CN104374904A - 一种靶向prc2抗肿瘤药物的筛选方法 - Google Patents
一种靶向prc2抗肿瘤药物的筛选方法 Download PDFInfo
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Abstract
本发明涉及一种抗PRC2依赖性肿瘤药物的筛选方法，特别的，本发明涉及以EED-EZH2相互作用为靶点的筛选方法，通过所述方法可筛选出能够拮抗EED-EZH2互作并能使EZH2降解的小分子化合物，该小分子化合物对PRC2依赖性肿瘤细胞有杀伤作用，可开发成为抗肿瘤药物。
Description
技术领域
本发明涉及一种抗PRC2依赖性肿瘤药物的筛选方法，所述方法以EED-EZH2相互作用为靶点，通过初筛和复筛，筛选出对EED-EZH2互作有拮抗并能使PRC2复合物组分有降解作用的小分子化合物，该小分子化合物可抑制PRC2复合物活性，降解EZH2，对PRC2依赖性的肿瘤细胞有杀伤作用，可用作抗肿瘤药物。
发明背景
癌症由多种因素引起，在世界范围内，它是第一致死因素。2008年统计的数据显示，大概有七百六十万人死于癌症(占所有死亡人数的13％)。急需新的，更有效的癌症疗法来改善这种状况。长期以来，表观遗传和基因突变被认为是癌症发生过程的两种不同机制。通过基因途径致癌相对比较直接：抑癌基因的突变和/或致癌基因引起的功能获得型突变或功能丧失型突变，及异常表达。通过表观遗传途径致癌相对来说没那么简单。它取决于染色质结构包括DNA甲基化，组蛋白变体和修饰，核小体重塑和一些小的非编码调节RNAs(Sharma et al.,2010)。在癌症的发生和发展中，表观基因组经历了很多改变，包括基因组范围DNA去甲基化(超甲基化)，CpG岛启动子甲基化程度增高，核小体空间结构改变及修饰。
进年来，对表观遗传失调在癌症中的作用的认识有所增强。由此，也给靶向性癌症疗法带来了新的机遇。表观遗传学主要是研究基因DNA序列不变的转录的可遗传变化。该领域的研究，也让我们发现了些选择性激活或抑制靶基因的药物。所以，表观遗传学也成为了一门发展很迅速的学科。虽然，我们对表观遗传学的认识还相对比较肤浅。但是，近来，我们也发现一些表观遗传调控因子，包括DNA甲基化，组蛋白修饰，染色质重塑和基因转录，在癌症的引起和发展中所起的作用。大量研究表明，异常的翻译后调控-组蛋白修饰，在很多疾病的发生和发展中有着重要作用。
科学家和药学界近来开发高效、特异的染色质相关蛋白抑制剂取得很大进步。而这些抑制剂主要靶向：组蛋白甲基转移酶、组蛋白去甲基酶、乙酰赖氨酸结合域。目前，大量的研究集中在靶向组蛋白甲基转移酶。
在癌症领域，在一些不同的肿瘤类型中发现的一些组蛋白甲基转移酶基因改变无疑引起了很多关注，也支撑了疾病中广泛被认为由基因驱动的表观遗传的失调的重要性。一些例子(像后面要讨论的甲基转移酶EZH2)，它的催化结构域，SET结构域的杂合子点突变导致相对野生型的功能获得性突变。使其更倾向于超甲基化，并且沉默抑癌基因和/或特异的分化相关基因。同样的，在其它肿瘤中(比方说，多发性骨髓瘤中NSD2的高表达)，染色体移位导致甲基转移酶的高表达，也导致异常的转录和扩增。相反地，由组蛋白甲基转移酶DOT1L引起的赖氨酸甲基化却使得白血病生成所需的多种基因持续的表达。因此，对于像EZH2或DOT1L的小分子抑制剂应该能减少或消除组蛋白甲基转移酶对特异位点赖氨酸的甲基化，并且逆转它的致癌性。现今，研究的比较多的抑制剂主要是针对EZH2。
EZH2(Enhancer of zeste homolog 2)是一种组蛋白甲基转移酶，它催化甲基从辅因子S腺苷甲硫氨酸转移到组蛋白H3的27位赖氨酸上，使得组蛋白H3的27位赖氨酸三甲基化。一些研究表明在一些实体瘤中，包括前列腺癌、乳腺癌、肾癌和肺癌。EZH2水平会升高并且使其靶基因沉默，从而导致预后差。
