Design, synthesis and degradation activity of BRD4-targeting ATTECs / 药学实践杂志

Objective To design and synthesize autophagic degraders targeting BRD4 based on autophagosome tethering compound (ATTEC) strategy and test their BRD4 degradation activity. Methods BRD4-targeting ATTECs were constructed by conjugating ispinesib that used as a LC3 ligand and JQ1 through a variety of alkane linkers. The final compounds were confirmed by 1H NMR, 13C NMR and ESI-MS, and their degradation activity in different cell lines were tested by Western Blot. Results Five BRD4-ATTEC molecules were successfully synthesized for the first time. Compound 4 showed moderate BRD4 degradation activity in different cell lines. Conclusion The novel BRD4 autophagic degraders were discovered, which expanded the applicability of targeted autophagic degradation via ATTEC.

On-demand integrated nano-engager converting cold tumors to hot via increased DNA damage and dual immune checkpoint inhibition

Cancer immunotherapy has become a promising strategy. However, the effectiveness of immunotherapy is restricted in "cold tumors" characterized with insufficient T cells intratumoral infiltration and failed T cells priming. Herein, an on-demand integrated nano-engager (JOT-Lip) was developed to convert cold tumors to hot via "increased DNA damage and dual immune checkpoint inhibition" strategy. JOT-Lip was engineered by co-loading oxaliplatin (Oxa) and JQ1 into liposomes with T-cell immunoglobulin mucin-3 antibodies (Tim-3 mAb) coupled on the liposomal surface by metalloproteinase-2 (MMP-2)-sensitive linker. JQ1 inhibited DNA repair to increase DNA damage and immunogenic cell death (ICD) of Oxa, thus promoting T cells intratumoral infiltration. In addition, JQ1 inhibited PD-1/PD-L1 pathway, achieving dual immune checkpoint inhibition combining with Tim-3 mAb, thus effectively promoting T cells priming. It is demonstrated that JOT-Lip not only increased DNA damage and promoted the release of damage-associated molecular patterns (DAMPs), but also enhanced T cells intratumoral infiltration and promoted T cell priming, which successfully converted cold tumors to hot and showed significant anti-tumor and anti-metastasis effects. Collectively, our study provides a rational design of an effective combination regimen and an ideal co-delivery system to convert cold tumors to hot, which holds great potential in clinical cancer chemoimmunotherapy.

Effect and Mechanism of Xianlian Jiedu Prescription Against Proliferation of Colorectal Cancer Cells in Hypoxic Microenvironment / 中国实验方剂学杂志

ObjectiveTo study the effect of Xianlian Jiedu prescription （XLJDP） on the activation of nuclear transcription factor-κB （NF-κB） signaling pathway induced by bromodomain-containing protein 4 （Brd4） in hypoxic microenvironment and to explore its mechanism in inhibiting the proliferation of colorectal cancer HT-29 cells. MethodThe human colorectal cancer HT-29 cells were cultured in a hypoxic incubator or normoxia incubator and treated with XLJDP at 0.8，1，1.2，1.6，3.2，6.4，and 12.8 g·L-1 for 48 h， respectively. Following the detection of cell vitality using methyl thiazolyl tetrazolium （MTT） colorimetry， the effects of XLJDP （1.25，2.5，and 5 g·L-1） on the cell mitochondrial membrane potential were determined using a fluorescent probe （JC-1）， and the apoptosis of colorectal cancer HT-29 cells was detected by flow cytometry. The cell colony formation assay and 5-ethynyl-2'-deoxyuridine （EDU） staining were conducted to test the proliferation of colorectal cancer HT-29 cells. The Western blot was carried out to measure the expression levels of Brd4 and its downstream relevant proteins such as c-Myc and hexamethylene bisacetamide-inducible protein 1 （HEXIM1）， as well as the effects of XLJDP on related proteins in the NF-κB signaling pathway. ResultCompared with the blank control group， XLJDP at 0.8，1，1.2，1.6，3.2，6.4，and 12.8 g·L-1 inhibited the vitality of colorectal cancer HT-29 cells （P<0.05 ， P<0.01）， with the median inhibitory concentration （IC50） under the hypoxic condition higher than that under the normoxia condition. Compared with the blank control group， XLJDP at 1.25，2.5，and 5 g·L-1 significantly decreased the mitochondria membrane potential， enhanced the apoptosis （P<0.05，P<0.01）， and lowered the number of cell colonies and also the EDU-positive cells （P<0.05， P<0.01）. The results of Western blot showed that compared with the blank control group， XLJDP at 1.25，2.5，and 5 g·L-1 down-regulated Brd4， c-Myc， p-NF-κB p65， and p-IκBα protein expression to varying degrees and up-regulated the expression of HEXIM1 （P<0.05， P<0.01）. ConclusionIn the hypoxic microenvironment， XLJDP inhibits the proliferation of colorectal cancer HT-29 cells regulated by Brd4， which may be related to its inhibition of the activation of NF-κB signaling pathway.

