Bromodomain Research Articles

On-Demand Local Immunomodulation via Epigenetic Control of Macrophages Using an Inflammation-Responsive Hydrogel for Accelerated Wound Healing.

Effective resolution of inflammation contributes to favorable tissue regenerative therapeutic outcomes. However, fine coordination of local immunomodulation in a timely manner is limited because of the lack of strategies for controlling disease dynamics. We developed an inflammation-responsive hydrogel (IFRep gel) as an effective therapeutic strategy for on-demand epigenetic modulation against disease dynamics in wound healing. The IFRep gel is designed to control drug release by cathepsins according to the state of inflammation for active disease treatment. The gel loaded with an inhibitor of the epigenetic reader bromodomain (BRD)4 regulates the translocation of nuclear factor erythroid 2 to the nucleus, where it promotes antioxidant gene expression to reverse the inflammatory macrophage state in vitro. In addition, on-demand BRD inhibition using the responsive hydrogel accelerates wound healing by controlling the early inflammatory phase and keratinocyte activation in vivo. Our data demonstrate the clinical utility of using the IFRep gel as a promising strategy for improving therapeutic outcomes in inflammation-associated diseases.

Genomic Identification, Evolution, and Expression Analysis of Bromodomain Genes Family in Buffalo.

Bromodomain (BRD) is an evolutionarily conserved protein–protein interaction module that is critical in gene regulation, cellular homeostasis, and epigenetics. This study aimed to conduct an identification, evolution, and expression analysis of the BRD gene family in the swamp buffalo (Bubalus bubalis). A total of 101 BRD protein sequences deduced from 22 BRD genes were found in the buffalo genome. The BRD proteins were classified into six groups based on phylogenetic relationships, conserved motifs, and conserved domains. The BRD genes were irregularly distributed in 13 chromosomes. Collinearity analysis revealed 20 BRD gene pairs that had remarkable homologous relationships between the buffalo and cattle, although no tandem or segmental duplication event was found in the buffalo BRD genes. Comparative transcriptomics using a 10x sequencing platform analysis showed that 22 BRD genes were identified in the Sertoli cells (SCs) at different developmental stages of buffalo. Further, the mRNA expression levels of bromodomain and the extraterminal (BET) family in SCs at the pubertal stage were higher than that at the prepubertal stage of buffalo. However, the SMARCA2, PHIP, BRD9, and TAF1 genes exhibited the opposite trend. The maturation process of SCs may be regulated by the BRD family members expressed differentially in SCs at different developmental stages of buffalo. In summary, our findings provide an understanding of the evolutionary, structural, and functional properties of the buffalo BRD family members, and further characterize the function of the BRD family in the maturation of SCs. It also provides a theoretical basis for further understanding in the future of the mechanism of SCs regulating spermatogenesis.

CBP/p300 bromodomain: new promising epigenetic target

CREB (cAMP responsive element binding protein) binding protein (CBP) and adenovirus E1A-associated 300 kDa protein (p300) are histone acetyltransferases, which are necessary for multiple cellular processes. Thus, CBP/p300 are promising potential antitumor targets. To date, despite various small molecule inhibitors of CBP/p300 bromodomain (BRD) having been reported, no specific inhibitor was approved by U.S. Food and Drug Administration (FDA). In this review, we described the discovery, optimization, binding mode evaluation, selectivity and potency evaluation, and therapeutic opportunities of our CBP/p300 bromodomain inhibitors, aiming to inspire new inhibitor design and advance drug discovery research in this field. One video presents the development of CBP/p300 bromodomain inhibitors.

Theoretical exploration of the binding selectivity of inhibitors to BRD7 and BRD9 with multiple short molecular dynamics simulations.

