CN111721932A - 以cd133为靶点的小分子化合物的筛选方法及其在制药中的应用 - Google Patents
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Abstract
本发明属于生化制药技术领域,涉及一种筛选抗肿瘤化合物的方法,包括将CD133与待测化合物孵育,使用SMM高通量筛选或者亲和性实验检测,比较实验组与对照组的信号差别。本发明以CD133蛋白为靶点,筛选到目标小分子化合物。本发明还提供了目标小分子化合物在制备抗肿瘤药物中的应用,通过CCK‑8实验,干细胞成球实验以及软琼脂克隆实验对筛到的化合物进行了生物活性检测,评估了肝癌治疗的有效性,并在分子水平上阐明了它与蛋白的作用机制和规律,为治疗肝癌的新药研发提供了基础。
Description
技术领域
本发明属于生化制药技术领域,涉及一种以CD133为靶点的抗肝癌药物筛选的方法及应用。
背景技术
肝癌(hepatocellular carcinoma,HCC)是一种最常见的原发性肝脏恶性肿瘤,发病率及死亡率呈逐年增长的趋势。目前我国肝癌的发病人数占据全国的半数以上,已成为严重威胁人们健康和生命的因素之一。肝癌在早期没有典型的症状,确诊时基本已经是肝癌的晚期,错过了最佳的治疗时期。目前临床上的治疗手段也比较有限,主要依赖于手术切除、化疗和少数的肝移植。但是治疗效果并不是那么的理想,而且化疗只能杀死部分肿瘤细胞,残余的肿瘤细胞容易再次死灰复燃形成新的病灶。因此,深入研究肝癌发生、发展的分子机制及寻找性价比高的治疗药物对于临床诊疗方案改进和病人预后的改善非常重要。
干细胞标志分子CD133蛋白又名Prominin-1,是一个五次跨膜的糖蛋白。在不同类型的肿瘤中均能发现CD133的异常表达。CD133+肿瘤细胞具有更强的自我更新能力、成瘤能力、放化疗抵抗能力和迁移能力等,在肿瘤的复发和转移中起重要作用,而肿瘤细胞的这些特点正是肿瘤难以治愈的主要原因。在多种人的肝癌细胞系中,以CD133分子为细胞表面标志物,分选出CD133+和CD133-细胞群体,经裸鼠皮下肿瘤形成比较发现,CD133+细胞具有更强的成瘤能力,同时通过干细胞成球实验、克隆形成实验和增殖比较发现,CD133+肿瘤细胞同样具有更强的体外增殖和自我更新能力。肝癌标本中分选出来的原代细胞中也有着CD133的表达,CD133的表达率大概为1.3%-13.6%,且分选出来的这部分CD133阳性细胞在干细胞培养基中的形成的细胞球明显大于阴性细胞,皮下肿瘤形成能力也更强,并且CD133表达高的病人预后更差。近年来,分子靶向治疗作为肿瘤治疗的新策略,以其特异性强,副作用小等治疗优势逐渐被推崇。研究表明CD133单克隆抗体可以通过抑制CD133+肝癌起始细胞生长起到治疗的作用。与CD133双特异性的融合毒蛋白靶向肿瘤干细胞,能够显著抑制小鼠异种移植肿块的生长。活性小分子化合物是指可以通过调节蛋白的生物功能引起细胞或生物体表型改变的一类化合物。许多上市药物都是通过对其结构修饰得到的,因此活性小分子化合物向来都是药物化学的研究热点。目前国内外还没有寻找到临床有效的CD133抑制剂,由于缺乏标准化的,有效的筛选方法,极大的限制了CD133抑制剂的筛选和应用。
发明内容
本发明的目的是提供一种以CD133蛋白为靶点的小分子化合物及其筛选方法和在制药中的应用。
本发明以CD133蛋白为靶点,利用小分子微阵列技术高通量筛选到了小分子化合物LDN193189,并通过体外激酶实验以及DARTS实验进行了验证;同时通过CCK-8实验,干细胞成球实验以及软琼脂克隆实验对筛到的化合物进行了生物活性检测,评估了肝癌治疗的有效性,并在分子水平上阐明了它与蛋白的作用机制和规律,本发明在上述基础上完成。