A BRD4 PROTAC nanodrug for glioma therapy via the intervention of tumor cells proliferation, apoptosis and M2 macrophages polarization

Glioma is a primary aggressive brain tumor with high recurrence rate. The poor efficiency of chemotherapeutic drugs crossing the blood‒brain barrier (BBB) is well-known as one of the main challenges for anti-glioma therapy. Moreover, massive infiltrated tumor-associated macrophages (TAMs) in glioma further thwart the drug efficacy. Herein, a therapeutic nanosystem (SPP-ARV-825) is constructed by incorporating the BRD4-degrading proteolytic targeting chimera (PROTAC) ARV-825 into the complex micelle (SPP) composed of substance P (SP) peptide-modified poly(ethylene glycol)-poly(d,l-lactic acid)(SP-PEG-PDLLA) and methoxy poly(ethylene glycol)-poly(d,l-lactic acid) (mPEG-PDLLA, PP), which could penetrate BBB and target brain tumor. Subsequently, released drug engenders antitumor effect via attenuating cells proliferation, inducing cells apoptosis and suppressing M2 macrophages polarization through the inhibition of IRF4 promoter transcription and phosphorylation of STAT6, STAT3 and AKT. Taken together, our work demonstrates the versatile role and therapeutic efficacy of SPP-ARV-825 micelle against glioma, which may provide a novel strategy for glioma therapy in future.

Discovery of novel 4-phenylquinazoline-based BRD4 inhibitors for cardiac fibrosis

Bromodomain containing protein 4 (BRD4), as an epigenetic reader, can specifically bind to the acetyl lysine residues of histones and has emerged as an attractive therapeutic target for various diseases, including cancer, cardiac remodeling and heart failure. Herein, we described the discovery of hit 5 bearing 4-phenylquinazoline skeleton through a high-throughput virtual screen using 2,003,400 compound library (enamine). Then, structure-activity relationship (SAR) study was performed and 47 new 4-phenylquinazoline derivatives toward BRD4 were further designed, synthesized and evaluated, using HTRF assay set up in our lab. Eventually, we identified compound C-34, which possessed better pharmacokinetic and physicochemical properties as well as lower cytotoxicity against NRCF and NRCM cells, compared to the positive control JQ1. Using computer-based molecular docking and cellular thermal shift assay, we further verified that C-34 could target BRD4 at molecular and cellular levels. Furthermore, treatment with C-34 effectively alleviated fibroblast activation in vitro and cardiac fibrosis in vivo, which was correlated with the decreased expression of BRD4 downstream target c-MYC as well as the depressed TGF-β1/Smad2/3 signaling pathway. Taken together, our findings indicate that novel BRD4 inhibitor C-34 tethering a 4-phenylquinazoline scaffold can serve as a lead compound for further development to treat fibrotic cardiovascular disease.