Bromodomain-containing proteins 7 and 9 (BRD7 and BRD9) have been considered as potential targets of clinical drug design toward treatment of human cancers and other diseases. Multiple short molecular dynamics simulations and binding free energy predictions were carried out to decipher the binding selectivity of three inhibitors 4L2, 5U6, and 6KT toward BRD7 and BRD9. The results show that 4L2 has more favorable binding ability to BRD7 over BRD9 compared to 5U6 and 6KT, while 5U6 and 6KT possess more favorable associations with BRD9 than BRD7. Furthermore, estimations of residue-based free energy decompositions further identify that four common residue pairs, including (F155, F44), (V160, V49), (Y168, Y57) and (Y217, Y106) in (BRD7, BRD9) generate obvious binding differences with 4L2, 5U6, and 6KT, which mostly drives the binding selectivity of 4L2, 5U6, and 6KT to BRD7 and BRD9. Dynamic information arising from trajectory analysis also suggests that inhibitor bindings affect structural flexibility and motion modes, which is responsible for the partial selectivity of 4L2, 5U6, and 6KT toward BRD7 and BRD9. As per our expectation, this study theoretically provides useful hints for design of dual inhibitors with high selectivity on BRD7 and BRD9.

Brd4 is required for chondrocyte differentiation and endochondral ossification.

Differentiation of multi-potent mesenchymal stromal cells (MSCs) is directed by the activities of lineage-specific transcription factors and co-factors. A subset of these proteins controls the accessibility of chromatin by recruiting histone acetyl transferases or deacetylases that regulate acetylation of the N-termini of H3 and H4 histone proteins. Bromodomain (BRD) proteins recognize these acetylation marks and recruit the RNA pol II containing transcriptional machinery. Our previous studies have shown that Brd4 is required for osteoblast differentiation in vitro. Here, we investigated the role of Brd4 on endochondral ossification in C57BL/6 mice and chondrogenic differentiation in cell culture models. Conditional loss of Brd4 in the mesenchyme (Brd4 cKO, Brd4fl/fl: Prrx1-Cre) yields smaller mice that exhibit alteration in endochondral ossification. Importantly, abnormal growth plate morphology and delayed long bone formation is observed in juvenile Brd4 cKO mice. One week old Brd4 cKO mice have reduced proliferative and hypertrophic zones within the physis and exhibit a delay in the formation of the secondary ossification center. At the cellular level, Brd4 function is required for chondrogenic differentiation and maturation of both ATDC5 cells and immature mouse articular chondrocytes. Mechanistically, Brd4 loss suppresses Sox9 levels and reduces expression of Sox9 and Runx2 responsive endochondral genes (e.g., Col2a1, Acan, Mmp13 and Sp7/Osx). Collectively, our results indicate that Brd4 is a key epigenetic regulator required for normal chondrogenesis and endochondral ossification.

The BET Protein Inhibitor Apabetalone Rescues Diabetes-Induced Impairment of Angiogenic Response by Epigenetic Regulation of Thrombospondin-1.

Aims: Therapeutic modulation of blood vessel growth holds promise for the prevention of limb ischemia in diabetic (DM) patients with peripheral artery disease (PAD). Epigenetic changes, namely, posttranslational histone modifications, participate in angiogenic response suggesting that chromatin-modifying drugs could be beneficial in this setting. Apabetalone (APA), a selective inhibitor of bromodomain (BRD) and bromodomain and extraterminal containing protein family (BET) proteins, prevents bromodomain-containing protein 4 (BRD4) interactions with chromatin thus modulating transcriptional programs in different organs. We sought to investigate whether APA affects angiogenic response in diabetes. Results: Compared with vehicle, APA restored tube formation and migration in human aortic endothelial cells (HAECs) exposed to high-glucose (HG) levels. Expression profiling of angiogenesis genes showed that APA prevents HG-induced upregulation of the antiangiogenic molecule thrombospondin-1 (THBS1). ChIP-seq and chromatin immunoprecipitation (ChIP) assays in HG-treated HAECs showed the enrichment of both BRD4 and active marks (H3K27ac) on THBS1 promoter, whereas BRD4 inhibition by APA prevented chromatin accessibility and THBS1 transcription. Mechanistically, we show that THBS1 inhibits angiogenesis by suppressing vascular endothelial growth factor A (VEGFA) signaling, while APA prevents these detrimental changes. In diabetic mice with hind limb ischemia, epigenetic editing by APA restored the THBS1/VEGFA axis, thus improving limb vascularization and perfusion, compared with vehicle-treated animals. Finally, epigenetic regulation of THBS1 by BRD4/H3K27ac was also reported in DM patients with PAD compared with nondiabetic controls. Innovation: This is the first study showing that BET protein inhibition by APA restores angiogenic response in experimental diabetes. Conclusions: Our findings set the stage for preclinical studies and exploratory clinical trials testing APA in diabetic PAD. Antioxid. Redox Signal. 36, 667-684.

