表观遗传修饰阅读器”reader”蛋白结构域靶点的发现

*注解: 将能识别特异性表观修饰并与其结合的蛋白喻为“读者或阅读器（readers）”

众所周知，小分子物质很难抑制蛋白质相互作用(Protein-Protein Interactions, PPI)。但由于PPI广泛存在及其生理重要性，因此发现和确认PPI药理学靶点就非常具有吸引力。目前虽有一些文献报道了PPI小分子抑制剂的例子，但仅有少数具有临床意义。不能有效鉴别这类靶点主要归因于PPI相互作用界面的本质特点。蛋白-小分子作用的接触面大约为300～1000Å2左右，而蛋白-蛋白相互作用的接触面则为1500～3000Å2之间。而且, 蛋白-蛋白相互接触的表面往往是平坦的，不会存在很多沟槽或者口袋, 使得小分子化合物找不到适合的结合位点。尽管如此，但成功鉴别这些相互作用的药物靶点对于人类健康事业极其重要。因此，近年来关于蛋白-蛋白相互作用小分子抑制剂的研究已经成为全球药物化学家关注的热点。

表观遗传及组蛋白密码

基因转录调控依赖于适当的转录因子结合到靶基因启动子区域。尽管调控基因表达的信号复杂多样，但其中一个主要的调控机制是通过DNA及组蛋白修饰来介导完成的。这些修饰标记以及参与修饰和去除修饰的相关蛋白(酶)，形成了转录调控的重要基础。近年采用质谱法鉴定出了上百种组蛋白翻译后修饰(Posttranslational modifications, PTMs)，其中最主要的PTMs存在于组蛋白的N-末端。虽然大多数修饰标记的生理学意义还未知，但一些关键的翻译后修饰已定义了明确的生物学活性。

可以说，到目前研究最深入的组蛋白翻译后修饰就是赖氨酸乙酰化。组蛋白赖氨酸乙酰化可弱化DNA-组蛋白相互作用，募集相关转录因子。该修饰是基因进行转录活化的标志。乙酰赖氨酸(Acetyllysine)可被称为溴结构域的蛋白基序(motif)选择性识别。目前在人类基因组中至少已鉴定出43种含溴结构域的蛋白质(如图1)，其中大多数为重要的转录调控因子。溴结构域一般由60－120个氨基酸残基组成。二级结构为双性α－螺旋，三级结构为左手上下四螺旋束状结构，含一个疏水穴(Hydrophobic pocket)。已发现人类某些疾病与溴结构域突变相关。

图1.含溴结构域蛋白聚类分析

尽管溴结构域在生物医学研究上获得重大关注，但其他阅读器”Reader”蛋白结构域异常也具有显著的生理学效应。基因转座使得甲基赖氨酸阅读器”reader”结构域从JARID1A或PHF23与Nucleoporin-98融合，导致维持细胞多能性的基因过量表达，而引起肿瘤发生。甲基赖氨酸阅读器蛋白结构域家族至少含有200个成员，分布于不同的亚家族。该大家族蛋白成员结构域各异，与甲基化赖氨酸的位置和甲基化状态具有高度特异性。与赖氨酸乙酰化相比,组蛋白甲基化的发生具有位点特异性。同样，许多甲基赖氨酸阅读器“Reader”也是与被修饰残基周围的氨基酸残基反应(其提供了序列特异性结构域)。这与赖氨酸甲基化在募集转录因子和其他染色质修饰酶等方面的作用密切相关。此外，其他的组蛋白翻译后修饰识别结构域也被广泛研究，包括结合磷酸化丝氨酸的BRCT家族、甲基化DNA结合蛋白(如MBD结构域)、甲基精氨酸结合蛋白(如ADD结构域)。一些阅读器“reader”结构域突变与某些疾病相关联，比如甲基CpG结合蛋白2(MeCP2)突变会破坏DNA结合基序(motif),会导致一种称为Rett综合症的X染色体连锁精神和生理发育迟滞症。BRCT结构域，识别磷酸化的组蛋白尾(histone tails),参与到DNA损伤应答元件的募集，BRCA1蛋白的BRCT结构域发生突变，会增加家族性患乳腺癌和卵巢癌的风险。

