Potent bromodomain and extraterminal domain inhibitor JAB-8263 suppresses MYC expression and exerts anti-tumor activity in colorectal cancer models

BACKGROUND The overexpression of the MYC gene plays an important role in the occurrence,development and evolution of colorectal cancer(CRC).Bromodomain and extraterminal domain(BET)inhibitors can decrease the function BET by recognizing acetylated lysine residues,thereby downregulating the expression of MYC.AIM To investigate the inhibitory effect and mechanism of a BET inhibitor on CRC cells.METHODS The effect of the BET inhibitor JAB-8263 on the proliferation of various CRC cell lines was studied by CellTiter-Glo method and colony formation assay.The effect of JAB-8263 on the cell cycle and apoptosis of CRC cells was studied by propidium iodide staining and Annexin V/propidium iodide flow assay,respectively.The effect of JAB-8263 on the expression of c-MYC,p21 and p16 in CRC cells was detected by western blotting assay.The anti-tumor effect of JAB-8263 on CRC cells in vivo and evaluation of the safety of the compound was predicted by constructing a CRC cell animal tumor model.RESULTS JAB-8263 dose-dependently suppressed CRC cell proliferation and colony formation in vitro.The MYC signaling pathway was dose-dependently inhibited by JAB-8263 in human CRC cell lines.JAB-8263 dose-dependently induced cell cycle arrest and apoptosis in the MC38 cell line.SW837 xenograft model was treated with JAB-8263(0.3 mg/kg for 29 d),and the average tumor volume was significantly decreased compared to the vehicle control group(P<0.001).The MC38 syngeneic murine model was treated with JAB-8263(0.2 mg/kg for 29 d),and the average tumor volume was significantly decreased compared to the vehicle control group(P=0.003).CONCLUSION BET could be a potential effective drug target for suppressing CRC growth,and the BET inhibitor JAB-8263 can effectively suppress c-MYC expression and exert anti-tumor activity in CRC models.

bromodomain结构域对溴区包含蛋白7亚细胞定位的影响

目的:探讨溴区包含蛋白7(BRD7)的亚细胞定位以及bromodomain结构域对BRD7亚细胞定位的影响。方法:首先采用GFP介导的亚细胞定位方法,在非洲绿猴肾COS7细胞中考察BRD7的亚细胞定位;然后分别构建bromodomain缺失型的BRD7重组体pCMV-Myc/BRD7△brd和pEGFP-C2/BRD7△brd,并通过GFP介导的亚细胞定位方法和间接免疫荧光方法,分别在COS7中考察bromodomain缺失型BRD7的亚细胞定位,从而探讨BRD7的bromodomain结构域对其亚细胞定位的影响。结果:成功构建了pCMV-Myc/BRD7△brd和pEGFP-C2/BRD7△brd。BRD7及bromodomain缺失型BRD7在COS7中均主要定位在细胞核,与染色质的分布存在一定程度的相似性。结论:bromodomain结构域的缺失并不影响BRD7的细胞核分布,但是在核内的分布模式发生了一定的改变。

基于Bromodomains靶体的小分子抑制剂的合成进展

Bromodomains是包含110个氨基酸的蛋白质功能结构域,可选择性识别组蛋白末端乙酰赖氨酸位点,参与表观遗传基因的读取和调控,影响一些疾病的发生。而基于Bromodomains靶体的小分子抑制剂的合成与应用,成为药物化学领域的研究热点。因此,对近三年该领域的合成进展进行归纳和总结,可为抗癌药物的发现提供研究思路。

Bromodomain

bromodomain是一进化上高度保守约 1 1 0个氨基酸的蛋白质功能结构域 ,可特异性识别组蛋白末端乙酰化的赖氨酸位点 ,通过染色质的组装和乙酰化而参与信号依赖性的、非基础性的基因转录调控 ;bromodomain亦可通过对转录因子等非组蛋白的乙酰化修饰而广泛参与细胞周期调控、细胞分化、信号转导等过程 ;bromodomain蛋白的改变通过组蛋白乙酰化的失调控参与了白血病等恶性肿瘤的发生 ,其与肿瘤关系的研究将为肿瘤治疗提供新的策略。

BET bromodomain蛋白小分子抑制剂研究进展

组蛋白赖氨酸乙酰化的识别是组蛋白乙酰化参与表观遗传调控的关键步骤,乙酰化的组蛋白赖氨酸可以被bromodomains(BRDs)结构域所特异性的识别,从而招募染色质调控因子到特定区域,协同完成基因表达调控。其中作用于bromodomain and extra-terminal(BET)蛋白家族的BRD结构域的小分子抑制剂在抗炎和抗肿瘤方面显示出巨大的潜力。本文通过对与BET bromodomain靶点相关的疾病、BET bromodomain结构、BET bromodomain小分子抑制剂的化学结构分类及其构效关系等多方面进行总结,为设计和开发高活性的BET bromodomain小分子抑制剂提供参考依据。