Design and Synthesis of Hybrid Compounds as Epigenetic Modifiers.

Epigenetic modifiers acting through polypharmacology mechanisms are promising compounds with which to treat several infectious diseases. Histone deacetylase (HDAC) enzymes, mainly class I, and extra-terminal bromodomains (BET) are involved in viral replication and the host response. In the present study, 10 compounds were designed, assisted by molecular docking, to act against HDAC class I and bromodomain-4 (BRD4). All the compounds were synthesized and characterized by analytical methods. Enzymatic assays were performed using HDAC-1, -4, and -11 and BRD4. Compounds (2–10) inhibited both HDAC class I, mainly HDAC-1 and -2, and reduced BRD4 activity. For HDAC-1, the inhibitory effect ranged from 8 to 95%, and for HDAC-2, these values ranged from 10 to 91%. Compounds (2–10) decreased the BRD4 activity by up to 25%. The multi-target effects of these compounds show desirable properties that could help to combat viral infections by acting through epigenetic mechanisms.

Abstract P255: Alvocidib synergizes with BRD4 inhibitors to improve cytotoxity in an AML cell line

Abstract The super enhancer complex (SEC) is a group of transcription regulatory proteins that coordinate the expression of genetic programs which determine cell identity and drive diseases such as cancer. In acute myeloid leukemia (AML), SECs have been shown to turn on transcriptional programs that drive tumorigenesis and progression. SEC-regulated transcription begins as Cyclin-Dependent Kinase 9 (CDK9) and Cyclin T1 are recruited from an inhibitory complex by bromodomain 4 (BRD4) and brought to the transcriptional start site of genes. CDK9 phosphorylates RNA polymerase II, releasing it from the SEC and leading to transcriptional elongation and gene expression. Alvocidib is a potent CDK9 inhibitor with clinical activity in AML from multiple Phase II studies. Additionally, BRD4 inhibitors have shown early promise in clinical studies with a focus on AML. Starting with the observation of close association of CDK9 and BRD4 and the central role of the SEC in AML, we previously showed that CDK9 inhibitors combined with bromodomain inhibitors inhibited the SEC more effectively than either of these compounds alone. In this study, we sought to quantify the synergy between alvocidib and each of the BRD4 inhibitors, OTX015, IBET762, and CPI-0610, in the MV-4-11 AML cell line. After determining the single agent IC50 value of each agent, 6x6 matrix combination assays were used to determine synergy between alvocidib and OTX015, IBET762, or CPI-0610. The matrix combination assays consisted of concentrations of each : 4x IC50, 2x IC50, 1x IC50, 0.5x IC50, 0.25x IC50, and 0.125x IC50 value. In the combination assays, the cells were incubated with alvocidib for 48 h, then a BRD4 inhibitor was added, and the cells were incubated with alvocidib and the BRD4 inhibitor for a further 48 h. Combination index (CI) values were calculated using the Chou-Talalay method, where CI values less than 1 indicate synergism and lower CI values indicating stronger synergism. Synergistic interactions were observed in MV-4-11 cells for all three combinations of alvocidib plus a BRD4 inhibitor. For the combination of alvocidib and OTX015, synergy was observed for 81% (29/36) of the tested combinations, with the strongest synergy (CI = 0.155) observed at 20 nM alvocidib and 1.472 µM OTX015. For the combination of alvocidib and IBET762, synergy was observed for 75% (27/36) of the tested combinations, with the strongest synergy (CI = 0.144) observed at 10 nM alvocidib and 6.288 µM IBET762. For the combination of alvocidib and CPI-0610, synergy was observed for 72% (26/36) of the tested combinations, with the strongest synergy (CI = 0.101) observed at 10 nM alvocidib and 5.312 µM CPI-0610. These results suggest that combination strategies might be rationally designed to improve tumor targeting effects while reducing adverse side effects. Clinical studies utilizing these combination strategies are the next steps to further explore this approach. Citation Format: Sal Sommakia, Ethika Tyagi, Yuta Matsumura, Jason M. Foulks, Steven L. Warner. Alvocidib synergizes with BRD4 inhibitors to improve cytotoxity in an AML cell line [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P255.