图2. 甲基赖氨酸阅读器结构域具结构多样性

表观遗传与药物发现

由于表观遗传修饰阅读器结构域的生理重要性，那么药理操纵这些蛋白相互作用(PPI)也为疾病管理提供了新的途径。最先进的一类以表观遗传阅读器结构域为靶点的抑制剂，来源于鉴定可提高肝细胞产生载脂蛋白A-1的分子表型筛选(phenotypic screens)。机理研究从筛选中鉴定出了先导化合物以及相关的化合物(+)-JQ1, 作为BET家族溴结构域的抑制剂。尽管ApoA-1水平升高对于心血管的好处仍存争议，但是BET溴结构域抑制已被证明能有效减缓NUT midline carubinas肿瘤(是一种罕见又致命的恶性皮肤癌，通常NMC患者的存活时间不足1年)生长。这一初始工作也有助于开发一种抗Midline carcinomas(NCT01587703)肿瘤的新型化疗药物，目前已经进入临床I期。另外一种BET溴结构域抑制剂，RVX-208，也作为糖尿病前期葡萄糖代谢药物进入临床II期。

由于BET家族溴结构域抑制剂的偶然发现，众多学术及企业界研究团体备受鼓舞，积极投身其他溴结构域靶点的发现。一个结构基因组学联盟项目就已经结晶解析出了很多溴结构域，并针对不同溴结构域筛选了选择性的小分子抑制剂(www.thesgc.org) [注:Cayman公司可提供很多此类小分子化合物]。其他阅读器结构域的小分子抑制剂也被鉴定出来，如以L3MBTL和L3MBTL3的甲基赖氨酸结合结构域为靶点小分子(如 UNC1215, Cayman货号# 13968)。

鉴于表观遗传修饰阅读器结构域在基因表达调控方面的重要性，那么这些蛋白基序家族则为药物研发提供了新的有前景的领域。实际上，目前研究发现BET溴结构域抑制剂不仅具有抗增值作用，还包括抗体病毒和抗炎症的作用。为了帮助全球从事该领域的科研人员更好的解析决读器蛋白结构域，Cayman公司可提供多种纯化的阅读器结构域、抑制剂筛选试剂盒以及化合物探针。随着该领域的深入研究，也希望有更多新型的表观修饰阅读器结构域抑制剂被鉴定出来，以此推动药物早期筛选的进程。

------------------------------------------------------------------------- 文: 欣博盛市场部 Harry 编译 -------------------------------------------------------------------------

研究工具推荐

蛋白甲基化修饰阅读器(Readers)---Tudor结构域及PHD锌指结构域-- 通常与di及tri甲基化赖氨酸反应，当成对存在时也称为“tandem Tudors”, 也可识别对称性甲基化精氨酸

编号	产品名称	识别位点	纯度
14134	JMJD2A tudor domains(humna recombinant)	Histone H3K4me,H3K9me3,and H3K20me2/3	≥90%
14136	SMN tudor domain(human recombinant)	Methylated Sm proteins,Which bind small nuclear RNA	≥90%
14073	TP53BP1 tudor-like region(human recombinant)	Dimethylated lysine 382 on p53 and histone H4K20me2	≥90%
14777	UHRF1 PHD domain(human recombinant)	Histone H3R2me0(unmetylated)	≥95%
14778	UHRF1 tudor-like region(human recombinant,GST-tagged)	Histone H3K9me3	≥95%
14779	UHRF1 tudor-like region(human recombinant,His-tagged)	Histone H3K9me3	≥95%

克罗莫结构域及异染色质蛋白(chromodomains and heterochromatin proteins) -- 结合mono, di-及tri-methyl H3K4/9/27

