CN114685641A - 一种抑制brca1/bard1复合体结合的多肽及其应用和防治癌症的药物 - Google Patents
一种抑制brca1/bard1复合体结合的多肽及其应用和防治癌症的药物 Download PDFInfo
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- CN114685641A CN114685641A CN202210175810.5A CN202210175810A CN114685641A CN 114685641 A CN114685641 A CN 114685641A CN 202210175810 A CN202210175810 A CN 202210175810A CN 114685641 A CN114685641 A CN 114685641A
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Abstract
本发明提供了一种抑制BRCA1/BARD1复合体结合的多肽及其应用和防治癌症的药物,属于蛋白多肽技术领域。所述多肽与BRCA1蛋白具有较强结合能力,将所述多肽递送至BRCA1阳性以及BARD1阳性癌细胞中,可有效抑制细胞的增殖,同时联合靶向药物PARP酶抑制剂奥拉帕利以及化疗药物卡铂,有靶向增敏和化疗增敏效应。可见,所述多肽可应用于在治疗癌症的药物中或制备抑制BRCA1蛋白结合BARD1蛋白的试剂中。相比于现有靶向药物,本发明提供的多肽药物精准抑制BRCA1/BARD1复合体蛋白介导的同源重组修复通路,可为多重耐药乳腺癌患者治疗提供新的治疗策略。
Description
技术领域
本发明属于蛋白多肽技术领域,具体涉及一种抑制BRCA1/BARD1复合体结合的多肽及其应用和防治癌症的药物。
背景技术
乳腺癌是发生在乳腺的上皮性恶性肿瘤,是威胁我国女性健康的头号杀手。乳腺癌早期阶段不易发现,中晚期患者临床主要表现为乳腺包块、乳头内陷、橘皮征、腋窝包块等症状。晚期患者还会累及骨、脑、肝、肺等部位出现相应症状。早中期乳腺癌主要通过手术、放化疗、内分泌、靶向等治疗获得长时间的无瘤生存甚至治愈,但晚期患者只能保守治疗减少痛苦。
目前公认的是,乳腺癌是一种受到基因和环境共同影响和调控的肿瘤。其中,同源重组修复在乳腺癌的基因损伤修复中有着重要的临床意义,鉴于多种化疗或者靶向药物都是通过制造肿瘤基因损伤杀灭肿瘤,因此靶向同源重组修复通路可以阻断癌细胞的修复。BRCA1/BARD1复合体是同源重组修复的起始分子,主要负责DNA双链损伤的精准修复。若通过干扰BRCA1/BARD1复合体的形成导致修复受阻,大量损伤DNA累积则会造成癌细胞死亡(见图1)。目前,临床靶向DNA修复药物如奥拉帕利是靶向DNA单链损伤修复药物,并无靶向DNA双链损伤的药物。不仅如此,由于BRCA1分子量较高(214kD),难以提纯分离,因此通过高通量筛选暂未找到合适的小分子抑制剂。
发明内容
有鉴于此,本发明的目的在于提供一种抑制BRCA1/BARD1复合体结合的多肽,所述多肽与BRCA1蛋白具有更高结合能力,有效封闭BRCA1与BARD1结合的关键氨基酸使BRCA1和BARD1无法结合,对BRCA1和BARD1阳性乳腺癌细胞增殖有效抑制,可用于制备治疗乳腺癌的药物。
本发明提供了一种抑制BRCA1/BARD1复合体结合的多肽,所述多肽的氨基酸序列如SEQ ID NO:1所示。
优选的,所述多肽与BRCA1蛋白的结合常数为11.9nM。
本发明提供了所述抑制BRCA1/BARD1复合体结合的多肽在制备预防和/或治疗癌症的药物中的应用。