Abstract 10474: The Bet Protein Inhibitor Apabetalone Rescues Diabetes Induced Impairment of Angiogenic Response

Introduction: Therapeutic modulation of blood vessel growth is a promising therapeutic strategy for the prevention of limb ischemia in diabetic (DM) patients with peripheral artery disease (PAD). Epigenetic changes, namely posttranslational histone modifications, participate in angiogenic response suggesting that chromatin-modifying drugs could be beneficial in this setting. Apabetalone (APA), a selective inhibitor of bromodomain (BRD) and extra-terminal (BET) proteins, prevents BRD4 interactions with chromatin thus modulating transcriptional programs in different organs. Hypothesis: We sought to investigate whether APA affects angiogenic response in diabetes. Methods: Primary human aortic endothelial cells (HAECs) were exposed to normal glucose (NG, 5 mM) or high glucose (HG, 20 mM) for 48 hours in presence of RVX-208 (20μM) or vehicle (DMSO). Streptozotocin-induced diabetic mice were orally treated with APA or vehicle for 5 days. Hindlimb ischemia was induced in diabetic mice and blood flow recovery analyzed at 30 minutes, 7, & 14 days by laser Doppler imaging. Gastrocnemius muscle samples from patients with and without diabetes were employed to translate our experimental findings. Results: APA restored tube formation and migration in human endothelial aortic cells (HAECs) exposed to HG levels. Expression profiling of angiogenesis genes showed that APA prevents HG-induced upregulation of the anti-angiogenic molecule thrombospondin (THBS1). ChIP-seq and chromatin immunoprecipitation (ChIP) assays in HG-treated HAECs showed the enrichment of both BRD4 and active regulatory marks (H3K27ac) on THBS1 promoter, whereas BRD4 inhibition by APA prevented chromatin accessibility and THBS1 transcription. Mechanistically, we show that THBS1 inhibits angiogenesis by suppressing VEGFA signaling, while APA prevents these detrimental changes. In diabetic mice with hindlimb ischemia, epigenetic editing by APA restored THSB1/VEGFA axis thus improving limb vascularization and perfusion. Finally, epigenetic regulation of THSB1 by BRD4/H3K27ac was also reported in DM patients with PAD as compared to non-diabetic controls. Conclusion: Our findings set the stage for clinical trials testing APA in DM patients with PAD.

Bromodomain 4 inhibition leads to MYCN downregulation in Wilms tumor.

Wilms tumor is the most common childhood kidney cancer. Two distinct histological subtypes of Wilms tumor have been described: tumors lacking anaplasia (the favorable subtype) and tumors displaying anaplastic features (the unfavorable subtype). Children with favorable disease generally have a very good prognosis, whereas those with anaplasia are oftentimes refractory to standard treatments and suffer poor outcomes, leading to an unmet clinical need. MYCN dysregulation has been associated with a number of pediatric cancers including Wilms tumor. In this context, we undertook a functional genomics approach to uncover novel therapeutic strategies for those patients with anaplastic Wilms tumor. Genomic analysis and in vitro experimentation demonstrate that cell growth can be reduced by modulating MYCN overexpression via bromodomain 4 (BRD4) inhibition in both anaplastic and nonanaplastic Wilms tumor models. We observed a time-dependent reduction of MYCN and MYCC protein levels upon BRD4 inhibition in Wilms tumor cell lines, which led to cell death and proliferation suppression. BRD4 inhibition significantly reduced tumor volumes in Wilms tumor patient-derived xenograft (PDX) mouse models. We suggest that AZD5153, a novel dual-BRD4 inhibitor, can reduce MYCN levels in both anaplastic and nonanaplastic Wilms tumor cell lines, reduces tumor volume in Wilms tumor PDXs, and should be further explored for its therapeutic potential.