来自Chinnaiyan实验室的开创性研究，揭示了在转移性前列腺癌中，EZH2和组蛋白H3的27位赖氨酸三甲基化高水平与预后差相关联。除此以外，组蛋白H3的27位赖氨酸去甲基酶UTX的失活突变也同样与转移性前列腺癌相关，这预示着组蛋白H3的27位赖氨酸的超甲基化在前列腺癌中起着重要的作用。其它研究也显示了EZH2的高表达及带来的靶基因的沉默与很多实体瘤的差的预后有同样的相关性。包括，乳腺，肾脏和肺。近些时候，EZH2的SET结构域的体细胞激活型突变，使组蛋白H3的27位赖氨酸三甲基化提高，在滤泡性淋巴瘤和弥散性大B细胞淋巴瘤中被鉴定出来了。结合起来，这些发现表明通过EZH2高表达或点突变而导致的组蛋白H3的27位赖氨酸三甲基化水平失调，沉默靶基因对于肿瘤的生长、存活都有重要作用，由此也突出了开发靶向此酶疗法的紧迫性。矛盾的是，EZH2的失活型突变也在骨髓增生异常综合征中被报道了，这也揭示了这个蛋白的抑癌作用。EZH2和组蛋白H3的27位赖氨酸甲基化在促进或是抑制肿瘤生成和/或维持中的作用似乎要依情况而定。鉴于潜在的毒性作用，在开发长期服用的治疗性抑制剂时要很谨慎。尽管有这些潜在的缺陷，但是很多制药和生物公司的研发团队已经开发了高效的、特异性的EZH2的小分子抑制剂。同样有很多研究兴趣在天然产物抑制剂。
EZH2，是PRC2(polycomb repressive complex 2)复合物中发挥催化作用的组分。PRC2包含四种核心亚单位：SUZ12(哺乳动物中的同源物为Su(z)12)，EZH2(哺乳动物中的同源物为E(z))，EED(哺乳动物ESC)和RbAP46/48(也称作RbBP4/7；哺乳动物中的同源物为P55)(Kuzmichev et al.,2002；Margueron andReinberg,2011)。这些组分都有很多功能。例如，SUZ12含有一个锌指模序，对于EZH2的催化活性是必须的(Pasini et al.,2004)。EZH2通过其保守的SET结构域而发挥组蛋白甲基转移酶作用。近来也发现了其类似物EZH1，同样能过介导H3K27三甲基化(Margueron et al.,2008；Shen et al.,2008)。EED是一种含WD-40重复序列的蛋白，它能与EZH2相互作用并通过所谓的芳香笼，也即由三个芳香族氨基酸(97位苯丙氨酸，364位色氨酸，365位酪氨酸)组成的结构结合三甲基化的组蛋白H3的27位赖氨酸，从而激活PRC2，是EZH2发挥甲基转移酶活性所必须的(Ketel et al.,2005；Kuzmichev et al.,2005)。总的来说，EZH2,EED和SUZ12三种组分是PRC2复合物发挥催化活性及后续的起始基因抑制功能所不可或缺的(Ketel et al.,2005；Sparmann and van Lohuizen,2006)。第四种组分，RbBP4/7，是PRC2与组蛋白尾巴发生联系所必须的(Kuzmichev et al.,2002)。除了核心亚单位外，PRC2还含有些其它因子，比方说JARID2,AEBP2,and PCL(Margueron and Reinberg,2011；Nekrasov et al.,2007；Peng et al.,2009；Shen et al.,2009)。这些组分的准确功能还不是很明白，但它们对PRC2酶活的发挥和PRC2与DNA的结合具有调节作用(Margueron and Reinberg,2011)。为了使癌症中的PRC2复合物失效，从而抑制无限制的细胞扩增，一些研究开始靶向EZH2和EED之间的相互作用，而这种相互作用对于酶活是必须的。采用了一种新的策略，通过干扰蛋白相互作用而解离PRC2复合物从而阻断EZH1和EZH2的活性。
在此种策略的启发下，本方法主要从EED开始，通过筛选能与EED结合的小分子天然产物，并进一步从中找到能干扰PRC2复合物形成的抑制剂，从而为开发一些针对于PRC2依赖性肿瘤的药物提供些借鉴。
发明内容