优选的,所述癌症为BRCA1和BARD1阳性的乳腺癌。
本发明提供了所述抑制BRCA1/BARD1复合体结合的多肽在制备抑制BRCA1蛋白和BARD1蛋白结合的试剂中的应用。
本发明提供了一种用于防治癌症的药物,包括所述多肽和递送系统。
优选的,所述递送系统包括以下一种:脂质体、纳米金和纳米硒。
优选的,所述药物为纳米硒-多肽。
优选的,所述药物还包括靶向药物PARP酶抑制剂和/或化疗药物。
本发明提供的抑制BRCA1/BARD1复合体结合的多肽,氨基酸序列如SEQ ID NO.1所示。本发明通过荧光偏振实验,检测多肽与BRCA1蛋白结合能力,多肽与BRCA1蛋白结合常数为11.9nM,表明所述多肽与BRCA1蛋白具有较强的结合能力,有效封闭BRCA1与BARD1结合的关键氨基酸使BRCA1和BARD1无法结合,对BRCA1和BARD1阳性乳腺癌细胞增殖有效抑制,可用于制备治疗乳腺癌的药物。本发明实施例将所述多肽递送至BRCA1和BARD1阳性乳腺癌细胞中,可有效抑制乳腺癌细胞的增殖,诱导细胞凋亡。可见,所述多肽可应用于在制备治疗乳腺癌的药物中或制备抑制BRCA1/BARD1蛋白结合的试剂中。
本发明提供了一种用于防治癌症癌的药物,包括所述多肽和递送系统。相比于现有靶向药物,本发明提供的药物定向干预DNA损伤修复过程可为乳腺肿瘤治疗提供新的治疗策略,不仅可以单药使用,还可以根据合成致死的原理,联用靶向药物PARP酶抑制剂奥拉帕利或者化疗药物卡铂,实现化疗或靶向增敏作用,填补了全球范围内靶向DNA双链损伤修复的PPI药物空白。
附图说明
图1为BRCA1/BARD1复合体诱导癌症发生机理图;
图2为本发明提供的抑制BRCA1/BARD1复合体结合的多肽与BRCA1亲和力检测结果;
图3为BRCA1/BARD1 Inhibitor药物体外增殖抑制活性检测结果;其中图3A为本实验中使用的不同细胞系BRCA1及BARD1蛋白检测结果,在MCF-10A、MDA-MB-231、HCC-1599、4T-1中BRCA1及BARD1为野生型,在MDA-MB-231中为缺失BRCA1基因缺失细胞系;图3B为多肽药物对于MCF-10A、MDA-MB-231、HCC-1599、4T-1以及MDA-MB-231中的生长抑制效果,多肽药物对于MCF-10A、MDA-MB-231、HCC-1599、4T-1展示出了良好的抑制增殖效果,但是在MDA-MB-231细胞系中无效果;图3C为多肽药物诱导4T-1细胞细胞周期停滞结果,多肽药物增加了处于G2期及S期细胞明显增多;图3D为多肽药物诱导4T-1细胞产生凋亡检测结果,高浓度下多肽药物有效诱导4T-1细胞发生凋亡;
图4为BRCA1/BARD1 Inhibitor药物细胞内抑制细胞内BRCA1/BARD1结合的结果,图4A为加入药物处理后检测DNA损伤标志物γ-H2X结果,证明药物处理后有效抑制了BRCA1蛋白介导的DNA损失修复过程;图4B为加入药物处理后检测BRCA1蛋白及BARD1蛋白共定位结果,证明了在加入药物处理后细胞内BRCA1蛋白及BARD1蛋白结合被有效抑制;
图5为动物实验检测多肽的药效,其中图5A为对4T-1裸鼠移植瘤药物处理过程实时监测结果;图5B为处理完成后对于肿瘤的照片结果;图5C为处理完成后各组肿瘤称重结果;图5D为对于药物处理后不同组的肿瘤进行HE染色观察;图5E为对于药物处理后不同组的肿瘤进行肿瘤标志物Ki-67染色观察;图5F为对于药物处理后不同组的肿瘤进行肿瘤标志物Tunel染色观察。
具体实施方式