Targeting the Bromodomain of BRG-1/BRM Subunit of the SWI/SNF Complex Increases the Anticancer Activity of Temozolomide in Glioblastoma.

Glioblastoma (GBM) is a deadly and incurable brain cancer with limited therapeutic options. PFI-3 is a small-molecule bromodomain (BRD) inhibitor of the BRM/BRG1 subunits of the SWI/SNF chromatin remodeling complex. The objective of this study is to determine the efficacy of PFI-3 as a potential GBM therapy. We report that PFI-3 binds to these BRDs when expressed in GBM cells. PFI-3 markedly enhanced the antiproliferative and cell death-inducing effects of temozolomide (TMZ) in TMZ-sensitive GBM cells as well as overcame the chemoresistance of highly TMZ-resistant GBM cells. PFI-3 also altered gene expression in GBM and enhanced the basal and interferon-induced expression of a subset of interferon-responsive genes. Besides the effects of PFI-3 on GBM cells in vitro, we found that PFI-3 markedly potentiated the anticancer effect of TMZ in an intracranial GBM animal model, resulting in a marked increase in survival of animals bearing GBM tumors. Taken together, we identified the BRG1 and BRM subunits of SWI/SNF as novel targets in GBM and revealed the therapeutic potential of applying small molecule inhibitors of SWI/SNF to improve the clinical outcome in GBM using standard-of-care chemotherapy.

The Functions of BET Proteins in Gene Transcription of Biology and Diseases.

The BET (bromodomain and extra-terminal domain) family proteins, consisting of BRD2, BRD3, BRD4, and testis-specific BRDT, are widely acknowledged as major transcriptional regulators in biology. They are characterized by two tandem bromodomains (BDs) that bind to lysine-acetylated histones and transcription factors, recruit transcription factors and coactivators to target gene sites, and activate RNA polymerase II machinery for transcriptional elongation. Pharmacological inhibition of BET proteins with BD inhibitors has been shown as a promising therapeutic strategy for the treatment of many human diseases including cancer and inflammatory disorders. The recent advances in bromodomain protein biology have further uncovered the complex and versatile functions of BET proteins in the regulation of gene expression in chromatin. In this review article, we highlight our current understanding of BET proteins’ functions in mediating protein–protein interactions required for chromatin-templated gene transcription and splicing, chromatin remodeling, DNA replication, and DNA damage repair. We further discuss context-dependent activator vs. repressor functions of individual BET proteins, isoforms, and bromodomains that may be harnessed for future development of BET bromodomain inhibitors as emerging epigenetic therapies for cancer and inflammatory disorders.