首先,本发明提供了一种筛选抗肿瘤化合物的方法:将CD133与待测化合物孵育,使用小分子微阵列技术或者亲和性实验检测,比较实验组与对照组的信号差别,具有显著差别的即目标化合物。
方法具体包括以下步骤:
制备靶分子CD133;
将CD133与待测化合物孵育;
使用SMM高通量筛选或者亲和性实验检测;
对比实验组和对照组的结果,筛选出目标化合物。
在高通量筛选步骤中,待测化合物在苯基异氰酸酯功能化载玻片和己基异氰酸酯功能化载玻片上复制,比较与CD133蛋白C端孵育前后的光折射情况,与蛋白结合后的小分子折射情况会发生变化。较好的,待测化合物与CD133孵育之前,暴露于含有BSA的PBS中以阻断未打印的异氰酸酯功能化表面。
在本发明的一个实施例中,使用DART亲和性实验进行筛选,其中待测化合物与CD133孵育,然后加入蛋白酶,比较加入蛋白酶前后CD133的性质,与CD133相互作用的化合物将减弱CD133的改变。
在本发明的优选实施例中,提供了以人CD133蛋白为靶点小分子微阵列SMM高通量筛选的方法,其特征如下:我们挑选了拥有3375种生物活性化合物库,包括1053种中药天然化合物(大部分来自草药)、1527种经食品药品监督管理局(FDA)批准的药物和795种已知抑制剂。将每种化合物溶解于二甲基亚砜中,浓度为10mM,并在苯基异氰酸酯功能化载玻片和己基异氰酸酯功能化载玻片上复制。在1×PBS中暴露于7600nM BSA中30分钟,以阻断未打印的异氰酸酯功能化表面;小分子固定在一个微阵列上,光照在上面会有折射。然后和纯化的CD133蛋白C端孵育过后,不和蛋白结合的小分子光的折射不会发生改变;而与蛋白结合后的小分子折射情况会发生变化。说明该化合物可能是一种靶向CD133蛋白的小分子抑制剂。
基于CD133蛋白与小分子化合物反应的亲和性进行筛选的方法,其特征如下:首先通过不同浓度的LDN193189(1mM,100μM,10μM)分别与表达纯化的CD133蛋白和肝癌细胞Huh7CD133+裂解液在4℃孵育2小时,孵育完成后,按照质量比1:100(1μg of pronase forevery 100μg of lysate)于各管中迅速加入相应的酶,室温(20-25℃)酶解15min。停止酶解:按质量比1:10加入0.5M EDTA(pH8.0)停止酶解反应。如果待筛选的小分子化合物加入细胞裂解液后与其靶蛋白CD133结合,从而降低了对蛋白酶的敏感性,并且随着浓度的增加,这种“保护”作用加强,说明二者间有相互作用。说明该化合物可能是一种靶向CD133蛋白的小分子抑制剂。
进一步,本发明提供了一种具有抗肿瘤活性的小分子化合物,所述的小分子化合物称为LDN193189,其结构如下:
所述的小分子化合物可以使用前述方法筛选获得,也可以根据分子结构人工合成。
相应的,本发明提供了一种抗肿瘤试剂盒,其活性成分是LDN193189。
进一步,本发明提供了所述的小分子化合物LDN193189在制备抗肿瘤药物中的应用。
本发明中,所述的抗肿瘤药物靶向CD133。
其中,小分子化合物LDN193189是抗肿瘤药物的活性成分。
较好的,所述的抗肿瘤药物为能够抑制肿瘤细胞增殖和自我更新能力的药物。
所述的抗肿瘤药物为能够抑制肿瘤细胞迁移的药物。
所述的抗肿瘤药物为能够诱导肿瘤细胞凋亡的药物。
所述的肿瘤可以是任何一种,较好的,所述的肿瘤源自CD133阳性或者表达量增高的细胞。
本发明提供了靶向肝癌细胞的CD133蛋白的药物,所述的药物可以是含有LDN193189小分子化合物的口服液、颗粒剂、片剂,硬胶囊剂、软胶囊剂、滴丸剂、注射剂、纳米制剂以及靶向制剂。
