Dynamics and Energetics of a Bromodomain in complex with bromosporine from Leishmania donovani

Leishmaniasis continues to be a neglected tropical disease, affecting people and animals and causing significant economic losses. Therefore, there is interest in the study and evaluation of new drug targets. In fact, it has been shown that by interfering with lysine-reading proteins such as bromodomain (BMD) there is a decrease in parasite survival. In this study, we researched the dynamics and energetics of the Leishmania donovani BMD in complex with bromosporin, which is considered to be a pan-inhibitor of BMDs, with the aim of understanding the molecular recognition mechanism. Molecular dynamics (MD) and non-equilibrium free energy calculation guided by steered molecular dynamics (SMD) simulations showed that the BMD has three flexible amino acid regions and bromosporin exhibiting various recognition states during the interaction. These results corroborate the promiscuity of bromosporin for energetically favourable sites, with the possibility of expanding its inhibition to other bromodomains. Furthermore, these results suggest that Van der Waals interactions have more relevance for complex recognition and residues ASN-87 and TRP-93 are key in forming hydrophobic and H-bond interactions, respectively. This research provides new insights for understanding the recognition mechanism, dynamics and energetics of the complex for the development of new therapeutic strategies.

La leishmaniasis sigue siendo una enfermedad tropical desatendida, que afecta a personas y animales y causa importantes pérdidas económicas. De ahí el interés por estudiar y evaluar nuevas dianas farmacológicas. De hecho, se ha demostrado que al interferir con proteínas lectoras de lisina como el bromodominio ("bromodomain", BMD) se produce una disminución de la supervivencia del parásito. En este artículo estudiamos la dinámica y la energética del BMD de Leishmania donovani en complejo con bromosporina, que se considera un pan-inhibidor de BMD, con el objetivo de comprender el mecanismo de reconocimiento molecular. Las simulaciones de dinámica molecular (DM) y el cálculo de energía libre de no-equilibrio guiado por dinámica molecular de estiramiento (DMS) mostraron que BMD tiene tres regiones de aminoácidos flexibles y la bromosporina presenta varios estados de reconocimiento durante la interacción. Estos resultados corroboran la promiscuidad de la bromosporina por sitios energéticamente favorables, siendo posible expandir su inhibición a otros bromodominios. Además, los resultados sugieren que las interacciones de Van der Waals tienen más relevancia para el reconocimiento del complejo y los residuos ASN-87 y TRP-93 son clave en la formación de interacciones hidrofóbicas y de puentes de hidrógeno, respectivamente. Esta investigación proporciona nuevos conocimientos para comprender el mecanismo de reconocimiento molecular, la dinámica y la energética del complejo para el desarrollo de nuevas estrategias terapéuticas.

A leishmaniose continua a ser uma doença tropical negligenciada, que afeta os seres humanos e os animais e causa perdas econômicas significativas. Daí o interesse em estudar e avaliar novos alvos de medicamentos. De fato, a interferência com proteínas leitoras de lisina, como o bromo domínio ("bromodomain", BMD), tem demonstrado diminuir a sobrevivência do parasita. Neste trabalho, estudamos a dinâmica e a energética do BMD de Leish-mania donovani em complexo com a bro-mosporina, considerada um pan-inibidor da BMDs, com o objetivo de compreender o mecanismo de reconhecimento molecular. As simulações de dinâmica molecular (MD) e cálculo de energia livre de não-equilíbrio guiada por dinâmica molecular esticamento (MDS) mostraram que o BMD tem três regiões de aminoácidos flexíveis e que a bromosporina apresenta vários estados de reconhecimento durante a interação. Esses resultados corroboram a promiscuidade da bromosporina para locais energeticamente favoráveis, possibilitando a expansão de sua inibição para outros bromodomínios. Além disso, os resultados sugerem que as interações de Van der Waals são mais relevantes no momento do reconhecimento do complexo e os resíduos ASN-87 e TRP-93 são fundamentais na formação de interações hidrofóbicas e de ligações de hidrogênio, respectivamente. Essa pesquisa fornece novos insights para compreender o mecanismo de reconhecimento, a dinâmica e a energética do complexo para o desenvolvimento de novas estratégias terapêuticas.