本发明提供了一种抑制BRCA1/BARD1复合体结合的多肽。由于BRCA1通过N端的两个α螺旋与BARD1形成稳定的二聚体复合物,根据BRCA1/BARD1的复合物结构的接触界面积计算,首选在BARD1上截出104位到115位氨基酸的多肽序列具备和BARD1的初步结合能力。在得到的多肽和BRCA1的复合物的结构上,进一步利用Rosetta系统对多肽和BRCA1的结合关键氨基酸进行虚拟饱和突变以得到一条远超BRCA1/BARD1结合能力的多肽,所述多肽的氨基酸序列如SEQ ID NO:1(CSLQELWEKLKKL)所示。
本发明对所述多肽的来源没有特殊限制,采用本领域所熟知的多肽来源即可。在本发明实施例中,所述多肽采用多肽固相合成方法合成。本发明对所述多肽固相合成方法没有特殊限制,采用本领域所熟知多肽合成方法即可,例如Fmoc多肽合成。Fmoc保护氨基酸购买自吉尔生化,HBTU和HIBT缩合剂来自苏州昊帆生物。
在本发明中,采用荧光偏振实验检测多肽与BRCA1蛋白结合能力,所述多肽与BRCA1蛋白的结合常数优选为11.9nM。多肽可以结合BRCA1的RING结构域的104位到115位氨基酸,而这段氨基酸是BARD1结合BRCA1所必须氨基酸序列。所述多肽药物与BRCA1的亲和力远超BARD1,从而实现阻止BARD1结合BRCA1。BARC1/BARD1复合体对于肿瘤DNA双链损伤后启动同源重组修复是必需的,因此抑制该复合体形成能够抑制肿瘤DNA修复,从而杀灭肿瘤细胞。
在本发明中,将所述多肽利用纳米硒递送至癌细胞中,能有效抑制细胞增殖。鉴于所述多肽靶向结合BRCA1,抑制乳腺癌细胞增殖的功能,本发明提供了所述抑制BRCA1/BARD1复合体结合的多肽在制备预防和/或治疗癌症的药物中的应用。
在本发明中,所述癌症优选表现为BRCA1和BARD1阳性的癌症,例如表现为BRCA1和BARD1阳性的乳腺癌。所述乳腺癌的细胞包括以下一种或几种乳腺细胞:MDA-MB-231,BT-549,HCC1599和4T1。
本发明提供了一种用于治疗乳腺癌的药物,包括所述多肽和递送系统。
本发明对所述递送系统的种类不做特殊限制,采用本领域所熟知的递送系统即可,例如纳米金、脂质体、纳米硒或其他本领域公知的纳米递送系统。在本发明实施例中,以纳米硒为递送系统为例,举例说明药物的药效。本发明对所述纳米硒-多肽药物的制备方法没有特殊限制,采用本领域所熟知的纳米硒偶联多肽的方法即可。所述药物经实验证实在细胞水平和动物水平表现出无毒性,具有较高的安全性。
在本发明中,所述药物还包括靶向药物PARP酶抑制剂和/或化疗药物。纳米硒-多肽药物联合靶向药物PARP酶抑制剂奥拉帕利以及化疗药物卡铂,有靶向增敏和化疗增敏效应。
下面结合实施例对本发明提供的一种抑制BRCA1和BARD1结合的多肽及其应用和防治癌症的药物进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实验原料的说明
试验材料分为合成部分:Fmoc氨基酸为上海吉尔生化购买,DIEA,HOBT,HBTU为在Sigma-Aldrich(默克生命科学旗下公司)购买。纳米硒制备部分为:壳聚糖,维生素C购买自阿拉丁试剂。
名词说明
BRCA1/BARD1 Inhibitor代表偶联了多肽药物及纳米硒递送系统的成品。
实施例1
使用蛋白质辅助设计的手段得到抑制BRCA1/BARD1结合的多肽
使用多肽固相合成方法按照氨基酸序列合成多肽药物(为自己实验室合成)。合成方法为一般Fmoc多肽合成。Fmoc保护氨基酸购买自吉尔生化,HBTU,HIBT缩合剂来自苏州昊帆生物。合成方法如下:
1)去保护:Fmoc保护的柱子和单体必须用一种碱性溶剂(piperidine)去除氨基的保护基团。
2)激活和交联:下一个氨基酸的羧基被一种活化剂所活化。活化的单体与游离的氨基反应交联,形成肽键。在此步骤使用大量的超浓度试剂驱使反应完成。循环:这两步反应反复循环直到合成完成。
3)洗脱和脱保护:多肽从柱上洗脱下来,其保护基团被一种脱保护剂(TFA)洗脱和脱保护,得到粗品。
采用高效液相色谱法和ESI-MS法分别对所述多肽进行表征。
多肽药物的氨基酸序列为CSLQELWEKLKKL(SEQ ID NO:1)。
实施例2
BRCA1/BARD1 Inhibitor纳米硒药物的制备
偶联方法为:1ml的实施例1合成的多肽,浓度1mg/ml,加入0.2ml50mM亚硒酸钠、0.6ml 5%壳聚糖,1.6ml 50mM维生素C,60℃加热30分钟,冷却至常温,即可得到偶联了纳米硒的多肽药物。
实施例3
BRCA1/BARD1 Inhibitor药物的细胞水平实验
1.细胞培养方法为:MDA-MB-231、BT-549、HCC1599和4T1细胞株的培养条件均为1640培养基,10%胎牛血清,5%CO2的饱和湿度,37℃贴壁培养,细胞培养密度最大至90%融合度。
2.荧光偏振检测多肽药物与BRCA1蛋白结合
荧光物质经单一平面的偏振光蓝光(波长485nm)照射后,可吸收光能跃入激发态;在恢复至基态时,释放能量并发出单一平面的偏振荧光(波长525nm)。偏振荧光的强度与荧光物质受激发时分子转动的速度成反比。大分子物质旋转慢,发出的偏振荧光强;小分子物质旋转快,其偏振荧光弱。利用这一现象建立了荧光偏振测定药物与BRCA1蛋白结合能力。
常规方法体外表达BRCA1蛋白并进行纯化,将多肽药物标记罗丹明对BRCA1蛋白经混合孵育进行荧光检测。在全黑96孔板中加入10nMBRCA1蛋白,多肽最高浓度从5μM浓度开始等倍稀释至9.7nM。BRCA1蛋白及荧光标记多肽共孵育10分钟后,检测荧光偏振值。
图2为多肽与BRCA1亲和力检测结果。所述多肽与BRCA1蛋白的结合常数优选为11.9nM。
3.药物抑制癌细胞增殖能力实验
Cell Counting Kit-8检测方法分析药物抑制癌细胞增殖能力。
Cell Counting Kit-8简称CCK-8试剂盒,是一种基于WST-8的广泛应用于细胞增殖和细胞毒性的快速高灵敏度检测试剂盒。WST-8是一种类似于MTT的化合物,在电子耦合试剂存在的情况下,可以被线粒体内的一些脱氢酶还原生成橙黄色的化合物。细胞增殖越多越快,则颜色越深;细胞毒性越大,则颜色越浅。对于同样的细胞,颜色的深浅和细胞数目呈线性关系。
在进行检测细胞活性的实验时,将MDA-MB-231、BT-549、HCC1599和4T1细胞以3×104cells/mL的密度,种入TC(Tissue Culture treated)处理的96孔板之中,每孔中加入100μl细胞液。细胞贴壁培养24h之后,在细胞内加入不同浓度实施例2制备的多肽药物(10000nM,5000nM,2500nM,1250nM,625nM,312.5nM,156.3nM,78.1nM)进行处理。处理48h之后,在每孔中均加入10μL CCK8试剂,并于37℃培养箱中孵育2小时。显色完成后,使用酶标仪检测用分光光度计测量每孔在450nm波长和690nm波长的吸光值。测得后,根据公式I校准每孔的吸光值。最后依据公式II计算细胞活力。
A=OD450-OD690公式I
细胞活力(%)=(A(加药)-A(背景)/A(对照)-A(空白)×100%公式II
经过多肽药物处理后,检测计算得到代表偶联了多肽及纳米金递送系统的多肽药物对于乳腺癌细胞的抑制增殖作用。