基于以CD133为靶点的小分子抑制剂是创新抗肝癌药物的开发方向之一。本发明提供了一种筛选抗肿瘤化合物的方法,即将CD133与待测化合物孵育,使用SMM高通量筛选或者亲和性实验检测,比较实验组与对照组的信号差别。本发明以CD133蛋白为靶点,筛选到了目标小分子化合物。本发明还提供了目标小分子化合物在制备抗肿瘤药物中的应用。通过CCK-8实验,干细胞成球实验以及软琼脂克隆实验对筛到的化合物进行了生物活性检测,评估了肝癌治疗的有效性,并在分子水平上阐明了它与蛋白的作用机制和规律,为治疗肝癌的新药研发提供了基础。
附图说明
图1.基于小分子微阵列技术的高通量筛选结果;通过比对小分子微阵列SMM与纯化的CD133蛋白孵育前后的图像筛选结果,筛到了与CD133蛋白结合的小分子LDN193189。这个化合物在两次实验均有反应信号,且背景信号为零或者相比于反应信号非常微弱的样品点。
图2.利用DARTS验证LDN193189与CD133蛋白之间的相互作用。图2A,LDN193189与原核表达的CD133蛋白孵育,并最后进行酶解实验。LDN193189的浓度与CD133条带的信号强度成正相关。而不加LDN193189,CD133都被蛋白酶水。图2B,LDN193189与Huh7CD133+肝癌细胞裂解液孵育并进行酶解实验。当小分子LDN193189加入细胞裂解液后,LDN193189与其靶蛋白CD133结合,从而降低了对蛋白酶的敏感性,并且随着LDN193189浓度的增加,这种“保护”作用加强。
图3.小分子化合物LDN193189对Huh7CD133+肝癌细胞的生长抑制作用。图3A为小分子化合物LDN193189在处理前后细胞培养形态。实验结果表明小分子LDN193189处理后Huh7细胞的形态显著改变,细胞密度降低,细胞固缩,体积变小。图3B为CCK8增殖实验,在化合物处理后24h,48h,72h进行CCK8检测。实验结果表明LDN193189对CD133+表达的细胞具有明显的抑制作用。
图4.小分子化合物LDN193189抑制剂CD133+的肝癌细胞的自我更新能力。图4A为Huh7CD133+肝癌细胞的细胞成球。结果如图所示,实验组LDN193189细胞形成的细胞球显著少于对照组,图4B为Prf5CD133+肝癌细胞的成球实验;实验组LDN193189细胞形成的细胞球直径明显比对照组小。图4C为Huh7CD133+的软琼脂克隆实验,同样的实验组干细胞成球的数量明显少于对照组,说明LDN193189能抑制CD133+的肝癌细胞的自我更新。
图5.小分子化合物LDN193189抑制CD133+肝癌细胞的迁移。Transwell实验结果表明,LDN193189处理后,Huh7CD133+迁移的细胞明显减少。
图6.小分子化合物LDN193189对细胞凋亡的影响。对照药物Dmso及小分子化合物处理48h后的Huh7CD133+细胞进行AnnexinV-FITC/PI双染,结果表明LDN193189可以诱导Huh7CD133+的肝癌细胞发生凋亡。
图7.小分子化合物LDN193189抑制肝癌发生的作用抑制。Western blot法检测经小分子化合物处理48h后,细胞中p-CD133,CD133以及肝癌细胞周期、凋亡、干性基因相关蛋白的表达情况。如图所示:小分子化合物LDN193189处理后,CD133的磷酸化水平明显降低,抗凋亡基因Bcl-2蛋白表达升高;周期相关基因CDK4,CDK6,P21基因蛋白表达水平下调,同时可以抑制P-src的表达水平,因此小分子化合物LDN193189抑制CD133的磷酸化,进而可以诱导肝癌细胞发生凋亡,引起细胞周期阻滞,并抑制p-src的异常激活。
具体实施方式
本发明提供的具体实施方式作详细说明,但本发明的实施并不仅限于此。
本实施例中未注明的具体条件和实验方法,通常按照《分子克隆实验指南》中所述常规条件,或者参照试剂生产商提供的条件实施。