Advances of structure and mechanisms of bromodomain-containing protein 4 and its related research in tumors / 生物工程学报

The bromodomain and extraterminal domain (Bet) family are the regulators of the epigenome and also the pivotal driving factors for the expression of tumor related genes that tumor cells depend on for survival and proliferation. Bromodomain-containing protein 4 (Brd4) is a member of the Bet protein family. Generally, Brd4 identifies acetylated histones and binds to the promoter or enhancer region of target genes to initiate and maintain expression of tumor related genes. Brd4 is closely related to the regulation of multiple transcription factors and chromatin modification and is involved in DNA damage repair and maintenance of telomere function, thus maintaining the survival of tumor cells. This review summarizes the structure and function of Brd4 protein and the application of its inhibitors in tumor research.

Effect of microglia inhibitor Pexidartinib on reconsolidation of remote contextual fear memory in mice / 新乡医学院学报

Objective To investigate the effect of microglia inhibitor Pexidartinib on reconsolidation of remote contextual fear memory in mice.Methods Twelve healthy C57BL/6 male mice were randomly divided into experimental group and control group,with 6 mice in each group.The mice in the two groups underwent contextual fear conditioning training in the contextual fear response box to establish the contextual fear models,and the freezing time of mice after each footshock was recorded.After 7 days,the mice in the experimental group were fed with food containing Pexidartinib formulation PLX3397,while the mice in the control group were fed with regular food until the end of behavioral experiment.On the 16th day after contextual fear conditioning training,the mice were put back into the contextual fear box for recalling the fear memory without any stimulation.The mice were taken out after 5 minutes of free exploration,and the freezing time of mice during this period was recorded.At 24 h after the fear memory was recalled,the mice were again placed in the contextual fear box,allowing them to explore freely for 3 minutes,and the freezing time of mice during this period was recorded;the fear response of mice was indicated by the percentage of freezing time.After the behavioral experiment,the mice were anesthetized by intraperitoneal injection of pentobarbital sodium,and three mice from the two groups were taken and rapidly opened the chest to expose their hearts,the perfusion needle was inserted into the left ventricle from the tip of heart and then was perfuse with 40 g·L-1 paraformaldehyde(pH=7.4)which precooled at 4 ℃ until the involuntary convulsion disappeared and the body limbs of mice were stiff.The brain tissue of mice was taken and fixed with paraformaldehyde solution,and then placed in sucrose solution for dehydration.The brain tissue of mice was coated with tissue embedding agent.The number of microglial cells in the hippocampus of mice in the two groups was detected by immunohistochemistry.The remaining mice in the two groups were taken and quickly decapitated to obtain brain tissues,and the hippocampus tissues of two sides were separated on ice.The expressions of phosphorylated bromodomain-containing protein 4(pBRD4),gasdermin D(GSDMD)and mixed lineage kinase domain-like protein(MLKL)in the hippocampus of mice were detected by Western blot.Results In the stage of contextual fear conditioning training,the percentage of freezing time of mice in two groups increased with the increase of the number of footshock(P＜0.05),but there was no significant difference in the percentage of freezing time of mice after the first,second,third,fourth and fifth footshock between the two groups(P＞0.05).There was no significant difference in the percentage of freezing time of mice between the two groups during recall period(P＞0.05).The percentage of freezing time of mice in the experimental group was significantly lower than that in the control group at the phase of memory test(P＜0.05).The number of microglia in CA1,CA3 and DG regions of hippocampus of mice in the experimental group was significantly lower than that in the control group(P＜0.05).The relative expressions of pBRD4,GSDMD and MLKL in hippocampus of mice in the experimental group were significantly lower than those in the control group(P＜0.05).Conclusion Microglia inhibitor Pexidartinib can injury the reconsolidation of remote contextual fear memory,which may be related to its inhibition of microglial cell activation and the down-regulation of the expressions of pBRD4,GSDMD and MLKL.