BRD4 inhibitor specifically inhibits the development of wild-type Kras differentiated thyroid carcinoma by regulating BRD4/miR-106b-5p/P21 axis / 国际肿瘤学杂志

Objective:To explore the influence of bromodomain-containing protein 4 (BRD4) inhibitor on wild-type Kras differentiated thyroid carcinoma (DTC) and its mechanism.Methods:The DTC cell line Kras WT TPC-1 was selected and the mutant Kras G12D TPC-1 cells were constructed. CCK-8 assay was used to detect the effect of BRD4 inhibitor JQ-1 on the proliferation activity of Kras WT TPC-1 cells. Kras WT TPC-1 cells were treated with 0.2 μmol/L JQ-1 (JQ-1 group), and a negative control group (NC group) was set. Transwell invasion assay and flow cytometry were used to detect the effect of JQ-1 on the invasion and apoptosis of Kras WT TPC-1 cells. The effect of JQ-1 on the expressions of BRD4, miR-106b-5p and P21, and the effect of P21 inhibitor UC2288 on the expressions of P21 and BRD4 were detected. Kras WT TPC-1 cells were divided into JQ-1+ NC-OE group, JQ-1+ p21-OE group (overexpression of p21) and JQ-1+ p21-OE+ miR-106b-5p mimic group (overexpression of p21 and miR-106b-5 at the same time), and the proliferation, invasion and apoptosis of cells in each group were detected. TPC-1 cells were divided into Kras WT group, Kras WT+ JQ-1 group, Kras G12D group and Kras G12D+ JQ-1 group, and the cell proliferation, invasion and apoptosis of each group were detected. Results:JQ-1 inhibited the proliferation activity of Kras WT TPC-1 cells in a dose-dependent and time-dependent manner. In the NC group and JQ-1 group, the numbers of cell invasion were 124.67±9.61 and 82.67±8.02, and the apoptosis rates were (5.91±0.34)% and (10.33±1.10)%, respectively, with statistically significant differences ( t=5.812, P=0.004; t=6.653, P=0.003). JQ-1 significantly inhibited the expressions of BRD4 and miR-106b-5p, and promoted the expression of P21 in Kras WT TPC-1 cells. UC2288 significantly inhibited P21 expression, but had no significant effect on BRD4 expression. In the JQ-1+ NC-OE group, JQ-1+ p21-OE group and JQ-1+ p21-OE+ miR-106b-5p mimic group, the proliferation activities at 24 h of Kras WT TPC-1 cells was 0.46±0.03, 0.35±0.04 and 0.44±0.03 ( F=8.720, P=0.017), and the proliferation activity of JQ-1+ p21-OE group was significantly lower than that of the JQ-1+ NC-OE group ( P<0.05). The numbers of cell invasion in the three groups were 83.00±9.17, 56.67±6.03 and 79.67±10.07 ( F=8.347, P=0.018), and the number of cell invasion in the JQ-1+ p21-OE group was significantly lower than that in the JQ-1+ NC-OE group ( P=0.009). The apoptosis rates of the three groups were (10.00±0.49)%, (15.39±1.14)% and (10.32±0.80)% ( F=37.764, P<0.001), and the apoptosis rate of the JQ-1+ p21-OE group was significantly higher than that in the JQ-1+ NC-OE group ( P<0.001). There were no significant differences in cell proliferation activity, invasion number and apoptosis rate between JQ-1+ p21-OE+ miR-106b-5p mimic group and JQ-1+ NC-OE group (all P>0.05). In Kras WT group, Kras WT+ JQ-1 group, Kras G12D group and Kras G12D+ JQ-1 group, the cell proliferation activities at 24 h were 0.50±0.05, 0.39±0.04, 0.68±0.08 and 0.64±0.05 ( F=17.776, P<0.001). Compared with the Kras WT group, cell proliferation activity in the Kras WT+ JQ-1 group was significantly decreased, while that in the Kras G12D group was significantly increased (both P<0.05). The numbers of cell invasion in the four groups were 129.33±11.50, 86.00±9.54, 161.67±13.01 and 146.33±13.20 ( F=22.598, P<0.001). Compared with the Kras WT group, the number of cell invasion in the Kras WT+ JQ-1 group was significantly decreased ( P=0.002), and that in the Kras G12D group was significantly increased ( P=0.010). The apoptosis rates in the four groups were (6.17±0.50)%, (10.42±0.73)%, (3.43±0.47)% and (3.41±0.32)% ( F=119.170, P<0.001). Compared with the Kras WT group, the apoptosis rate in the Kras WT+ JQ-1 group was significantly increased ( P<0.001), and that in the Kras G12D group was significantly decreased ( P<0.001). There were no significant differences in cell proliferation activity, invasion number and apoptosis rate between Kras G12D+ JQ-1 group and Kras G12D group (all P>0.05). Conclusion:BRD4 inhibitor can specifically inhibit the development of wild-type Kras DTC via regulating the molecular axis of BRD4/miR-106b-5p/P21, but has no significant effect on the proliferation, invasion and apoptosis of mutant Kras DTC tumor cells.