实施例1.小分子微阵列高通量筛选
小分子微阵列(SmallMoleculeMicroarray,SMM)是近十年来迅速发展的另一种高通量技术。SMM是指在固态表面上通过点样(或打印)然后固定各类有机小分子所形成的高密度微阵列,能够一次性、一步式地同时分析成千上万个生物化学相互作用。小分子化合物微阵列:主要用于药物筛选和药物发现。化合物微阵列具有识别和评估小分子的能力,因此在制药行业中,它显得比其它技术更为有用。
本发明制备了3375种生物活性化合物(的小分子微阵列,包括1053种中药天然化合物(大部分来自草药)、1527种经食品药品监督管理局(FDA)批准的药物和795种已知抑制剂。将每种化合物溶解于二甲基亚砜中,浓度为10mM,并在苯基异氰酸酯功能化载玻片和己基异氰酸酯功能化载玻片上复制。在1×PBS中暴露于7600nM BSA中30分钟,以阻断未打印的异氰酸酯功能化表面;小分子固定在一个微阵列上,光照在上面会有折射。然后和纯化的CD133蛋白C端孵育过后,不和蛋白结合的小分子光的折射不会发生改变;而与蛋白结合后的小分子折射情况会发生变化。
实施例2.DARTS技术筛选小分组和蛋白相互作用
DARTS作为一种新的药物靶标鉴定方法,主要通过蛋白酶水解未被药物结合的蛋白质,从而验证药物与靶蛋白的结合的一种方法。我们首先通过不同浓度的LDN193189(1mM,100μM,10μM)分别与表达纯化的CD133蛋白和肝癌细胞Huh7CD133+裂解液在4℃孵育2小时,孵育完成后,按照质量比1:100(1μg of pronase for every 100μg of lysate)于各管中迅速加入相应的酶,室温(20-25℃)酶解15min。停止酶解:按质量比1:10加入0.5MEDTA(pH8.0)停止酶解反应。当小分子LDN193189加入细胞裂解液后,LDN193189与其靶蛋白CD133结合,从而降低了对蛋白酶的敏感性,并且随着LDN193189浓度的增加,这种“保护”作用加强,可以看出,LDN193189的浓度与CD133条带的信号强度成正相关。而不加LDN193189,CD133都被蛋白酶水解而检测不到条带信号。
实施例3.细胞水平的抗肝癌活性检测
1.细胞形态学观察
将CD133+表达Huh7的细胞制成单细胞悬液,按3x104个/ml,接种于6孔板中,每孔2ml,设给药组和阴性对照组,培养48h后,放置倒置显微镜下观察细胞的生长状态并拍照。
2.CCK8增殖实验
利用96孔板进行CCK8细胞增殖检测,将CD133+表达Huh7的细胞消化悬浮于含有血清的培养基中,吹打均匀调整细胞密度为3x104个/ml,每孔接种3000个,置于37℃,5%CO2及饱和湿度条件下进行培养,待细胞贴壁后进行化合物处理,在96孔板中每孔加入3000个细胞,分别取24h,48h,72h进行CCK8检测检测。结果表明,加入小分子化合物LDN193189能够抑制肿瘤细胞增殖。
3.干细胞成球实验
将贴壁培养CD133+表达Huh7肝癌吸去上清再加入消化液,作用2min,.2000rpm离心5min吸去上清,用含有20ng/mL EGF(Chemicon)、20ng/mL FGF-2(Chemicon)、2μg/mLheparin(Sigma)、1:50的比例加入不含维生素A的B27(Gibco)、100μg/ml青霉素及50μg/ml链霉素的DMEM/F12培养基(Gibco)洗涤细胞,2000rpm离心5分钟,吸去上清,用DMEM/F12培养基重复洗涤细胞2次,用细胞计数仪计算细胞数量。将对照组和实验组的细胞等量(100个每孔)分到低吸附的96孔板(Corning),每孔细胞悬浮于100μl。每3天补充30μlDMEM/F12培养基,培养2周后,在显微镜下拍照,统计形成细胞球的数量和直径,以直径大于100μm的细胞球作为有效的结果。小分子化合物LDN193189能够减弱肿瘤细胞自我更新作用。
