CN114685641B - 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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- CN114685641B CN114685641B CN202210175810.5A CN202210175810A CN114685641B CN 114685641 B CN114685641 B CN 114685641B CN 202210175810 A CN202210175810 A CN 202210175810A CN 114685641 B CN114685641 B CN 114685641B
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- brca1
- polypeptide
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- protein
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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、***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***。
优选的,所述递送***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***。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***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***。
本发明对所述递送***的种类不做特殊限制,采用本领域所熟知的递送***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***。在本发明实施例中,以纳米硒为递送***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***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***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***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%***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***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 (7)
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.权利要求1所述抑制BRCA1/BARD1复合体结合的多肽在制备治疗癌症的药物中的应用,所述癌症为BRCA1和BARD1阳性的乳腺癌。
3.权利要求1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。
4.一种用于治疗BRCA1和BARD1阳性的乳腺癌的药物,其特征在于,包括权利要求1所述多肽和递送***。
5.根据权利要求4所述药物,其特征在于,所述递送***包括以下一种:脂质体、纳米金和纳米硒。
6.根据权利要求4所述药物,其特征在于,所述药物为纳米硒-多肽。
7.根据权利要求4~6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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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003052100A1 (en) * | 2001-12-18 | 2003-06-26 | Universite De Geneve | Use of bard1 inhibitors for promoting cell survival |
EP1365034A2 (en) * | 2002-05-21 | 2003-11-26 | Bayer Aktiengesellschaft | Methods and compositions for the prediction, diagnosis, prognosis, prevention and treatment of malignant neoplasia |
CN101585882A (zh) * | 2009-06-25 | 2009-11-25 | 中山大学 | 一种用于治疗乳腺癌的融合蛋白及其应用 |
CN106153944A (zh) * | 2010-08-17 | 2016-11-23 | 日内瓦大学 | 肺癌和结直肠癌中的bard1同工型、其检测方法及其应用 |
CN108602855A (zh) * | 2015-12-22 | 2018-09-28 | 伊玛提克斯生物技术有限公司 | 用于乳腺癌和其他癌症免疫治疗的肽和肽组合物 |
CN113912679A (zh) * | 2021-10-12 | 2022-01-11 | 西安交通大学医学院第一附属医院 | 一种靶向雄激素受体的多肽及其应用和防治***癌的药物 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20120040896A1 (en) * | 2008-04-11 | 2012-02-16 | The Regents Of The University Of Colorado | Compositions, methods and uses for modulation of brca 1 |
-
2022
- 2022-02-25 CN CN202210175810.5A patent/CN114685641B/zh active Active
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003052100A1 (en) * | 2001-12-18 | 2003-06-26 | Universite De Geneve | Use of bard1 inhibitors for promoting cell survival |
EP1365034A2 (en) * | 2002-05-21 | 2003-11-26 | Bayer Aktiengesellschaft | Methods and compositions for the prediction, diagnosis, prognosis, prevention and treatment of malignant neoplasia |
CN101585882A (zh) * | 2009-06-25 | 2009-11-25 | 中山大学 | 一种用于治疗乳腺癌的融合蛋白及其应用 |
CN106153944A (zh) * | 2010-08-17 | 2016-11-23 | 日内瓦大学 | 肺癌和结直肠癌中的bard1同工型、其检测方法及其应用 |
CN108602855A (zh) * | 2015-12-22 | 2018-09-28 | 伊玛提克斯生物技术有限公司 | 用于乳腺癌和其他癌症免疫治疗的肽和肽组合物 |
CN113912679A (zh) * | 2021-10-12 | 2022-01-11 | 西安交通大学医学院第一附属医院 | 一种靶向雄激素受体的多肽及其应用和防治***癌的药物 |
Non-Patent Citations (4)
Title |
---|
BRCA1-associated protein 1 interferes with BRCA1/BARD1 ring heterodimer activity;Hiroyuki et al;Cell, Tumor, and Stem Cell Biology;20081231;全文 * |
BRCA1与乳腺癌的研究进展;查国松;左文述;;中国普外基础与临床杂志;20170725(第07期);全文 * |
BRCA1结合蛋白BRAP的研究进展;赵楠楠;平毅;;世界最新医学信息文摘;20180323(第24期);全文 * |
Interaction of BARD1 and HP1 is required for BRCA1 retention at sites of DNA damage;Wenwen Wu et al;Molecular and Cellular Pathobiology;20150331;全文 * |
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