本发明涉及一种靶向PRC2抗肿瘤药物的筛选方法，包括：(a)使待检样品与EED接触，选出与EED能结合的初筛样品；(b)将初筛样品与EED-EZH2互作(也可称为EED-EZH2复合物)接触，若所述样品能拮抗EED-EZH2互作，则该样品为抗PRC2依赖性肿瘤阳性药物。所述方法以EED-EZH2相互作用为靶点。
其中优选的，上述方法还包括步骤(c)验证步骤(b)筛选获得的样品对PRC2依赖性肿瘤细胞的抑制效果。优选的，上述方法的步骤(a)中应用生物分子相互作用仪筛选，如生物分子相互作用仪BIACORE3000。优选的，上述方法的步骤(b)中采用竞争性Co-IP模型筛选出可以拮抗EED-EZH2互作的小分子化合物。
本发明还涉及通过上述方法筛选获得的小分子化合物。优选的，本发明涉及小分子化合物2D7或1E7。本发明还涉及一种组合物，含有上述方法筛选获得的小分子化合物以及药学上可接受的载体或辅料。优选的，本发明涉及含有小分子化合物2D7或1E7的组合物，所述组合物还含有药学上可接受的载体或辅料。
本发明还涉及上述筛选获得的小分子化合物(特别是小分子化合物2D7或1E7)或上述组合物(含有小分子化合物2D7或1E7)在制备抗肿瘤药物、或降解EZH2药物中的应用。
其中优选的，在初筛之前，先表达纯化EED蛋白：用大肠杆菌BL21表达原核表达质粒pGEX-4T-1-EED，得到的带GST标签的EED融合蛋白比较容易用Glutathione Sepharose 4B珠子进行纯化，GST标签和EED蛋白之间含凝血酶的酶切位点，使得能够从Glutathione Sepharose 4B珠子上直接切下EED，保证了所获得的EED蛋白的纯度。采用的是原核表达，获得了较高浓度的EED蛋白。同时偶联在CM5芯片上的EED相对较耐NaOH洗脱，也就是说用此方法获得的EED稳定性较高。
接着，利用生物分子相互作用仪BIACORE3000筛选待检样品，初筛出能与EED结合的小分子化合物：应用BIACORE3000进行小分子与蛋白相互作用分析。相比传统的酵母双杂，ELISA，FRET，Co-IP，ChIP，Western，EMSA等方法，有实时监控，无需标记，强亲和力，弱亲和力及瞬间相互作用都可以检测，完全活性的互作分析，自动化高、省时省力等优点。实验证明，初筛得到的小分子化合物，第一批挑18个，进行下一步复筛，有效小分子化合物有2个，其中一个在文献中有报道证实，1E7即EGCG为报道的具有抗癌作用的小分子化合物，并对EZH2和EED有降解作用。我们对2D7即Wedelolactone的相似作用进行了初步验证。说明此方法的筛选有效性还是挺高的。
接着，采用竞争性Co-IP模型复筛拮抗EED-EZH2互作的小分子化合物：竞争性CO-IP模型，对于筛选拮抗EED-EZH2互作的小分子化合物的复筛准确性高。虽然只是体外的实验，但是跟体内的作用效果还是很符合的。此模型不仅能筛选出能与EZH2竞争结合EED的小分子化合物，也能筛选出能直接降解EZH2或是EED的小分子化合物，而这些小分子化合物的共同效果是能阻碍EZH2-EED的互作。从而使其靶向性强。
最后，对筛选获得的小分子化合物的抗肿瘤作用或降解EZH2或EED进行验证：对筛选到的两种小分子化合物的作用的初步验证。对内源EZH2的降解是靶向EZH2-EED互作的一个比较直接的衡量指标，它操作简单，标准明确。目前抗肿瘤药物体外筛选的方法很多,主要有MTT比色法、3H-T dR掺入法、酸性磷酸酶法(APA)、ATP生物发光法、SRB法以及高通量筛选等,其中MTT比色法因其具有简单、快速、精确、不涉及使用放射性元素,且结果与同位素掺入法一致等优点,在很多实验室被广泛使用作为抗肿瘤药物体外筛选的常用方法。