Bromodomain and extra-terminal inhibitors emerge as potential therapeutic avenues for gastrointestinal cancers

Gastrointestinal(GI)cancers,including colorectal cancer,pancreatic cancer,liver cancer and gastric cancer,are severe social burdens due to high incidence and mortality rates.Bromodomain and extra-terminal(BET)proteins are epigenetic readers consisting of four conserved members(BRD2,BRD3,BRD4 and BRDT).BET family perform pivotal roles in tumorigenesis through transcriptional regulation,thereby emerging as potential therapeutic targets.BET inhibitors,disrupting the interaction between BET proteins and acetylated lysines,have been reported to suppress tumor initiation and progression in most of GI cancers.In this review,we will demonstrate how BET proteins participate in the GI cancers progression and highlight the therapeutic potential of targeting BET proteins for GI cancers treatment.

基于多向药理学的BET Bromodomain抑制剂及降解剂研究进展

溴结构域和末端外结构域(BET)Bromodomain已成为可用于治疗癌症和其他人类疾病的新靶标。目前已发现了几类有效的选择性小分子BET Bromodomain抑制剂,且多种已处于临床开发中。临床前和临床数据已证明BET Bromodomain抑制剂有良好的前景,但也存在耐药性等潜在缺陷。目前人们正在尝试将具有不同作用机制的靶标与BET Bromodomain组合,开发基于多向药理学的BET Bromodomain抑制剂及降解剂。本文综述了激酶/BET小分子抑制剂、组蛋白去乙酰化酶/BET小分子抑制剂及BET蛋白降解剂,为后续针对BET蛋白更深入的研究提供思路。

灰葡萄孢Bromodomain转录因子家族成员鉴定及表达分析

为了解灰葡萄孢Bromodomain转录因子家族成员的功能,本研究利用生物信息学方法对灰葡萄孢Bromodomain转录因子家族成员进行理化性质、染色体定位、系统发育、基因结构、保守结构域以及分生孢子发育时期及侵染时期的表达规律分析;并利用qPCR技术检测灰葡萄孢Bromodomain家族基因在NaCl和刚果红处理后的表达变化。结果显示,灰葡萄孢中包含8个Bromodomain家族基因,系统发育分析将它们分为4个亚家族,Bromodomain家族基因在分生孢子的不同发育和侵染时期呈现不同的表达规律,在NaCl和刚果红处理后都呈现下调表达,表明了Bromodomain家族基因在灰葡萄孢生长发育、侵染植物以及胁迫应答过程中可能具有重要的功能。本研究为深入探讨Bromodomain转录因子家族基因的功能和灰葡萄孢的防治奠定基础。

抑制剂8Q9和8QC与bromodomain结构域蛋白4作用机理的分子动力学研究

Bromodomain结构域蛋白4(bromodomain-containing protein 4,BRD4)已成为治疗多种疾病药物设计的重要靶标.最近在实验上发现了几种有效的靶向BRD4的抑制剂,但具体的抑制机理尚不清楚.此工作采用分子动力学模拟,动态相关性分析和结合自由能计算研究抑制剂8Q9和8QC与BRD4(1)的结合模式.分子动力学分析表明抑制剂结合对BRD4(1)的结构柔性产生重大影响.同时动态相关性分析进一步表明抑制剂结合极大地改变了BRD4(1)的运动模式.结合自由能计算结果表明范德华相互作用是抑制剂与BRD4(1)结合的主要驱动力.采用基于残基的自由能分解方法评估了分离残基对抑制剂结合的贡献,数据表明氢键相互作用和疏水相互作用是影响抑制剂与BRD4(1)结合的关键因素.本研究有望为设计和开发靶向BRD4的抑制剂提供有意义的理论指导.

Structural insights into selective histone H3 recognition by the human Polybromo bromodomain 2

The Polybromo （PB） protein functions as a key component of the human PBAF chromatin remodeling complex in regulation of gene transcription. PB is made up of modular domains including six bromodomains that are known as acetyl-lysine binding domains. However, histone-binding specificity of the bromodomains of PB has remained elusive. In this study, we report biochemical characterization of all six PB bromodomains＇ binding to a suite of lysine-acetylated peptides derived from known acetylation sites on human core histones. We demonstrate that bromodomain 2 of PB preferentially recognizes acetylated lysine 14 of histone H3 （H3K14ac）, a post-translational mark known for gene transcriptional activation. We further describe the molecular basis of the selective H3K14ac recognition of bromodomain 2 by solving the protein structures in both the free and bound forms using X-ray crystallography and NMR, respectively.