Sevoflurane attenuates myocardial ischemia/reperfusion injury by up-regulating microRNA-99a and down-regulating BRD4

Purpose: It has been explored that sevoflurane (Sevo) is cardioprotective in myocardial ischemia/reperfusion injury (MI/RI) and mediates microRNA (miRNA) expression that control various physiological systems. Enlightened by that, the work was programmed to decode the mechanism of Sevo and miR-99a with the participation of bromodomain-containing protein 4 (BRD4). Methods: MI/RImodel was established on mice. MI/RI modeled mice were exposed to Sevo or injected with miR-99a or BRD4-related vectors to identify their functions in cardiac function, pathological injury, cardiomyocyte apoptosis, inflammation, and oxidative stress in MI/RI mice. MiR-99a and BRD4 expression in myocardial tissues were tested, and their relation was further validated. Results: MiR-99a was down-regulated, and BRD4 was up-regulated in MI/RI mice. Sevo up-regulated miR-99a to inhibit BRD4 expression in myocardial tissues of MI/RI mice. Sevo improved cardiac function, relieved myocardial injury, repressed cardiomyocyte apoptosis, and alleviated inflammation and oxidative stress in mice with MI/RI. MiR-99a restoration further enhanced the positive effects of Sevo on mice with MI/RI. Overexpression of BRD4 reversed up-regulation of miR-99a-induced attenuation of MI/RI in mice. Conclusions: The work delineated that Sevo up-regulates miR-99a to attenuate MI/RI by inhibiting BRD4.

BRD4 inhibitors kill ABC-DLBCL cells by targeting BTK / 中国病理生理杂志

AIM:To investigate the effect of bromodomain-containing protein 4(BRD4)inhibitors on the via-bility and apoptosis of activated B cell-like diffuse large B-cell lymphoma(ABC-DLBCL)cells and the molecular mecha-nism. METHODS:The ABC-DLBCL cells were treated with BRD4 inhibitors JQ1 and I-BET-762,and Bruton tyrosine kinase(BTK)inhibitor ibrutinib. The viability and death of the cells were determined by CCK-8 assay and PI staining,re-spectively. The mRNA levels of BTK,phospholipase Cγ(PLCγ),LYN,SYK,interleukin-6(IL-6),MYC,protein ki-nase Cβ(PKCβ),mucosa-associated lymphoid tissue lymphoma translocation protein 1(MALT1),MYC and RELA were detected by real-time PCR. The protein levels of BTK,PLCγ,MYC and RELA were determined by Western blot. Super-enhancer around BTK gene was revealed by bioinformatics analysis. RESULTS:The ABC-DLBCL cells were sensitive to BRD4/super-enhancer inhibitors such as JQ1 and I-BET-762. Both JQ1 and I-BET-762 inhibited the chronic active B-cell receptor(BCR)/nuclear factorκB(NFκB)signaling through reducing the transcription of BTK,but they had minimal ef-fect on other components in BCR/NFκB signaling. Interestingly,there was no super-enhancer around BTK gene,and the inhibitory effect of JQ1 was likely due to disruption of BRD4 binding within BTK gene. Inhibition of BRD4 had synergic ef-fect with BTK inhibitor ibrutinib. Moreover,inhibition of BRD4 induced significant cell death in ibrutinib-resistant ABC-DLBCL cells. CONCLUSION:Inhibitors of BRD4 induce ABC-DLBCL cell death via blocking BCR/NFκB signaling and has synergic effect with BTK inhibitor. Inhibition of BRD4 might be a promising strategy for treatment of ABC-DLBCL,es-pecially ibrutinib-resistant ABC-DLBCL.

Effect of JQ1 combined with siPD-L1 on proliferation and apoptosis of oral squamous cell carcinoma cells / 中国医师杂志