正确的研究方法和科学的思路在抗肿瘤药物的筛选中起关键的作用,针对机制的筛选系统是比较理想的，现在很多集中在与肿瘤发生，转移，发展相关的蛋白激酶、肿瘤细胞中阻止细胞程序性死亡的基因如Bcl-2、p53基因,基质金属蛋白酶等都成为抗肿瘤药物筛选的靶标。PRC2复合物与肿瘤相关性的研究已比较明确，靶向EZH2-EED互作从而影响PRC2复合物的功能，从而重新激活被沉默的抑癌基因对于抗肿瘤有很好的效果及靶向性。本方法以EZH2-EED相互为靶点，靶向性强。应用分子相互作用分析仪BIACORE3000筛选与EED结合的小分子化合物，灵敏度较高，自动化好，操作简单。拮抗EZH2-EED互作的体外筛选模型，与体内的一致性较高。其中筛到的小分子化合物出现在被发表的文献中，也证实了此筛选方法的有效性。为今后的抗肿瘤药物的筛选提供了很好的借鉴。本发明筛选出的另一个小分子化合物也被验证对内源EZH2具有降解作用，对PRC2依赖性肿瘤细胞有很好的杀伤作用。
附图简介
现将本发明用有关附图来描述，其中：
图1：用凝血酶酶切后，收集的流出物，期间加入PBS冲洗，分管收集的EED蛋白液，分别上样，跑胶考染，对于理论值大小下面的两条蛋白条带，切胶后再进行质谱鉴定；
图2：考染理论值大小下面的两条蛋白带的质谱鉴定结果，其中(A)代表图1中上面那条带的MS结果，其中上：质谱鉴定目标蛋白条带结果，划线部分为与hEED氨基酸序列相匹配的部分，下：以上序列于NCBI数据库中比对结果，其中hEED得分最高；(B)代表图1中下面那条带的MS结果，其中上：质谱鉴定目标蛋白条带结果，划线部分为与hEED氨基酸序列相匹配的部分，下：以上序列于NCBI数据库中比对结果，其中hEED得分最高。
图3：此图为502种小分子化合物(实测499个)，用BIACORE3000检测结合EED的反应值汇总。横坐标是化合物编号，从1-499，纵坐标是BIACORE 3000测得的反应值(RU)，箭头标示的为发现的能靶向EZH2-EED互作的两种小分子化合物，1E7已被报道，2D7为新发现的；
图4：为体外筛选能干扰EED-EZH2互作的模型，小分子化合物竞争性的与EED结合，从而使Co-IP下来的EZH2减少；
图5：用竞争性Co-IP实验筛选出1E7、2D7的结果图；
图6：此图为用BIACORE3000对2D7、1E7与EED结合的动力学进行测定后的处理结果，左图为2D7的结果，右图为1E7的结果；
图7：2D7对白血病细胞K562，肝癌细胞HepG2，内源EZH2的降解作用删掉；
图8：在白血病细胞K562，肝癌细胞HepG2，不同浓度的2D7对其增殖影响的MTT试验，LO2为正常肝细胞，作为对照。
实施例
本发明下面所述是以实施例说明，本发明不限于这样的实施例。
对于本发明所述方法的实施例如下：
1、表达纯化EED蛋白。
取本实验室保存的pGEX-4T-1-EED原核表达质粒(100ng)，加入100μL感受态细胞E.coli BL21(DE3)(由康为世纪公司购买)中，冰上放置20min后42℃热刺激90s，再继续于冰上放置2min。向感受态细胞中加入800μL新鲜配置的无抗生素LB培养基(0.5％酵母提取物,1％胰蛋白胨,1％氯化钠,pH 7.0，121℃灭菌20min)后，37℃、200rpm摇床培养45min。取200μL培养后菌液均匀涂布在含50mg/L氨苄(后面用到的抗性平板及液体培养基终浓度均相同)的LB平板(0.5％酵母提取物,1％胰蛋白胨,1％氯化钠,1.5％琼脂粉，pH 7.0，121℃灭菌20min后倒皿)上，置于37℃隔水式恒温培养箱中培养,待其长出单菌落,挑单菌落至4ml含氨苄的LB液体培养基中,放摇床上37℃，250rpm，摇过夜，按体积比1：100接入200ml含氨苄的LB液体培养基中，摇到OD600至0.6-0.8，按终浓度0.2mM(母液1M)加入IPTG诱导，放摇床上28℃，150rpm，摇过夜，5000rpm,5min离心收集菌体，PBS洗三遍，用15ml PBS(KH2PO4 0.24g/L，Na2HPO4 1.44g/L，NaCl 8.0g/L，KCl 0.2g/L，pH7.4)重悬，加入终浓度1mM的蛋白酶抑制剂PMSF，用超声波细胞粉碎机，程序设定，工作时间8s,间隙时间15s,工作次数30，超声功率200W，在冰上超声裂解，重复三次，8000rpm，15min离心，收集上清再离心，8000rpm 