Functional characterization of the catalytic and bromodomain of FgGCN5 in development,DON production and virulence of Fusarium graminearum

FgGCN5,a GCN5 homolog in Fusarium graminearum,plays a critical role in hyphal vegetative growth,asexual and sexual reproduction,deoxynivalenol(DON)biosynthesis and plant infection.For nuclear localized GCN5,four conserved sequence motifs(I-IV)are presented in the catalytic domain and a bromodomain in the carboxy-terminus.As a lysine acetyltransferase,conserved negatively charged residues are present to neutralize the protons from lysine substrates.However,the role of conserved motifs/domains and residues in FgGCN5 are unclear.Here,we generated deletion mutant strains for each the conserved motifs/domains and a glutamate residue 130(E130)replacement mutant.Deletion of each conserved motif in the catalytic domain and replacement of E130 site resulted in manifold defects in hyphae growth,asexual and sexual development,DON biosynthesis,and plant infection.Phenotypic defects in the mutant strains were similar to deletion mutants.The deletion of the bromodomain led a significant reduction in DON production and virulence,with no effects on hyphae growth,asexual or sexual reproduction.FgGCN5 was further found to localize to the nucleus in conidia and hyphae cells.In conclusion,FgGCN5 encodes a nuclear localized acetyltransferase.The conserved motifs in the catalytic domain and E130 are essential for correct functions of the gene.The conserved bromodomain is impotant for DON production and pathogen virulence.This was the first report to identify the functions of conserved motifs/domains in FgGCN5,which will contribute to our understanding of the mechanism(s)by which FgGCN5 regulates F.graminearum.

BET bromodomain蛋白在人胚胎干细胞造血分化中的作用研究

目的:探讨BET bromodomain蛋白在人胚胎干细胞造血分化中的作用。方法:应用人胚胎干细胞单层造血分化体系,通过免疫荧光技术、流式细胞术及实时定量PCR技术,研究BET bromodomain蛋白抑制剂I-BET151对造血分化的影响,并在不同分化阶段添加I-BET 151探讨其在造血分化过程中的作用阶段。结果:I-BET151能够显著抑制CD43^+造血干/祖细胞的产生,并且在APLNR^+侧板中胚层生成、CD34^+CD31^+生血内皮产生及内皮细胞生血转化3个阶段添加I-BET 151对CD43^+造血干/祖细胞的产生均有显著抑制作用。结论:人胚胎干细胞造血分化过程中添加BET bromodomain蛋白抑制剂I-BET 151,能够抑制CD43^+造血干/祖细胞的产生,并进一步证实BET

Discovery of a small-molecule bromodomain-containing protein 4 inhibitor that induces AMP-activated protein kinase-modulated autophagy-associated cell death in breast cancer

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 antiproliferative 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,immunocytochemistry,si RNA and GFP-m RFP-LC3 plasmid transfections,as well as immunohistochemistry and immunofluorescence.Potential mechanisms were discovered by i TRAQ-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 autophagyassociated cell death(ACD)by BRD4-AMPK interaction,and thus activating AMPK-m TOR-ULK1-modulated autophagic pathway in breast cancer cells.Interestingly,the i TRAQ-based proteomics analyses revealed that 8f induced ACD pathways,involved in HMGB1,VDAC1/2 and e EF2.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.

Bromodomains识别并结合配体的机理研究

关于酵母重组蛋白内的Bromodomain识别乙酰化赖氨酸的研究近年来受到广泛的关注,但是其识别配体并与之相紧密结合的机理有待进一步的研究。本文采用2015年开发的结合位点拓扑学方法(FCTM)和分子动力学模拟的方式对Bromodomains识别并结合配体的机理进行了充分研究,其中分子动力学模拟时间达24 ns。通过FCTM方法发现结合位点的几何结构具有高度的凹性,且其alphaspace达到了131。分子动力学模拟的结果显示:在模拟的过程中结合位点表面的脯氨酸(Pro66)始终对配体保持着强的分子间相互作用,同时pocket内的水分子分布对配体的氢键网络也一直存在影响。以上结果表明Bromodomains识别并结合配体有两个重要因素:蛋白结构域自身的几何结构和配体受到来自于结合位点表面的氨基酸分子相互作用和pocket内水分子的氢键网络作用。