Progress of research on inhibition strategy of bromodomain-containing protein 4 and its application in tumor therapy / 中国药科大学学报

@#Bromodomain-containing protein 4 (BRD4), a new target for tumor therapy, is the most important member of the bromodomain and extra-terminal family. The overexpression of BRD4 is associated with genesis and development of various cancers.Used either alone or in combination with other treatments such as chemotherapy, photothermal therapy and immunotherapy, the BRD4 inhibitors or degraders exhibited excellent antitumor effects, providing a new direction in tumor treatment. In this review, the structure and function of BRD4, the inhibition strategies of BRD4, the application in tumor combination therapy and drug resistance are introduced, which provides reference for targeting BRD4 in tumor therapy.

The poly(ADP-ribosyl)ation of BRD4 mediated by PARP1 promoted pathological cardiac hypertrophy

The bromodomain and extraterminal (BET) family member BRD4 is pivotal in the pathogenesis of cardiac hypertrophy. BRD4 induces hypertrophic gene expression by binding to the acetylated chromatin, facilitating the phosphorylation of RNA polymerases II (Pol II) and leading to transcription elongation. The present study identified a novel post-translational modification of BRD4: poly(ADP-ribosyl)ation (PARylation), that was mediated by poly(ADP-ribose)polymerase-1 (PARP1) in cardiac hypertrophy. BRD4 silencing or BET inhibitors JQ1 and MS417 prevented cardiac hypertrophic responses induced by isoproterenol (ISO), whereas overexpression of BRD4 promoted cardiac hypertrophy, confirming the critical role of BRD4 in pathological cardiac hypertrophy. PARP1 was activated in ISO-induced cardiac hypertrophy and facilitated the development of cardiac hypertrophy. BRD4 was involved in the prohypertrophic effect of PARP1, as implied by the observations that BRD4 inhibition or silencing reversed PARP1-induced hypertrophic responses, and that BRD4 overexpression suppressed the anti-hypertrophic effect of PARP1 inhibitors. Interactions of BRD4 and PARP1 were observed by co-immunoprecipitation and immunofluorescence. PARylation of BRD4 induced by PARP1 was investigated by PARylation assays. In response to hypertrophic stimuli like ISO, PARylation level of BRD4 was elevated, along with enhanced interactions between BRD4 and PARP1. By investigating the PARylation of truncation mutants of BRD4, the C-terminal domain (CTD) was identified as the PARylation modification sites of BRD4. PARylation of BRD4 facilitated its binding to the transcription start sites (TSS) of hypertrophic genes, resulting in enhanced phosphorylation of RNA Pol II and transcription activation of hypertrophic genes. The present findings suggest that strategies targeting inhibition of PARP1-BRD4 might have therapeutic potential for pathological cardiac hypertrophy.

Development of hedgehog pathway inhibitors by epigenetically targeting GLI through BET bromodomain for the treatment of medulloblastoma

Medulloblastoma (MB) is a common yet highly heterogeneous childhood malignant brain tumor, however, clinically effective molecular targeted therapy is lacking. Modulation of hedgehog (HH) signaling by epigenetically targeting the transcriptional factors GLI through bromodomain-containing protein 4 (BRD4) has recently spurred new interest as potential treatment of HH-driven MB. Through screening of current clinical BRD4 inhibitors for their inhibitory potency against glioma-associated oncogene homolog (GLI) protein, the BRD4 inhibitor

Design, synthesis, and biological evaluation of quinazolin-4(3H)-one derivatives co-targeting poly(ADP-ribose) polymerase-1 and bromodomain containing protein 4 for breast cancer therapy