Objective:To investigate the effects of bromodomain and extraterminal domain (BET) inhibitor JQ1 combined with siPD-L1 on the proliferation and apoptosis of oral squamous cell carcinoma (OSCC) Scc-25 cells and its mechanism.Methods:Scc-25 cells were cultured in vitro and treated with different concentrations of JQ1 (0, 0.2, 1, 5 μmol/L). Cell proliferation was detected by cell count kit-8 (CCK-8) assay; the expression levels of programmed cell death ligand 1(PD-L1) and forkhead box M1(FoxM1) protein were detected by Western blot. Appropriate concentration of JQ1 was selected for subsequent experiments. Scc-25 cells were divided into four groups: control group (without any treatment), siPD-L1 group (transfected with siPD-L1), JQ1 group (added JQ1 after transfected with non-specific siRNA), and combined treatment group (added JQ1 after transfected with siPD-L1). CCK-8 assay was used to detect the proliferation ability of Scc-25 cells in each group. Western blot was used to detect the expression levels of cleaved caspase-3, PD-L1 and FoxM1, and flow cytometry was used to detect the apoptosis rate of cells in each group. Results:With the increase of JQ1 concentration, the proliferation ability of SCC-25 cells and the expression levels of PD-L1 and FoxM1 decreased gradually (all P<0.01). JQ1 concentration of 1 μmol/L had obvious inhibitory effect on cell proliferation and the expression levels of PD-L1 and FoxM1, so JQ1 concentration of 1 μmol/L was selected for subsequent experiments. The proliferation ability of Scc-25 cells, the expression of PD-L1 and FoxM1 proteins in JQ1 group, siPD-L1 group and combination treatment group were significantly lower than those in the control group (all P<0.01), and the expression of cleaved caspase-3 protein and the rate of apoptosis were significantly higher than those in the control group (all P<0.01); Moreover, the effect of the combination treatment group was more significant than that of siPD-L1 group, JQ1 group (all P<0.01). Conclusions:The combination of JQ1 and siPD-L1 could effectively inhibit the proliferation and promotes the apoptosis of OSCC Scc-25 cells, and its mechanism may be related to the suppression of PD-L1 and FoxM1 signaling pathways.

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.

Autophagy enhanced by curcumin ameliorates inflammation in atherogenesis via the TFEB-P300-BRD4 axis

Disturbance of macrophage-associated lipid metabolism plays a key role in atherosclerosis. Crosstalk between autophagy deficiency and inflammation response in foam cells (FCs) through epigenetic regulation is still poorly understood. Here, we demonstrate that in macrophages, oxidized low-density lipoprotein (ox-LDL) leads to abnormal crosstalk between autophagy and inflammation, thereby causing aberrant lipid metabolism mediated through a dysfunctional transcription factor EB (TFEB)-P300-bromodomain-containing protein 4 (BRD4) axis. ox-LDL led to macrophage autophagy deficiency along with TFEB cytoplasmic accumulation and increased reactive oxygen species generation. This activated P300 promoted BRD4 binding on the promoter regions of inflammatory genes, consequently contributing to inflammation with atherogenesis. Particularly, ox-LDL activated BRD4-dependent super-enhancer associated with liquid-liquid phase separation (LLPS) on the regulatory regions of inflammatory genes. Curcumin (Cur) prominently restored FCs autophagy by promoting TFEB nuclear translocation, optimizing lipid catabolism, and reducing inflammation. The consequences of P300 and BRD4 on super-enhancer formation and inflammatory response in FCs could be prevented by Cur. Furthermore, the anti-atherogenesis effect of Cur was inhibited by macrophage-specific Brd4 overexpression or Tfeb knock-out in Apoe knock-out mice via bone marrow transplantation. The findings identify a novel TFEB-P300-BRD4 axis and establish a new epigenetic paradigm by which Cur regulates autophagy, inhibits inflammation, and decreases lipid content.

Progress on the BRD4 of super enhancer regulatory element in hematologic oncology / 国际儿科学杂志

Abnormal transcription of oncogenes driven by super enhancers was found to be critical for maintaining tumor cell identity.The expression of oncogenes can be effectively suppressed by inhibiting the key regulator that super enhancers regulate oncogene transcription.Bromodomain protein 4(BRD4)is a key protein to recognize the super enhancer regulatory elements, which can bind to acetylated histones or non-histones to regulate gene transcription.The abnormal expression of BRD4 is closely related to the malignant development of a variety of hematologic oncology.Targeting BRD4 can effectively control the malignant development of hematologic tumors.In recent years, BRD4-targeted drugs in hematologic oncology have received extensive attention, and they showed good antitumor effects either as a single drug or in combination with other drugs.In this paper, in order to provide a new understanding of the occurrence of leukemia and treatment of hematologic oncology, the biological functions of BRD4 as well as the molecular drugs targeting BRD4 are reviewed.