编号	产品名称	识别位点	纯度
14768	CBX1(human recombinant)	Chromodomain recognizes histones H3K9me2 and H3K9me3,with a pref- erence for H3K9me3,While the chro- moshadow domain is responsible for homodimerization and interaction with a nuclear of non-histone chrom- atin-associated proteins	≥95%
14769	CBX2 chromodomain(human recombinant)	Histone H3K9me3 or H3K27me3	≥90%
11235	CBX5(human recombinant)	Chromodomain recognizes histones H3K9me2 and H3K9me3,with a pre- ference for H3K9me3,While the chro- moshadow domain facilitates protein-proteins interactions	≥95%
14772	CHD1 chromodomains(human recombinant)	Histone H3K4me2/3	≥90%
14773	CHD2 chromodomains(human recombinant)	Histone H3K4me	≥90%

MBT(恶性脑瘤The malignant brain tumor)域--主要结合mono- 及di-甲基化赖氨酸，通常以多个MBT-重复序列(2,3或4)存在，其仅结合甲基化赖氨酸

编号	产品名称	识别位点	纯度
14775	L3MBTL1 MBT domains(human r ecombinant;GST-tagged)	Histones H3K4me1 and H4K20me2	≥90%

WD40重复蛋白(WD40 Repeat-containing Proteins)

编号	产品名称	识别位点	纯度
10628	EED(human recombinant)	Forms part of the Polycomb repressive com- plex2; transcriptional repression by PRC2- mediated H3K27me3 is dependent on EED binding to repressive histone marks	≥95%
10947	RbBP5(human recombinant)	Binds directly to with underphosphorylated tumor suppressor retinoblastoma protein; interacts directly with WDR5 contribution to the activation of the MLL1 core proteins complex	≥90%
10944	WDR5(human recombinant)	Contains seven WD40 repeats and binds histone H3 by recognizing the first three amino acids of the N-terminal tail; binding to a conserved arginine-containing motif in MLL-1 promotes the assembly and activity of MLL core complex.	≥95%

组蛋白乙酰化修饰阅读器（Readers）：溴结构域(Bromodomains)