BRD7结构功能域Bromodomain的研究以及一个新的BRD7交互作用蛋白的鉴定

溴区包含蛋白7(BRD7)是采用cDNA代表性差异分析方法克隆的一个新基因.研究证实了BRD7能够与乙酰化的组蛋白3结合,其识别位点在组蛋白3的14位氨基酸;并且证实了溴区结构域(Bromodomain)缺失型的BRD7突变体失去了与乙酰化组蛋白3的结合能力.Bromodomain是在进化上高度保守的功能结构域,该结构域在空间构象上具有鲜明的特征:包括4个左手、呈反向平行排列的“螺旋(αz,αA,αB,αC)”以及2个“连结环”(ZAloop,BCloop).通过生物信息学等综合分析,预测BRD7可能具有上述特征.依据上述分析结果,构建了BRD7的Bromodomain相关缺失突变体,通过肽段结合实验分析上述突变体与乙酰化组蛋白3结合的能力.结果表明,ZAloop与BCloop的完整性对于BRD7结合乙酰化的组蛋白3有着重要的意义.同时通过免疫荧光分析,证实了ZAloop与BCloop的完整性能够影响BRD7的亚细胞定位.最后,证实了BRD7与CBP可能存在交互作用.CBP不仅具有乙酰化转移酶活性(HATs),能够对组蛋白末端进行乙酰化修饰,并且作为一种重要的细胞转录因子广泛参与细胞的各种生物学活动.

原虫的bromodomain研究进展

原生动物寄生虫感染是当今全球面临的最紧迫的健康问题之一,且某些种类寄生虫可造成巨大的经济损失。耐药性寄生虫的不断产生,迫切需要揭示其分子致病机理,希望找到新的潜在药物靶点。通过干扰赖氨酸乙酰化破坏基因表达对原生动物寄生虫(疟原虫、弓形虫、锥虫等)的存活具有重要影响,赖氨酸乙酰化的bromodomain蛋白已经成为基因表达的关键调控因子,甚至有希望成为新一类药物靶标。虫体bromodomain蛋白质在寄生虫的生存活力、侵袭和生长阶段转换中发挥重要作用,并且bromodomain抑制剂具有作为新型抗寄生虫药的功效。论文对代表性寄生虫的bromodomain蛋白等相关方面的研究进展做一综述。

Bromodomain小分子抑制剂的研究进展

草甘膦是一种高效、低毒的灭生性除草剂,在世界范围内得到广泛应用。本文概述了IDA法和甘氨酸法草甘膦母液的主要成分,总结了草甘膦的主要回收方法,指出了现有研究的不足之处,为进一步研究提供参考。

BET inhibitors potentiate melanoma ferroptosis and immunotherapy through AKR1C2 inhibition

Dear Editor,Ferroptosis,an iron-dependent form of cell death driven by overwhelming lipid peroxidation,represents a vulnerability in cancers,and therapeutic strategies to further potentiate ferroptosis hold great potential for melanoma treatment.

Discovery of CECR2 Bromodomain Inhibitors with High Selectivities over BPTF Bromodomain

Cat's eye syndrome chromosome candidate 2 bromodomain(CECR2 BRD)and Bromodomain PHD transcription factor bromodomain(BPTF BRD)are the same subfamily proteins,both of which are highly conserved in sequence and binding pockets.Challenges remain in the development of small molecules to inhibit one of the two bromodomains(BRDs),in view of each subtype may possess unique physiological and pathological functions.There is still a lack of effective selective inhibitors of CECR2 BRD,which makes it difficult to fully understand the pathogenesis of CECR2-BRD in diseases,especially cancers.Herein,we report our efforts to discover a series of highly selective CECR2 BRD inhibitors over BPTF BRD based on TP-248.Structure-based molecular optimization led to the discovery of DC-CEi-26,whose IC_(50) for CECR2 BRD was 96.7±14.9 nmol/L and selectivity was up to 590×over BPTF BRD.DC-CEi-26 showed weak potencies for other classic BRDs in different subfamily,which may serve as a chemical probe for CECR2 BRD biological research.

Bromodomains:表观遗传医学新靶点及其抑制剂

Bromodomains(BRDs)是一类能够特异性识别乙酰化赖氨酸残基的保守蛋白结构域,存在于染色质及与转录相关的蛋白中,其功能包括染色质重塑和转录调控,并在细胞内由乙酰化介导的蛋白-蛋白相互作用中发挥极为重要的作用,是多种疾病(包括癌症、炎症和自身免疫病)的表观遗传医学靶点。介绍BRDs的生物学功能、结构及分类,主要从BETbromodomain抑制剂和非BETbromodomain抑制剂两个方面对BRDs抑制剂的研究进展作一综述,为高活性和选择性的BRDs抑制剂研发提供参考。