Effect and mechanism of bromodomain-containing protein 4 inhibitor JQl on formaldehyde solution induced inflammatory pain in mice / 解剖学报

Objective To explore the expression and role of bromodomain-containing protein 4(BRD4) in spinal cord of mice which suffered inflammatory pain induced by formaldelryde solution. Methods Thirty-two ICR mice were randomly divided into normal saline group and formaldehyde injection 5 minutes, 30 minutes and 60 minutes groups, with 8 mice in each group. The expression of BRD4 protein and mRNA in spinal cord of mice in each group were detected by Western blotting (n = 4/group) and Real-time PCR (n = 4/group); 66 mice were randomly divided into formaldelryde injection group, vehicle (DMSO) plus formaldelryde injection group and 6. 25, 12. 5, 25 and 50 mg/kg JQl injection plus formaldelryde solution group, with 11 mice in each group. The effect of BRD4 inhibitor JQl on spontaneous pain in each group was observed (n= 11/group); Immunohistochemistry (n= 3/group), Real-time PCR (n = 4/group) or Western blotting (n= 4/group) were used to detect the effects of 25 mg/kg JQ1 on the expression of c-fos and glutamate receptor 2 (GluR2) in the spinal cord of model mice. Results The result showed that levels of BRD4 protein (P<0. 01) and mRNA in spinal cord increased significantly 30 min and 60 min after formaldehyde solution injection (P<0. 05). The behavioral test showed that both 25 mg/kg and 50 mg/kg JQf administration could reduce the second phase spontaneous pain compared with the solvent (DMSO) group (P < 0 . 05). Furthennore, immunohistochemistry and Real-time PCR result showed that 25 mg/kg JQf injection could significantly reduce positive numbers (P<0. 01) and high mRNA expression of c-fos in mouse spinal cord induced by formaldehyde solution (P < 0 . 05), and the Western blotting result showed that 25 mg/kg JQf administration could significantly reduce the expression of glutamate receptor GluR2 (P < 0. OOf). Conclusion BRD4 may play an important role in the induction of central sensitization of inflammatory pain, and JQf may alleviate inflammatory pain behavior by inhibiting the formation of central sensitization of pain.

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.

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

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.

Effect of BRWD3 on lymph node metastasis in breast cancer / 解放军医学杂志

Objective: To explore the effect of the bromodomain and WD repeat domain containing 3 (BRWD3) on lymph node metastasis in breast cancer and its mechanism. Methods: In vitro cell invasion experiments were used to explore the effect of BRWD3 on the invasion phenotype of breast cancer cell lines. Mouse lymph node metastasis model and lung metastasis model were used to investigate the role of BRWD3 in regulating breast cancer lymph node metastasis and lung metastasis in BALB/c nude mice. The BRWD3 co-expressed genes were searched through cBioPortal databases to analyze the biological functions and pathways of BRWD3, constructed a BRWD3 molecular regulatory network, which is examined with Western blotting analysis partly. The public breast cancer dataset and the KMPLOT analysis platform was used to analyze the expression of BRWD3 in breast cancer and the relationship between the expression of BRWD3 and breast cancer prognosis. Results: In vitro experiments showed that knockdown of BRWD3 significantly enhanced the invasion and migration of breast cancer cells (P<0.01), but did not promote their proliferation. The lymph node metastasis model demonstrated that knockdown of BRWD3 dramatically enhanced lymph node metastasis of breast cancer. Interestingly, the lung metastasis model showed that BRWD3 did not affect the mouse lung metastasis ability. Further functional clustering GO analysis of the co-expressed genes of BRWD3 suggested that they are mainly involved in gene expression regulation, DNA damage repair, chromosome organization and modification, ubiquitination, etc. Meanwhile, KEGG enrichment analysis showed that they were involved in signaling pathways such as ubiquitination, oxidative phosphorylation, MAPK, etc. Besides, via the Western blotting experiment, it was found that knockdown of BRWD3 increased the phosphorylation of ERK1/2. Moreover, BRWD3 expression in breast cancer with lymph node metastasis is significantly lower than in patients without lymph node metastasis. Further, the survival analysis in KMPLOT found that the prognosis of patients with low expression of BRWD3 was poor, which was significantly lower than that of patients with high expression of BRWD3. Conclusions: BRWD3 can regulate breast cancer invasion in vitro and lymph node metastasis in vivo. Afterward, the prognosis of patients with low expression of BRWD3 is poor. Meanwhile, ubiquination, oxidative phosphorylation, MAPK pathway, etc. were the possible regulation pathways of BRWD3, which provide a new theoretical basis for the research and application of molecular markers related to breast cancer lymph node metastasis.