@#This study was aimed to design the first dual-target small-molecule inhibitor co-targeting poly (ADP-ribose) polymerase-1 (PARP1) and bromodomain containing protein 4 (BRD4), which had important cross relation in the global network of breast cancer, reflecting the synthetic lethal effect. A series of new BRD4 and PARP1 dual-target inhibitors were discovered and synthesized by fragment-based combinatorial screening and activity assays that together led to the chemical optimization. Among these compounds, 19d was selected and exhibited micromole enzymatic potencies against BRD4 and PARP1, respectively. Compound 19d was further shown to efficiently modulate the expression of BRD4 and PARP1. Subsequently, compound 19d was found to induce breast cancer cell apoptosis and stimulate cell cycle arrest at G1 phase. Following pharmacokinetic studies, compound 19d showed its antitumor activity in breast cancer susceptibility gene 1/2 (BRCA1/2) wild-type MDA-MB-468 and MCF-7 xenograft models without apparent toxicity and loss of body weight. These results together demonstrated that a highly potent dual-targeted inhibitor was successfully synthesized and indicated that co-targeting of BRD4 and PARP1 based on the concept of synthetic lethality would be a promising therapeutic strategy for breast cancer.

Effects of BRD4 inhibitor on histone crotonylation, proliferation and migration of prostate cancer cells / 上海交通大学学报（医学版）

Objective: To investigate the effects of bromodomain-containing protein 4 (BRD4) inhibitor I-BET762 on histone crotonylation, proliferation and migration of prostate cancer cells. Methods: Three human prostate cancer cell lines, i.e., LNCaP, C4-2B and PC-3, were respectively treated with I-BET762 of half maximal inhibitory concentration. Histone crotonylation modification and acetylase expression were detected by Western blotting. CCK-8, transwell migration test and scratch test were used to detect the effects of I-BET762 on proliferation and migration of LNCaP, C4-2B and PC-3 cells. Results: I-BET762 inhibited the expression of histone acetylase P300 and GCN5, and reduced the histone crotonylation modification. Transwell migration test and scratch test showed that I-BET762 could inhibit the migration of prostate cancer cell lines LNCaP, C4-2B and PC-3 (all P<0.01); CCK-8 test showed that the proliferation of three prostate cancer cell lines was inhibited by I-BET762. Conclusion: In prostate cancer cells, I-BET762 can reduce the histone crotonylation and also inhibit cell proliferation and migration.

Formaldehyde induced acute inflammation pain leads to enhanced expression of BRD 4 in spinal cord of mice / 基础医学与临床

Objective To investigate the expression of BRD 4 in the spinal cord and its relationship with acute in-flammation pain induced by formaldehyde in mice.Methods Thirty-six mice were randomly divided into three groups:control group,formaldehyde group and indomethacin+formaldehyde group;25 μL 1%formaldehyde was injected into the right plantar to establish the acute inflammationpain model,while the indomethacin(20 mg/kg) was injected intraperitoneally 1 hour before formaldehyde injection.Then,all the mice were video recored for 1h to observe the spontaneous pain.Then,cell localization of BRD4 in the spinal cord of normal mice was determined by immunofluorescence assasy.The expression of BRD4 in spinal cord was detected by immunohistochemistry and Western blot.Results Immunofluorescence showed that BRD 4 was mainly co-locolized with the neuronal marker NeuN in the spinal cord of normal mice.Formaldehyde injection could induce two-phase spontaneous pain, while indomethacin intervention could only decrease the second phase pain(P<0.05).Furthermore,formaldehyde injec-tion led to significantly enhanced expression of BRD 4 in bilateral spinal cord,which was remarkbly inhibited by in-domethacin(P<0.05).Conclusions Up-regulation of BRD4 in spinal dorsal horn may be involved in the acute in-flammatory pain.