编号	产品名称	功能	纯度
14489	ASH1L bromodomain (humanre combinant)	ASH1L is a histone methyltransferase that regulates mammalian Hox gene expression, which plays an important role in haemato- poietic development in mammals.	≥90%
14490	ATAD2 bromodomain (human recombinant)	ATAD2 is an AAA+ ATPase-containing nuclear transcriptional coactivator for estrogen and androgen receptors. Its bromodomain associates with acetylated lysine 14 on histone H3 to regulate the genes required for cell cycle progression.	≥90%
11917	BAZ2B bromodomain (human recombinant)	A rare allele of BAZ2B has been identified to be a predictor of Sudden Cardiac Death.	≥80%
11918	BPTF bromodomain (human recombinant)	BPTF is the largest component of the NURF chromation remodeling complex.Its bromodomain recognizes acetylation of lysines in histone 4.	≥80%
11507	BRD1 bromodomain (human recombinant)	BRD1 has been identified as a susceptibility gene in neurological disorders, such as schizophrenia and bipolar affective disorder.	≥95%
11071	BRD2 bromodomain1 (human recombinant; GST-tagged)	A member of the BET protein family, which plays a key role in many processes. The bromodomains of BRD2 bind acetylated histone tails, coupling histone acetylation marks to the transcriptional regulation of target promoters.	≥95%
11070	BRD2 bromodomain 2 (human recombinant;GST-tagged)	See Item No. 11071 for details.	≥95%
11069	BRD2 bromodomains 1 and 2 (human recombinant;GST-tagged)	See Item No. 11071 for details.	≥90%
11285	BRD3 bromodomain 1 (human recombinant)	The BET proteins play a key role in many cellular processes, including inflammatory gene expression, mitosis, and viral/host interactions.The bromodomains of BRD3 bind acetylated histone tails, coupling histone acetylation marks to the transcriptional regulation of target promoters.	≥95%
14658	BRD3 bromodomain 2 (human recombinant)	See Item No. 11285 for details.	≥95%
14864	BRD3 bromodomain 1 and 2 (human recombinant)	See Item No. 11285 for details.	≥95%
11068	BRD4 bromodomain 1 (human recombinant; GST-tagged)	The BET proteins play a key role in many cellular processes, including inflammatory gene expression, mitosis, and viral/host interactions.The bromodomains of BRD4 bind acetylated histone tails, serving to couple histone acetylation marks to the transcriptional regulation of target promoters.	≥60%
11720	BRD4 bromodomain 1 (human recombinant; His-tagged)	See Item No. 11068 for details.	≥95%
11066	BRD4 bromodomain 2 (human recombinant; GST-tagged)	See Item No. 11068 for details.	≥95%
11721	BRD4 bromodomain 2 (human recombinant; His-tagged)	See Item No. 11068 for details.	≥95%
14822	BRD4 bromodomains 1 and 2 (human recombinant; aa2-477)	See Item No. 11068 for details.	≥70%
11052	BRD4 bromodomains 1 and 2 (human recombinant; aa49-460)	See Item No. 11068 for details.	≥90%
14491	BRD7 bromodomain (human recombinant)	BRD7 is subunit of the polybromo-associated BRG1-associated factor(PBAF)-specific component of the switch/sucrose nonfermen- table chromation-remodeling complex.It has a tumor suppressor role by acting as a cofactor for p53 and regulating breast cancer tumori- genicity.	≥90%
11509	BRD9 bromodomain (human recombinant)	Human BRD9 contains a single bromodomain and has five isoforms that are produced by alternative splicing.	≥95%
11548	BRDT bromodomain 1 (human recombinant)	BRDT shares homology with RING3 protein. The two bromodomains of BRDT recognize acetylated histone H4, Loss of BRDT leads to defects in spermatogenesis.	≥95%
11649	BRDT bromodomain 2 (human recombinant)	See Item No. 11548 for details.	≥95%
14492	BRDT bromodomains 1 and 2 (human recombinant)	See Item No. 11548 for details.	≥95%
11284	BRG1 bromodomain (human recombinant)	BRG1 is a member of the SWI/SNF proteins family, which forms part of the large ATP- dependent chromatin remodeling complex that is requires for transciptional activation of genes normally repressed by chromatin.	≥95%
11289	BRM bromodomain (human recombinant)	BRM is a member of the SWI/SNF proteins family, which forms part of the large ATP- dependent chromatin remodeling complex that is requires for transciptional activation of genes normally repressed by chromatin.	≥95%
11650	BRPF3 bromodomain 1 (human recombinant)	BRPF3 is a component of the MOZ/MORF histone acetytransferase(HAT) complex.The addition of BRPF proteins to MOZ/MORF increases its HAT activity.This product contains the first bromodomain of BRPF3.	≥95%
14133	CECR2 bromodomain (human recombinant)	CECR2 is a transcription factor that forms a heterodimeric complex with the ATP-dependent chromation remodeler SNF2L forming the CERF， which plays a critical role in neurula- tion. The bromodomain of CECR2 has strong γ-H2AX inhibition activity suggesting that CEC- R2 may play a role in DNA damage response.	≥95%
11288	CREB-binding protein bromodomain (human recombinant)	The CREBBP bromodomain has been shown to modulate the stability and function of the tumor suppressor protein p53. CREBBP brom- odomain recognizes the acetylated lysine residue 382 on p53.	≥95%
11920	PCAF bromodomain (human recombinant)	p300/CBP-associated factor(PCAF) is a tran- scriptional coactivator that works both as a histone lysine acetytransferase, through its HAT domain, and as an acety-lysine reader throung its conserved bromodomain located directly C-terminal to the HAT domain.The PCAF bromodomain binds acetylated histone H3 and H4 as well as non-histone targets.	≥95%
11652	Polybromo-1D bromodomain 1 (human recombinant)	PBRM1 contains six bromodomains and is a component of the SWI/SNF complex, PBAF. PBAF is target to acetylated sites in chromatin by the PBRM1 bromodomains, where it plays a role in cell cycle regulation and tumor suppression.	≥95%
14659	SP140 PHD and bromodomain (human recombinant)	The C-terminal PHD and bromodomain regions of SP140 work in concert to bind to chromatin and regulate gene transcription. Nuclear bodies, which are involved in the pathogenesis of acute promyelocytic leukemia and viral infection, consist of several components,such as Sp100, NDP52,PML, and SP140.	≥95%
11922	TAF1 bromodomain 1 (human recombinant)	TAF1 is a component of transcription factor IID, and binds to core promoter sequences at the transcription start site. TAF1 helps control transcription by both its kinase and histone acetyltranferase enzymatic activities. This protein product contains the first bromodomain of TAF1.	≥95%
14495	TAF1 bromodomain 2 (human recombinant)	Contains the second bromodomain of TAF1	≥95%
14494	TAF1 bromodomains 1 and 2 (human recombinant)	Contains the first and second bromodomains of TAF1	≥90%
11653	TRIM24 bromodomain (human recombinant)	TRIM24 is a transcriptional confactor whose inactivation leads to hepatocellular carcinoma in mice. The N-terminal TRIM domain of TRIM- 24 binds ligand-bound nuclear receptors, while its tandem C-terminal plant homeo-domain and bromodomain target TRIM24 to acetylated histones in chromatin.	≥95%
14660	TRIM28 PHD and bromodomain (human recombinant)	TRIM28, a KRAB associated protein, in- teracts with SETD1 and other methyltrans- ferases for gene silencing of endogenous retroviruses during embryogenesis.The PHD finger of TRIM28 plays an important role in gene silencing.The PHD domain and bromo- domain of TRIM28 work together to facilitate lysine SUMOylation, which is a requirement for TRIM28 activity in gene silencing.	≥85%
14661	TRIM33 PHD and bromodomain (human recombinant)	TRIM33 is a multi-domain regulator of transcription that is necessary for emb- ryogenesis. TRIM33 is targeted to DNA by its tandem PHD and bromodomain, which bind histone 3 at H3K9me3 and H3K18ac,respectively.	≥80%
11549	WDR9 bromodomain 2 (human recombinant)	WDR9 pessesses two bromodomain motifs and eight WD repeats.It is also known to interact with BRG1 (SMARCA4).	≥85%