INHIBITION OF BRD PROTEINS SUPPRESSES THE PHENOTYPE OF UTERINE FIBROIDS VIA REGULATION OF N6-METHYLADENOSINE REGULATORS

Bromodomain (BRD)-containing proteins are involved in many biological processes, most notably epigenetic regulation of transcription, and BRD protein dysfunction has been linked to many diseases including tumorigenesis. However, the role of BRD proteins in the pathogenesis of uterine fibroids (UFs), the most common pelvic tumors in women of reproductive age, is unknown. The present study aimed to determine the expression pattern of BRD proteins in UFs and matching myometrium and assess the effect of BRD inhibitors on UF phenotype and epitranscriptomic changes. Human UFs (n=22) and adjacent myometrium tissues (MyoF, n=7) were collected at the time of hysterectomy at the University of Chicago. Western blot (WB) was performed to determine the levels of BRD proteins in UFs and myometrium. Fourteen BRD inhibitors from the Epigenetic Chemical Probe Library provided by the University of Illinois at Chicago were used to determine the cell proliferation by MTT assay. Two BRD inhibitors (I-BRD9 and TP-472) were further used to explore their mechanistic actions. The student’s t-test was used to determine the significant difference. Our studies demonstrated that among 22 UFs analyzed, 96% (21/22, p<0.01), 55% (12/22), 82% (18/22, p<0.01) exhibited the upregulation of BRD2, BRD3, and BRD9 respectively, suggesting the aberrant BRD protein expression may contribute to the pathogenesis of UFs. To examine the biological impact of BRD inhibition on the UFs, 14 BRD inhibitors were screened to identify specific and potent BRD inhibitors in UF cells. 43% (6/14) of these BRD inhibitors selectively inhibited UF, but not myometrial cell proliferation. Furthermore, targeting BRD proteins with BRD inhibitors (I-BRD9 and TP-472) decreased the protein levels of extracellular matrix proteins (Type I Collagen, fibronectin), PCNA (cell proliferation marker), and Bcl-2 (anti-apoptotic marker) in a dose- and time-dependent manner in UF cells. Notably, inhibition of BRD proteins with I-BRD9 and TP-472 downregulated the expression of METTL3, YTHDC2, and YTHDF2, which are the key regulators for N6-methyladenosine (m6A) modification, indicating the tight link between BRD proteins and epitranscriptomic landscape. These results demonstrate for the first time that BRD proteins are aberrantly expressed in UFs. BRD inhibitors suppress the UF phenotype while concomitantly altering the expression of the central m6A regulators.

Binding specificity and function of the SWI/SNF subunit SMARCA4 bromodomain interaction with acetylated histone H3K14

Bromodomains (BD) are conserved reader modules that bind acetylated lysine residues on histones. Although much has been learned regarding the in vitro properties of these domains, less is known about their function within chromatin complexes. SWI/SNF chromatin-remodeling complexes modulate transcription and contribute to DNA damage repair. Mutations in SWI/SNF subunits have been implicated in many cancers. Here we demonstrate that the BD of Caenorhabditis elegans SMARCA4/BRG1, a core SWI/SNF subunit, recognizes acetylated lysine 14 of histone H3 (H3K14ac), similar to its Homo sapiens ortholog. We identify the interactions of SMARCA4 with the acetylated histone peptide from a 1.29 Å-resolution crystal structure of the CeSMARCA4 BD–H3K14ac complex. Significantly, most of the SMARCA4 BD residues in contact with the histone peptide are conserved with other proteins containing family VIII bromodomains. Based on the premise that binding specificity is conserved among bromodomain orthologs, we propose that loop residues outside of the binding pocket position contact residues to recognize the H3K14ac sequence. CRISPR-Cas9-mediated mutations in the SMARCA4 BD that abolish H3K14ac binding in vitro had little or no effect on C. elegans viability or physiological function in vivo. However, combining SMARCA4 BD mutations with knockdown of the SWI/SNF accessory subunit PBRM-1 resulted in severe developmental defects in animals. In conclusion, we demonstrated an essential function for the SWI/SNF bromodomain in vivo and detected potential redundancy in epigenetic readers in regulating chromatin remodeling. These findings have implications for the development of small-molecule BD inhibitors to treat cancers and other diseases.

Retraction Note: A biomimetic assay platform for the interrogation of antigen-dependent anti-tumor T-cell function

Overcoming tumor-mediated immunosuppression and enhancing cytotoxic T-cell activity within the tumor microenvironment are two central goals of immuno-oncology (IO) drug discovery initiatives. However, exploratory assays involving immune components are often plagued by low-throughput and poor clinical relevance. Here we present an innovative ultra-high-content assay platform for interrogating T-cell-mediated killing of 3D multicellular tumor spheroids. Employing this assay platform in a chemical genomics screen of 1800 annotated compounds enabled identification of small molecule perturbagens capable of enhancing cytotoxic CD8+ T-cell activity in an antigen-dependent manner. Specifically, cyclin-dependent kinase (CDK) and bromodomain (BRD) protein inhibitors were shown to significantly augment anti-tumor T-cell function by increasing cytolytic granule and type II interferon secretion in T-cells in addition to upregulating major histocompatibility complex (MHC) expression and antigen presentation in tumor cells. The described biotechnology screening platform yields multi-parametric, clinically-relevant data and can be employed kinetically for the discovery of first-in-class IO therapeutic agents. Jeremy To et al. develop an ultra-high-content assay platform used to interrogate T-cell-mediated killing of 3D multicellular tumor spheroids. They screen 1,800 annotated compounds to identify small molecules that enhance cytotoxic CD8+ T-cell activity in an antigen-dependent manner, demonstrating the utility of this assay platform.