5min，再取上清，加入填充有终体积1mLGlutathione Sepharose 4B珠子的的GST蛋白纯化柱，柱子密封，在4℃环境室的转子上旋转4h让蛋白与珠子充分结合，接着让上清流过柱子，再密封，将凝血酶(sigma T4648 1000u)500ul(储液浓度为100units/mL)与1500ul binding buffer(NaCl 8.2g/L,KCl 0.2g/L,Na2HPO4 3.6g/L,KH2PO4 0.2g/L,pH 7.3)混匀，加入珠子中，室温，低速摇床上过夜，次日收集流出物，再向珠子中加入1.5m L bindingbuffer并分管收集流出物，并用考马斯亮蓝法定性是否还有蛋白，重复该操作(再加1.5mL binding buffer,收集流出物)，直至考马斯亮法测定液不变色止，停止收集，以上所得的收集液即为所需蛋白，跑胶考染并质谱鉴定。考染及质谱鉴定结果见图1和2。所有收集液用10KD的蛋白浓缩管浓缩(操作按照Ultra-15Centrifugal Filter Devices for volumes up to 15mL User Guide进行)，用微量紫外可见·荧光分光光度计e-spect(Malcom)测定其浓度为4.152mg/mL。
2、利用生物分子相互作用仪BIACORE3000筛选，初筛出能与EED结合的小分子化合物。
将纯化的EED蛋白通过氨基偶联的方式偶联到CM5(GE Healthcare)芯片上。简言之，根据供应商的说明书，将羧甲基化的葡聚糖生物传感器芯片(CM5，GE Healthcare)用N-乙基-N’-(3-二甲基氨基丙基)-碳化二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)活化。将EED用10mM乙酸钠pH5.0稀释至100μg/ml，之后以5μL/分钟的流速注射，以达到约10000反应单位(RU)的偶联蛋白质。在蛋白质注射后，注射IM乙醇胺以封闭未反应的基团。将502个从天然植物等来源中分离的单体化合物，用binding buffer稀释后按顺序加入96孔板，设定BIACORE3000程序，自动进样(具体操作按Biacore 3000Instrument Handbook进行)。挑选RU值20以上的小分子化合物，做下一步复筛。小分子化合物过机数据见图3。
3、拮抗EED-EZH2互作的小分子化合物复筛。
用实验室保存的带myc标签的EZH2真核表达质粒，和构建在pCNDA-4.0载体的EED真核表达质粒(带myc和his双标签)，共转染至293T细胞(转染步骤要求按英格恩生物公司Entranster TM-H转染试剂使用说明进行)，转染后24h，4℃，3000rpm，5min,离心收集细胞，用PBS重悬洗去残余DMEM培养基，4℃，3000rpm，5min,离心弃上清，菌体用加有终浓度1mM的蛋白酶抑制剂PMSF1×cell lysis buffer(购自cell signaling的cell lysis buffer(10×)Catalog:9803)冰上裂解，30min，4℃，12000rpm，15min，离心收集上清，加入anti-His(TA-02,中杉金桥)抗体，4℃环境室转子上旋转1h，加入Protein G Plus-Agrose(sc-2002)，混匀后均分几管，分别按5um/L的终浓度加入初筛获得的的小分子化合物，加入等量的DMSO的管作为对照。