DNA甲基修饰阅读器 (Readers)

编号	产品名称	识别位点	纯度
11286	MBD2 (human recombinant; methyl binding domain aa 150-220	5-Methylcytosine in promoters on CpG islands	≥95%
11287	MeCP2 (human recombinant; methyl binding domain aa 77-166	5-Methylcytosine in promoters on CpG islands; high affinity binding is facilit-ated by DNA fragments containing A/T bases[A/T]≥4) adj- acent to the methyl-CpG.	≥95%

溴结构域TR-FRET分析试剂盒/Bromodomain TR-FRET Assay Kits
--- 可用于高通量快速鉴定溴结构域/乙酰化肽相互作用的抑制剂

图. 溴结构域TR-FRET(时间分辨率荧光共振能量转移)分析试剂盒原理示意图。该检测利用铕螯合物标记的重组溴结构域作为供体，乙酰化的组蛋白肽与链亲和素-APC偶联作为受体。铕螯合物受激发后释放光子或传递给APC分子

编号	产品名称
600710	BAZ2B bromodomain TR-FRET Assay Kit
600500	BRD2 bromodomain 1 TR-FRET Assay Kit
600510	BRD2 bromodomain 2 TR-FRET Assay Kit
600810	BRD2 bromodomains 1 and 2 TR-FRET Assay Kit
600630	BRD3 bromodomain 1 TR-FRET Assay Kit
600820	BRD3 bromodomains 1 and 2 TR-FRET Assay Kit
600640	BRD3 bromodomain 2 TR-FRET Assay Kit
600520	BRD4 bromodomain 1 TR-FRET Assay Kit
600530	BRD4 bromodomain 2 TR-FRET Assay Kit
600830	BRD4 bromodomains 1 and 2 TR-FRET Assay Kit
600650	BRDT bromodomain 1 TR-FRET Assay Kit
600720	BRG1 bromodomain TR-FRET Assay Kit
600730	BRM bromodomain TR-FRET Assay Kit
600850	CBP bromodomain TR-FRET Assay Kit
600870	TAF-1 bromodomain 1 TR-FRET Assay Kit
600930	TAF-1 bromodomains 1 and 2 TR-FRET Assay Kit