Selectively targeting individual bromodomain: Drug discovery and molecular mechanisms

Bromodomain-containing proteins include bromodomain and extra-terminal (BET) and non-BET families. Due to the conserved bromodomain (BD) module between BD-containing proteins, and especially BETs with each member having two BDs (BD1 and BD2), the high degree of structural similarity makes BD-selective inhibitors much difficult to be designed. However, increasing evidences emphasized that individual BDs had distinct functions and different cellular phenotypes after pharmacological inhibition, and selectively targeting one of the BDs could result in a different efficacy and tolerability profile. This review is to summarize the pioneering progress of BD-selective inhibitors targeting BET and non-BET proteins, focusing on their structural features, biological activity, therapeutic application and experimental/theoretical mechanisms. The present proteolysis targeting chimeras (PROTAC) degraders targeting BDs, and clinical status of BD-selective inhibitors were also analyzed, providing a new insight into future direction of bromodomain-selective drug discovery.

Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain.

The ATPase Family, AAA domain-containing protein 2 (ATAD2) bromodomain (BRD) has a canonical bromodomain structure consisting of four α-helices. ATAD2 functions as a co-activator of the androgen and estrogen receptors as well as the MYC and E2F transcription factors. ATAD2 also functions during DNA replication, recognizing newly synthesized histones. In addition, ATAD2 is shown to be up-regulated in multiple forms of cancer including breast, lung, gastric, endometrial, renal, and prostate. Furthermore, up-regulation of ATAD2 is strongly correlated with poor prognosis in many types of cancer, making the ATAD2 bromodomain an innovative target for cancer therapeutics. In this study, we describe the recognition of histone acetyllysine modifications by the ATAD2 bromodomain. Residue-specific information on the complex formed between the histone tail and the ATAD2 bromodomain, obtained through nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallography, illustrates key residues lining the binding pocket, which are involved in coordination of di-acetylated histone tails. Analytical ultracentrifugation, NMR relaxation data, and isothermal titration calorimetry further confirm the monomeric state of the functionally active ATAD2 bromodomain in complex with di-acetylated histone ligands. Overall, we describe histone tail recognition by ATAD2 BRD and illustrate that one acetyllysine group is primarily engaged by the conserved asparagine (N1064), the “RVF” shelf residues, and the flexible ZA loop. Coordination of a second acetyllysine group also occurs within the same binding pocket but is essentially governed by unique hydrophobic and electrostatic interactions making the di-acetyllysine histone coordination more specific than previously presumed.

Retinal Inflammation, Oxidative Stress, and Vascular Impairment Is Ablated in Diabetic Mice Receiving XMD8-92 Treatment.