Epigenetic strategies synergize with PD-L1/PD-1 targeted cancer immunotherapies to enhance antitumor responses

Immunotherapy strategies targeting the programmed cell death ligand 1 (PD-L1)/programmed cell death 1 (PD-1) pathway in clinical treatments have achieved remarkable success in treating multiple types of cancer. However, owing to the heterogeneity of tumors and individual immune systems, PD-L1/PD-1 blockade still shows slow response rates in controlling malignancies in many patients. Accumulating evidence has shown that an effective response to anti-PD-L1/anti-PD-1 therapy requires establishing an integrated immune cycle. Damage in any step of the immune cycle is one of the most important causes of immunotherapy failure. Impairments in the immune cycle can be restored by epigenetic modification, including reprogramming the environment of tumor-associated immunity, eliciting an immune response by increasing the presentation of tumor antigens, and by regulating T cell trafficking and reactivation. Thus, a rational combination of PD-L1/PD-1 blockade and epigenetic agents may offer great potential to retrain the immune system and to improve clinical outcomes of checkpoint blockade therapy.

Diversified Application of Barcoded PLATO (PLATO-BC) Platform for Identification of Protein Interactions / 基因组蛋白质组与生物信息学报·英文版

Proteins usually associate with other molecules physically to execute their functions. Identifying these interactions is important for the functional analysis of proteins. Previously, we reported the parallel analysis of translated ORFs (PLATO) to couple ribosome display of full-length ORFs with affinity enrichment of mRNA/protein/ribosome complexes for the "bait" molecules, followed by the deep sequencing analysis of mRNA. However, the sample processing, from extraction of precipitated mRNA to generation of DNA libraries, includes numerous steps, which is tedious and may cause the loss of materials. Barcoded PLATO (PLATO-BC), an improved platform was further developed to test its application for protein interaction discovery. In this report, we tested the antisera-antigen interaction using serum samples from patients with inclusion body myositis (IBM). Tripartite motif containing 21 (TRIM21) was identified as a potentially new IBM autoantigen. We also expanded the application of PLATO-BC to identify protein interactions for JQ1, single ubiquitin peptide, and NS5 protein of Zika virus. From PLATO-BC analyses, we identified new protein interactions for these "bait" molecules. We demonstrate that Ewing sarcoma breakpoint region 1 (EWSR1) binds to JQ1 and their interactions may interrupt the EWSR1 association with acetylated histone H4. RIO kinase 3 (RIOK3), a newly identified ubiquitin-binding protein, is preferentially associated with K63-ubiquitin chain. We also find that Zika NS5 protein interacts with two previously unreported host proteins, par-3 family cell polarity regulator (PARD3) and chromosome 19 open reading frame 53 (C19orf53), whose attenuated expression benefits the replication of Zika virus. These results further demonstrate that PLATO-BC is capable of identifying novel protein interactions for various types of "bait" molecules.

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.

Progress on the relationship between BPTF and development of tumors in children / 国际儿科学杂志

The gene of bromodomain PHD finger transcription factor (BPTF) is expressed in all tissues and its protein regulate the transcription process.Overexpression and mutations of BPTF are found in a variety of tumors,which demonstrate poor prognosis.It is also reported that BPTF plays an important role in the development of childhood malignancy.The study aims to summarize the latest studies of tumors in children associated with BPTF.

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.

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.