4.软琼脂克隆实验
1)取适当生长状态的细胞,消化为单细胞悬液,使用细胞计数仪计数,用培液吹打成单细胞悬液备用。
2)准备浓度为1.2%和0.7%的低熔点琼脂糖溶液,高温高压灭菌后,保持在40℃使琼脂糖溶液不会凝固。
3)将1.2%的低熔点琼脂糖溶液与预热到40℃的2×DMEM培养基(含有20%的胎牛血清和2倍工作浓度的抗生素)以1:1混匀,铺入6孔板的底层,冷却凝固后即为底层平板。
4)按1:1比例混匀0.7%的琼脂糖与2×DMEM培养基(含2倍工作浓度抗生素和20%胎牛血清),再向其中加入含有(1um化合物处理的3X104个细胞量的细胞悬液,彻底混匀,加入已制备好的底层平板上,即为制备好的双琼脂层。待上层琼脂凝固后,转移入细胞培养箱中培养了16天,期间观察。
5)使用成像***采集图像,计算细胞克隆数、克隆形成率。
结果显示,加入小分子化合物LDN193189,细胞克隆数和克隆形成率均降低。
5.凋亡检测
将CD133+表达Huh7的细胞制成单细胞悬液,按3x104个/ml,接种于6孔板中,每孔2ml,设给药组和阴性对照组,培养48h后,加入无EDTA的胰酶进行消化,悬浮于有血清的培养基中。然后用PBS清洗2次(1000rpm离心2min).加入利用500ul的1xAnnexin-Bingdingbuffer重悬细胞,然后加入5ul的Annexin和PI工作液,于室温避光孵育,15min利用流式细胞仪进行检测。
结果显示,小分子化合物LDN193189能够促进肿瘤细胞发生凋亡。
Claims (13)
1.一种筛选抗肿瘤化合物的方法,其特征在于,将CD133与待测化合物孵育,使用SMM高通量筛选或者亲和性实验检测,比较实验组与对照组的信号差别,确定目标化合物;该方法包括步骤:
(1)制备靶分子CD133;
(2)将CD133与待测化合物孵育;
(3)使用SMM高通量筛选或者亲和性实验检测;
(4)对比实验组和对照组的结果,筛选出目标化合物。
2.如权利要求1所述的方法,其特征在于,步骤(3)中待测化合物在苯基异氰酸酯功能化载玻片和己基异氰酸酯功能化载玻片上复制,比较与CD133蛋白C端孵育前后的光折射情况,与蛋白结合后的小分子折射情况会发生变化。
3.如权利要求2所述的方法,其特征在于,待测化合物与CD133孵育之前,暴露于含有BSA的PBS中以阻断未打印的异氰酸酯功能化表面。
4.如权利要求1所述的方法,其特征在于,待测化合物与CD133孵育,然后加入蛋白酶,比较加入蛋白酶前后CD133的性质,与CD133相互作用的化合物将减弱CD133的改变。
5.权利要求1所述的方法在制备或者筛选抗肿瘤药物中的应用。
7.如权利要求6所述的小分子化合物,其特征在于,所述的小分子化合物采用权利要求1所述的方法筛选获得。
8.一种抗肿瘤试剂盒,其特征在于,该试剂盒的活性成分是权利要求7所述的小分子化合物。
9.权利要求7所述的小分子化合物在制备抗肿瘤药物中的应用。
10.如权利要求9所述的应用,其特征在于,所述的抗肿瘤药物靶向CD133。
11.如权利要求9所述的应用,其特征在于,所述的抗肿瘤药物是抑制肿瘤细胞增殖、肿瘤细胞迁、促进肿瘤细胞凋亡或者自我更新的药物。
12.如权利要求9所述的应用,其特征在于,所述的肿瘤源自CD133阳性或者表达量增高的细胞。
13.如权利要求9所述的应用,其特征在于,所述的肿瘤源自CD133阳性的肝癌细胞。
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