonstrates how these small molecule inhibitors could be optimized for therapeutic use in a parasite-specific manner.

Aire controls immunologic tolerance by inducing a battery of thymic transcripts encoding proteins characteristic of peripheral tissues. Its unusually broad effect is achieved by releasing RNA polymerase II paused just downstream of transcriptional start sites. We explored Aire's collaboration with the bromodomain-containing protein, Brd4, uncovering an astonishing correspondence between those genes induced by Aire and those inhibited by a small-molecule bromodomain blocker. Aire:Brd4 binding depended on an orchestrated series of posttranslational modifications within Aire's caspase activation and recruitment domain. This interaction attracted P-TEFb, thereby mobilizing downstream transcriptional elongation and splicing machineries. Aire:Brd4 association was critical for tolerance induction, and its disruption could account for certain point mutations that provoke human autoimmune disease. Our findings evoke the possibility of unanticipated immunologic mechanisms subtending the potent antitumor effects of bromodomain blockers.

Neural stem cells and progenitor cells (NPCs) are increasingly appreciated to hold great promise for regenerative medicine to treat CNS injuries and neurodegenerative diseases. However, evidence for effective stimulation of neuronal production from endogenous or transplanted NPCs for neuron replacement with small molecules remains limited. To identify novel chemical entities/targets for neurogenesis, we had established a NPC phenotypic screen assay and validated it using known small-molecule neurogenesis inducers. Through screening small molecule libraries with annotated targets, we identified BET bromodomain inhibition as a novel mechanism for enhancing neurogenesis. BET bromodomain proteins, Brd2, Brd3, and Brd4 were found to be downregulated in NPCs upon differentiation, while their levels remain unaltered in proliferating NPCs. Consistent with the pharmacological study using bromodomain selective inhibitor (+)-JQ-1, knockdown of each BET protein resulted in an increase in the number of neurons with simultaneous reduction in both astrocytes and oligodendrocytes. Gene expression profiling analysis demonstrated that BET bromodomain inhibition induced a broad but specific transcription program enhancing directed differentiation of NPCs into neurons while suppressing cell cycle progression and gliogenesis. Together, these results highlight a crucial role of BET proteins as epigenetic regulators in NPC development and suggest a therapeutic potential of BET inhibitors in treating brain injuries and neurodegenerative diseases.