BRD4 interacts with PML/RARα in acute promyelocytic leukemia / 医学前沿

Bromodomain-containing 4 (BRD4) has been considered as an important requirement for disease maintenance and an attractive therapeutic target for cancer therapy. This protein can be targeted by JQ1, a selective small-molecule inhibitor. However, few studies have investigated whether BRD4 influenced acute promyelocytic leukemia (APL), and whether BRD4 had interaction with promyelocytic leukemia-retinoic acid receptor α (PML/RARα) fusion protein to some extent. Results from cell viability assay, cell cycle analysis, and Annexin-V/PI analysis indicated that JQ1 inhibited the growth of NB4 cells, an APL-derived cell line, and induced NB4 cell cycle arrest at G1 and apoptosis. Then, we used co-immunoprecipitation (co-IP) assay and immunoblot to demonstrate the endogenous interaction of BRD4 and PML/RARα in NB4 cells. Moreover, downregulation of PML/RARα at the mRNA and protein levels was observed upon JQ1 treatment. Furthermore, results from the RT-qPCR, ChIP-qPCR, and re-ChIP-qPCR assays showed that BRD4 and PML/RARα co-existed on the same regulatory regions of their target genes. Hence, we showed a new discovery of the interaction of BRD4 and PML/RARα, as well as the decline of PML/RARα expression, under JQ1 treatment.

Abrupt Dyskeratotic and Squamoid Cells in Poorly Differentiated Carcinoma: Case Study of Two Thoracic NUT Midline Carcinomas with Cytohistologic Correlation

Cytologic diagnosis of nuclear protein in testis (NUT) midline carcinoma (NMC) is important due to its aggressive behavior and miserable prognosis. Early diagnosis of NMC can facilitate proper management, and here we report two rare cases of thoracic NMC with cytohistologic correlation. In aspiration cytology, the tumor presented with mixed cohesive clusters and dispersed single cells, diffuse background necrosis and many neutrophils. Most of the tumor cells had scanty cytoplasm and medium-sized irregular nuclei, which had fine to granular nuclear chromatin. Interestingly, a few dyskeratotic cells or squamoid cell clusters were present in each case. Biopsy specimen histology revealed more frequent squamous differentiation, and additional immunohistochemistry tests showed nuclear expression of NUT. Because this tumor has a notorious progression and has been previously underestimated in terms of its prevalence, awareness of characteristic findings and proper ancillary tests should be considered in all suspicious cases.

BRD4 inhibition suppresses cell growth, migration and invasion of salivary adenoid cystic carcinoma

BACKGROUND: Bromodomain-containing protein 4 (BRD4) inhibition is a new therapeutic strategy for many malignancies. In this study, we aimed to explore the effect of BRD4 inhibition by JQ1 on in vitro cell growth, migration and invasion of salivary adenoid cystic carcinoma (SACC). METHODS: The human normal epithelial cells and SACC cells (ACC-LM and ACC-83) were treated with JQ1 at concentrations of 0, 0.1, 0.5 or 1 µM. Cell Counting Kit-8 (CCK-8) assay was performed to evaluate cell proliferation. Cell apoptosis and cell cycle distribution was evaluated by Flow cytometry. Immunofluorescence staining was used to examine the expression of BRD4 in SACC cells. The quantitative real-time polymerase chain reaction (qRT-PCR) assay and western blot assay were performed to examine messenger RNA (mRNA) and protein levels in SACC cells. Wound- healing assay and transwell assay were used to evaluate the activities of migration and invasion of SACC cells. RESULTS: JQ1 exhibits no adverse effects on proliferation, cell cycle and cell apoptosis of the normal human epithelial cells, while suppressed proliferation and cell cycle, and induced apoptosis of SACC cells, down-regulated the mRNA and protein levels of BRD4 in SACC cells, meanwhile reduced protein expressions of c-myc and BCL-2, two known target genes of BRD4. Moreover, JQ1 inhibited SACC cell migration and invasion by regulating key epithelial-mesenchymal transition (EMT) characteristics including E-cadherin, Vimentin and Twist. CONCLUSIONS: BRD4 is an important transcription factor in SACC and BRD4 inhibition by JQ1 may be a new strategy for SACC treatment.