结果见图3。在MCF-10A、MDA-MB-231、HCC-1599、4T-1中BRCA1及BARD1为野生型,在MDA-MB-231中为缺失BRCA1基因缺失细胞系。多肽药物对于MB231乳腺癌细胞的IC50为0.156μM,对于BT-549乳腺癌细胞的IC50为1.00μM,对于HCC-1599乳腺癌细胞的IC50为0.169μM,多肽药物对于MCF-10A、MDA-MB-231、HCC-1599、4T-1展示出了良好的抑制增殖效果,但是在MDA-MB-231细胞系中无效果。多肽药物诱导4T-1细胞细胞周期停滞结果,多肽药物增加了处于G2期及S期细胞明显增多。多肽药物诱导4T-1细胞产生凋亡检测结果,高浓度下多肽药物有效诱导4T-1细胞发生凋亡。
4.为了研究BRCA1/BARD1 Inhibitor药物对BRCA1及BARD1蛋白的抑制能力,采用免疫荧光染色(IHC)分析BRCA1以及BARD1。其具体实验过程为:
1)在培养板中将已爬好细胞的玻片用PBS浸洗3次,每次3min;
2)用4%的多聚甲醛固定爬片15min,PBS浸洗玻片3次,每次3min;
3)0.5%TritonX-100(PBS配制)室温通透20min;
4)PBS浸洗玻片3次,每次3min,吸水纸吸干PBS,在玻片上滴加正常山羊血清,室温封闭30min;
5)吸水纸吸掉封闭液,不洗,每张玻片滴加足够量的稀释好的一抗并放入湿盒,4℃孵育过夜;
6)加荧光二抗:PBST浸洗爬片3次,每次3min,吸水纸吸干爬片上多余液体后滴加稀释好的荧光二抗,湿盒中37℃孵育1h,PBST浸洗切片3次,每次3min;
7)复染核:滴加DAPI避光孵育5min,对标本进行染核,PBST 5min 4次洗去多余的DAPI;
8)用吸水纸吸干爬片上的液体,用含抗荧光淬灭剂的封片液封片,然后在荧光显微镜下观察采集图像。
结果见图4。γ-H2X为DNA损伤修复过程中的关键标志物。结果证明药物处理后有效抑制了γ-H2X蛋白量,证明了多肽药物抑制了BRCA1蛋白介导的DNA损失修复过程。图4B为加入药物处理后使用激光共聚焦检测BRCA1蛋白及BARD1蛋白共定位结果,证明了在加入药物处理后细胞内BRCA1蛋白及BARD1蛋白结合被有效抑制。
实施例4
动物水平上验证BRCA1/BARD1 Inhibitor药物的作用
1.为了评价BRCA1/BARD1 Inhibitor药物在体内的治疗效果,建立了皮下移植4T-1乳腺癌小鼠模型。将20只小鼠随机分为4组,每3天给予PBS、5mg/ml BRCA1/BARD1Inhibitor药物、2.5mg/ml BRCA1/BARD1 Inhibitor药物、2.5mg/ml BRCA1/BARD1Inhibitor药物与卡铂联用。持续观察,并记录肿瘤体积,当肿瘤体积增长至50~100mm3时进行药物注射。肿瘤体积计算按公式IV完成。所有药物均为隔天注射,注射方式为腹腔注射,注射体积为100μl,注射同时记录肿瘤大小变化情况。药物处理14天后,解刨小鼠,将肿瘤部位以及其它主要器官取出并进行HE染色等处理和TUNEL实验。
肿瘤体积(V)=长×宽2/2公式III。
2.HE(HEMATOXYLIN-EOSIN STAINING)染色方法
1)组织固定
将取下的新鲜组织,放入预先配好的10%福尔马林的固定液中,使组织、细胞的蛋白质变性凝固,以防止细胞死后的自溶或细菌的分解,从而保持细胞本来的形态结构。
2)组织脱水
将固定之后的组织修剪为25mm×25mm×5mm,纯水冲洗去掉组织中的固定液。之后将组织按照低浓度至高浓度,逐渐将组织内水份替换为酒精。再将组织块置于既溶于酒精,又溶于石蜡的透明剂二甲苯中,以二甲苯替换出组织块的中酒精,才能浸蜡包埋。