接着将其放到4℃环境室转子上旋转过夜，4℃，3000rpm,3min，离心后去上清，用PBS重悬ProteinG珠子，放到4℃环境室转子上旋转10min，重复离心，旋转，共三次，最后4℃，3000rpm，3min,离心，小心去掉上清，向Protein G珠子中加入2×loading buffer(每20mL体系，1.0mol/LTris-HCl(pH 6.8)4mL，20％SDS 6mL,甘油4mL,β-巯基乙醇1.2mL,0.1％溴酚蓝0.8mL,ddH2O 4mL)30ul，沸水浴中煮5min，冰上冷却，12000rpm,1min，离心取上清跑8％的SDS-PAGE胶，用c-Myc抗体(9E10)(sc-40)做western blot，对比对照组与加入小分子化合物组的myc-ezh2的条带，带明显变弱的化合物为复筛获得的可以拮抗EED-EZH2互作的小分子化合物。筛选主要采用竞争性Co-IP模型(见图4)，筛选实验获得2种小分子化合物，编号1E7和2D7，结果见附图5，并对其结合EED的动力学用BIACORE3000进行了测定。结果见附图6。
4、对筛选到的两种小分子化合物的作用的初步验证。
(1)小分子化合物2D7对内源EZH2的降解作用。
实验用细胞系为本实验室保存的白血病细胞系K562和人肝癌细胞系HepG2。前一天铺六孔板，第二天长至80％汇合度时，分别按照0，10uM,50uM,100uM和0，10uM，25uM,50uM终浓度加入小分子化合物2D7，刺激24h后，用细胞刮刀刮下，并吸入离心管中，4℃，5000rpm,3min，离心去上清，收集细胞，用PBS重悬，再离心去上清，收集细胞，加入80ul，1×cell lysisbuffer含终浓度1mM的蛋白酶抑制剂PMSF冰上裂解，30min,4℃，12000rpm，10min，离心，取上清，加入5×loading buffer,沸水浴中煮5min,冰上放置冷却，短暂离心，上样，跑SDS-PAGE电泳胶，用western blot检测内源EZH2(#3147Scell signaling)表达量，结果见图7。
(2)小分子化合物2D7对肿瘤细胞的杀伤作用。
用MTT法对小分子化合物2D7对K562细胞和HepG2细胞的杀伤作用进行初步评价，LO2为正常肝细胞作为对照。本实验用的为凯基MTT细胞增殖及细胞毒性检测试剂盒。操作步骤按照说明进行。
1.在96孔板加入细胞100μL/孔(约1×104)，置37℃5％CO2细胞培养箱培养24小时。
2.加入适当浓度的受试化合物。(0，3.125uM,6.25uM,12.5uM,25uM,50uM，100uM)
3.将96孔板在37℃，含5％CO2空气及100％湿度的细胞培养箱中孵育适当时间48h。
4.将5×MTT用Dilution Buffer稀释成1×MTT。
5.每孔加50μL 1×MTT，在37℃孵育4小时，使MTT还原为甲月赞。
6.吸出上清液，每孔加150μL DMSO使甲月赞溶解，用平板摇床摇匀。
7.酶标仪在490nm波长处检测每孔的光密度。
8.结果分析：以小分子化合物浓度为横坐标，490nm波长下的光吸收值为纵坐标做曲线图。
小分子作用效果见图8。
Claims (9)
1.一种靶向PRC2抗肿瘤药物的筛选方法，包括：
(a)使待检样品与EED接触，选出与EED能结合的初筛样品；
(b)将初筛样品与EED-EZH2互作接触，若所述样品能拮抗EED-EZH2互作，则该样品为抗PRC2依赖性肿瘤阳性药物。
2.如权利要求1所述方法，其中还包括步骤(c)：验证步骤(b)筛选获得的样品对PRC2依赖性肿瘤细胞的抑制效果。
3.如权利要求1所述方法，其中步骤(a)中应用生物分子相互作用仪筛选。
4.如权利要求1所述方法，其中步骤(b)中采用竞争性Co-IP模型筛选。
5.权利要求1-6任一所述方法筛选获得的小分子化合物。
6.如权利要求5所述的小分子化合物，其为2D7或1E7。
7.组合物，含有权利要求1-4任一所述方法筛选获得的小分子化合物以及药学上可接受的载体或辅料。
8.如权利要求7所述的组合物，其中的小分子化合物为2D7或1E7。
9.权利要求5或6的小分子化合物或权利要求7或8的组合物在制备抗肿瘤药物、或降解EZH2药物中的应用。
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