Discovery of a small-molecule bromodomain-containing protein 4 inhibitor that induces AMP-activated protein kinase-modulated autophagy-associated cell death in breast cancer / 中国药理学与毒理学杂志

OBJECTIVE To discover a small-molecule bromodomain-containing protein 4 (BRD4) inhibitor that induces AMP- activated protein kinase- modulated autophagy- associated cell death in breast cancer and exploreits potential mechanisms. METHODS BRD4 interactors were analyzed by PPI network prediction and The Cancer Genome Atlas (TCGA) analysis. The interaction between BRD4 and AMPK was confirmed by co- immunoprecipitation assay. Novel BRD4 inhibitors were designed and synthesized based upon pharmacophore analysis of BRD4 (1), then screened by anti-proliferative activity and Alpha Screen of BRD4 (1). The selectivity of the best candidate compound 8f was validated by co-crystallization, FRET assay and co-immuno precipitation assay. The mechanisms of 8f were investigated by fluorescence microscopy, electron microscopy, Western blotting, immunocy?tochemistry, siRNA and GFP-mRFP-LC3 plasmid transfections, as well as immunohistochemistry and immunofluorescence. Potential mechanisms were discovered by iTRAQ- based proteomics analysis and the therapeutic effect of 8f was assessed by xenograft breast cancer mouse and zebrafish models. RESULTS We identified that BRD4 interacted with AMPK, which was remarkably downregulated in breast cancer. We next designed and synthesized 49 candidate compounds, and eventually discovered a selective small-molecule inhibitor of BRD4 (8f). Subsequently, 8f was discovered to induce autophagy-associated cell death (ACD) by BRD4- AMPK interaction, and thus activating AMPK- mTOR- ULK1-modulated autophagic pathway in breast cancer cells. Interestingly, the iTRAQ- based proteomics analyses revealed that 8f induced ACD pathways, involved in HMGB1, VDAC1/2 and eEF2. Moreover, 8f displayed a therapeutic potential on both xenograft breast cancer mouse and zebrafish models. CONCLUSION We discovered a novel small-molecule inhibitor of BRD4 that induces BRD4-AMPK-modulated ACD in breast cancer, which may provide a candidate drug for future cancer therapy.

Design, synthesis and evaluation of a novel BRD4 protein inhibitors / 药学学报

Bromodomain-containing proteins (BCPs) can specifically recognize acetylated lysine (KAc) in histones and other substrate proteins. Recently, several kinase inhibitors were found to inhibit bromodomains, such as the PLK1 inhibitor BI-2536 and the JAK2 inhibitor TG101209, which bind to BRD4 with IC50 values of 25 nmol·L-1 and 130 nmol·L-1, respectively. To obtain potent BRD4 inhibitors from inhibitor BI-2536, we used dihydroquinoxalin-2(1H)-one to replace the 7,8-dihydropteridin-6(5H)-one in BI2536. By exploring the structure-activity relationships of the new dihydroquinoxalin-2(1H)-one structures, we obtained a novel phenyl side chain series of BRD4 inhibitors. We identified several potent BRD4 inhibitors, especially compounds 16, 22, 28 and 29, which had IC50 values below 100 nmol·L-1 in fluorescence anisotropy (FA) assays, indicating this series of compounds are worth to fruther investigation.

BRD4 scilencing plus gemcitabine may be a novel therapy for triple-negative breast cancer / 中国癌症杂志

Background and purpose:Breast cancer has the highest morbidity and mortality rate in women worldwide. Triple-negative breast cancer (TNBC) has no speciifc target and has low survival rate. Recent studies have veriifed BRD4 could promote tumor progression. This study aimed to detect the expression level of BRD4 in TNBC after treatment with gemcitabine, and to reveal the effect ofBRD4 silencing plus gemcitabine as a treatment for TNBC. Methods:The expression ofBRD4 in TNBC cell lines treated with gemcitabine was detected by reverse transcription PCR (RT-PCR) and Western blot. The effect of BRD4 silencing plus gemcitabine in TNBC was illustratedin vitro and in vivo.Results:The expression ofBRD4 in TNBC was signiifcantly increased after treatment with gemcitabine.In vitro,BRD4 knockdown signiifcantly lowered the IC50 value. The apoptotic rate of TNBC was signiifcantly increased in theBRD4 silencing plus gemcitabine group compared to the other. The growth rate of tumorin vivo was signiifcantly lowered in the BRD4 silencing plus gemcitabine group.Conclusion:BRD4 may play an important role in the drug resistance to gemcitabine in TNBC.BRD4 silencing plus gemcitabine may be a novel treatment strategy for TNBC.