3)组织包埋
将已透明的组织块置于已溶化的石蜡中,放入溶蜡箱保温。待石蜡完全浸入组织块后,静止冷却凝固成块即成。
4)组织切片
将包埋好的蜡块固定于切片机上,切成薄片,一般为5~8μm厚。
5)切片染色
组织染色前,需用二甲苯重新脱去切片中的石蜡,之后经由高浓度到低浓度酒精,最后浸入纯水中脱去酒精。之后开始染色,将切片置于苏木精水溶液中染色10min。再将切片酸水及氨水中分色,各数秒钟即可。纯水冲洗之后,放入70%和90%酒精中脱水各10min后,将切片入酒精伊红染色液染色2min。
6)切片再脱水
按照上述组织脱水的方式将染色后的切片再重新脱水。
7)封片
将已透明的切片滴上树胶,盖上盖玻片封片。之后显微镜观察并拍照。
3.TUNEL(TDT-MEDIATED DUTPNICK END LABELING)实验方法
1)组织固定及切片
同上述HE染色固定及切片方法。
2)蛋白酶K处理
在脱水处理完成的切片上,滴加溶解于pH 7.4的Tris-HCl缓冲液中20μg/ml不含DNase的蛋白酶K,37℃孵育20min,之后用PBS重复洗涤三次。
3)标记反应
在酶处理后的切片上滴加50μl的TUNEL反应混合液,在湿盒中37℃孵育60分钟。之后使用PBS洗5min×3次。
4)信号转导
擦干切片周围的水分,加入50μl的转化剂即POD溶液,湿盒中37℃孵育20min。
5)DAB显色
加入约100μl的DAB底物溶液,室温25℃孵育10min,PBS洗5min×3次。
6)封片。
将染色完全的切片滴上树胶,盖上盖玻片封片。之后显微镜观察并拍照。
结果如图5A~图5F所示,BRCA1/BARD1 Inhibiror药物可有效抑制裸鼠模型中4T-1移植瘤的生长,并且可有效增强化疗药物卡铂的效果,起到化疗增敏的效果。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 西安交通大学医学院第一附属医院
<120> 一种抑制BRCA1/BARD1复合体结合的多肽及其应用和防治癌症的药物
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Cys Ser Leu Gln Glu Leu Trp Glu Lys Leu Lys Lys Leu
1 5 10
Claims (9)
1.一种抑制BRCA1/BARD1复合体结合的多肽,其特征在于,所述多肽的氨基酸序列如SEQ ID NO:1所示。
2.根据权利要求1所述多肽,其特征在于,所述多肽与BRCA1蛋白的结合常数为11.9nM。
3.权利要求1或2所述抑制BRCA1/BARD1复合体结合的多肽在制备预防和/或治疗癌症的药物中的应用。
4.根据权利要求3所述应用,其特征在于,所述癌症为BRCA1和BARD1阳性的乳腺癌。
5.权利要求1或2所述抑制BRCA1/BARD1复合体结合的多肽在制备抑制BRCA1蛋白和BARD1蛋白结合的试剂中的应用。
6.一种用于防治癌症的药物,其特征在于,包括权利要求1或2所述多肽和递送系统。
7.根据权利要求6所述药物,其特征在于,所述递送系统包括以下一种:脂质体、纳米金和纳米硒。
8.根据权利要求7所述药物,其特征在于,所述药物为纳米硒-多肽。
9.根据权利要求6~8任意一项所述药物,其特征在于,所述药物还包括靶向药物PARP酶抑制剂和/或化疗药物。
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