The global number of diabetics continues to rise annually. As diabetes progresses, almost all of Type I and more than half of Type II diabetics develop diabetic retinopathy. Diabetic retinopathy is a microvascular disease of the retina, and is the leading cause of blindness in the working-age population worldwide. With such a significant health impact, new drugs are required to halt the blinding threat posed by this visual disorder. The cause of diabetic retinopathy is multifactorial, and an optimal therapeutic would halt inflammation, cease photoreceptor cell dysfunction, and ablate vascular impairment. XMD8-92 is a small molecule inhibitor that blocks inflammatory activity downstream of ERK5 (extracellular signal-related kinase 5) and BRD4 (bromodomain 4). ERK5 elicits inflammation, is increased in Type II diabetics, and plays a pathologic role in diabetic nephropathy, while BRD4 induces retinal inflammation and plays a role in retinal degeneration. Further, we provide evidence that suggests both pERK5 and BRD4 expression are increased in the retinas of our STZ (streptozotocin)-induced diabetic mice. Taken together, we hypothesized that XMD8-92 would be a good therapeutic candidate for diabetic retinopathy, and tested XMD8-92 in a murine model of diabetic retinopathy. In the current study, we developed an in vivo treatment regimen by administering one 100 μL subcutaneous injection of saline containing 20 μM of XMD8-92 weekly, to STZ-induced diabetic mice. XMD8-92 treatments significantly decreased diabetes-mediated retinal inflammation, VEGF production, and oxidative stress. Further, XMD8-92 halted the degradation of ZO-1 (zonula occludens-1), which is a tight junction protein associated with vascular permeability in the retina. Finally, XMD8-92 treatment ablated diabetes-mediated vascular leakage and capillary degeneration, which are the clinical hallmarks of non-proliferative diabetic retinopathy. Taken together, this study provides strong evidence that XMD8-92 could be a potentially novel therapeutic for diabetic retinopathy.

The signal transducer CD24 suppresses the germ cell program and promotes an ectodermal rather than mesodermal cell fate in embryonal carcinomas.

Testicular germ cell tumors (GCTs) are stratified into seminomas and nonseminomas. Seminomas share many histological and molecular features with primordial germ cells, whereas the nonseminoma stem cell population—embryonal carcinoma (EC)—is pluripotent and thus able to differentiate into cells of all three germ layers (teratomas). Furthermore, ECs are capable of differentiating into extra‐embryonic lineages (yolk sac tumors, choriocarcinomas). In this study, we deciphered the molecular and (epi)genetic mechanisms regulating expression of CD24, a highly glycosylated signaling molecule upregulated in many cancers. CD24 is overexpressed in ECs compared with other GCT entities and can be associated with an undifferentiated pluripotent cell fate. We demonstrate that CD24 can be transactivated by the pluripotency factor SOX2, which binds in proximity to the CD24 promoter. In GCTs, CD24 expression is controlled by epigenetic mechanisms, that is, histone acetylation, since CD24 can be induced by the application histone deacetylase inhibitors. Vice versa, CD24 expression is downregulated upon inhibition of histone methyltransferases, E3 ubiquitin ligases, or bromodomain (BRD) proteins. Additionally, three‐dimensional (3D) co‐cultivation of EC cells with microenvironmental cells, such as fibroblasts, and endothelial or immune cells, reduced CD24 expression, suggesting that crosstalk with the somatic microenvironment influences CD24 expression. In a CRISPR/Cas9 deficiency model, we demonstrate that CD24 fulfills a bivalent role in differentiation via regulation of homeobox, and phospho‐ and glycoproteins; that is, it is involved in suppressing the germ cell/spermatogenesis program and mesodermal/endodermal differentiation, while poising the cells for ectodermal differentiation. Finally, blocking CD24 by a monoclonal antibody enhanced sensitivity toward cisplatin in EC cells, including cisplatin‐resistant subclones, highlighting CD24 as a putative target in combination with cisplatin.

Identification of isoform/domain-selective fragments from the selection of DNA-encoded dynamic library

DNA-encoded chemical library (DEL) has emerged to be a powerful ligand screening technology in drug discovery. Recently, we reported a DNA-encoded dynamic library (DEDL) approach that combines the principle of traditional dynamic combinatorial library (DCL) with DEL. DEDL has shown excellent potential in fragment-based ligand discovery with a variety of protein targets. Here, we further tested the utility of DEDL in identifying low molecular weight fragments that are selective for different isoforms or domains of the same protein family. A 10,000-member DEDL was selected against sirtuin-1, 2, and 5 (SIRT1, 2, 5) and the BD1 and BD2 domains of bromodomain 4 (BRD4), respectively. Albeit with modest potency, a series of isoform/domain-selective fragments were identified and the corresponding inhibitors were derived by fragment linking.