CN106589062A - 靶向Grb2蛋白的抗肿瘤共价多肽抑制剂及其制备方法和应用 - Google Patents
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Abstract
本发明涉及靶向Grb2蛋白的抗肿瘤共价多肽抑制剂及其制备方法和应用。所述的共价多肽抑制剂的氨基酸序列为：VPPPVPPRRX，其中X为(2S)‑2‑氨基‑3‑[(2‑氯乙酰)氨基]丙酸。所述的制备方法为Fmoc固相多肽合成法，X的原料为Fmoc‑Dap(Mtt)‑OH和氯乙酸。本发明的共价多肽抑制剂对Grb2蛋白有极高的亲和性，故其会避免“脱靶”共价反应，因此治疗效果好，克服了现有技术中共价多肽药物存在毒副作用的难题。本发明为共价抑制剂药物研发提供了一条新思路，将有助于乳腺癌等疾病的治疗。
Description
技术领域
本发明涉及分子生物学和医药技术领域，具体地说，涉及一种靶向Grb2蛋白的抗肿瘤共价多肽抑制剂及其制备方法和应用。
背景技术
共价抑制剂是现有药物抑制剂中唯一可完全沉默或永久关闭致病蛋白的一类药物，其作用机理是共价抑制剂能与目标蛋白中特定某一个或几个氨基酸残基发生共价反应，从而抑制目标蛋白的相关活性，以此达到治疗疾病的目的。相较于常规非共价药物，共价药物用量少且效果持久。近年来美国FDA和我国CFDA已通过一些此类药物的审批并应用于临床。由于共价抑制剂的独特治疗优势，它也被用于肿瘤治疗研究中，并已得到了一些显著的效果。如Neubig和其同事利用一珠一化合物(one-bead one-compound，OBOC)库筛选方法得到了一个可与调节G蛋白信号蛋白4(RGS4)共价结合的多肽，通过共价抑制RGS4达到了调控肿瘤相关蛋白G蛋白的目的，进而抑制G蛋白偶联受体相关信号。Taunton和其同事开发了一种可与乳腺癌细胞MDA-MB-231中核糖体蛋白激酶RSK2共价结合的共价抑制剂，并取得了良好的抑瘤效果。虽然共价药物在抑制蛋白生物活性方面显示了强大作用，但一些共价抑制剂会与细胞中非特异的蛋白发生反应，这种“脱靶”共价反应会给机体带来很强的毒副作用，严重阻碍了共价抑制剂的发展。
生长因子受体结合蛋白2(Growth factor receptor bound protein 2，Grb2)是一类衔接蛋白，其由一个中间的SH2(Src homology 2)结构域和位于两端的SH3(Srchomology 3)结构域组成。研究发现Grb2在乳腺癌中呈高表达状态，其表达水平与***转移及预后相关。在乳腺癌细胞中Grb2与蛋白Sos(son of sevenless)相互作用并通过调控受体酪氨酸激酶(receptor tyrosine kinase，RTK)信号通路来促进肿瘤细胞的侵袭和转移。
靶向Grb2蛋白的共价抑制剂类药物在治疗乳腺癌中同样存在“脱靶”问题，造成药物毒副作用较大，且治疗效果欠佳。由此可见，有必要设计一种与Grb2特异发生共价反应、不易脱靶的药物。
发明内容
本发明的目的是针对现有技术中的不足，提供一种靶向Grb2蛋白的抗肿瘤共价多肽抑制剂。
本发明的再一的目的是，提供所述的共价多肽抑制剂的制备方法。
本发明的另一的目的是，提供所述的共价多肽抑制剂的用途。
为实现上述第一个目的，本发明采取的技术方案是：
一种靶向Grb2蛋白的抗肿瘤的共价多肽抑制剂，所述的共价多肽抑制剂的氨基酸序列为：VPPPVPPRRX，其中X为(2S)-2-氨基-3-[(2-氯乙酰)氨基]丙酸。
为实现上述第二个目的，本发明采取的技术方案是：
所述的共价多肽抑制剂的制备方法，所述的制备方法为Fmoc固相多肽合成法。
X的原料为Fmoc-Dap(Mtt)-OH和氯乙酸。
为实现上述第三个目的，本发明采取的技术方案是：
所述的共价多肽抑制剂在制备治疗Grb2蛋白介导的疾病的药物中的应用。
所述的Grb2蛋白介导的疾病为肿瘤。
所述的肿瘤为乳腺癌。
本发明优点在于：
本申请的发明人在研究过程中发现在Grb2的N端SH3结构域(Grb2N-SH3，PDB:1GBQ)中有一个半胱氨酸残基(Cys32)并且其具有反应活性的巯基基团暴露于外侧，非常靠近它的相互作用蛋白Sos。Sos来源的多肽片段VPPPVPPRRR与Grb2有较高的亲和性，并且此片段C端的Arg(底部标有横线)与Grb2N-SH3的Cys32非常接近，于是用一非天然氨基酸X(带有亲核基，其中X为(2S)-2-氨基-3-[(2-氯乙酰)氨基]丙酸)取代Arg，X会与Grb2蛋白的N端SH3结构域中的Cys32发生亲核取代反应，从而可以将此多肽抑制剂共价结合到Grb2蛋白上，以此可以达到调控Grb2下游的信号通路，进而抑制肿瘤细胞生长的目的。这一新的多肽VPPPVPPRRX(命名为RP)作为Grb2的共价抑制剂，是迄今唯一一种针对乳腺癌细胞Grb2蛋白的共价多肽抑制剂。因RP与Grb2有极高的亲和性，故其会避免“脱靶”共价反应，在达到抑制乳腺癌细胞生长的同时也不会产生其他毒副作用。本发明为共价抑制剂药物研发提供了一条新思路，将有助于乳腺癌的治疗。
附图说明
图1.共价多肽抑制剂RP的化学结构式。
图2.RP序列VPPPVPPRRX的质谱鉴定结果。
图3.RP与Grb2N-SH3蛋白共价结合的质谱结果。
图4.RP对乳腺癌细胞生长活性的影响检测结果。
图5.RP对乳腺癌细胞迁移的影响检测结果。
图6.RP对乳腺癌细胞凋亡的影响检测结果。
具体实施方式
下面结合附图对本发明提供的具体实施方式作详细说明。
实施例1
一、共价多肽抑制剂RP的合成及表征
1方法
1.1共价多肽抑制剂RP的合成
1)采用Fmoc化学固相多肽合成，使用Rink Amide-ChemMatrix树脂，用5倍过量的Fmoc保护的氨基酸和缩合试剂(HBTU/HOBt)合成多肽，其中X的前体用Fmoc-Dap(Mtt)-OH。
2)多肽N端第一个氨基酸Fmoc脱保护后用乙酸酐反应保护。
3)将X前体的Mtt用1％TFA选择性脱保护，加入氯乙酸，将侧链修饰成酰氯。
4)用95％TFA将多肽从树脂上切除，用冰***沉淀，得到的初步多肽用HPLC分离纯化。
1.2共价多肽抑制剂RP的表征
经HPLC分离纯化的多肽用MALDI-TOF MS来检测证实。
2结果
共价多肽抑制剂RP的序列为VPPPVPPRRX，其中X为(2S)-2-氨基-3-[(2-氯乙酰)氨基]丙酸。共价多肽抑制剂RP的化学结构式见图1。RP序列VPPPVPPRRX的质谱鉴定结果见图2。
二、共价多肽抑制剂RP与蛋白Grb2N-SH3体外共价结合检测
1方法
1.1构建pGEX-6P-3-Grb2N-SH3原核表达质粒
用Trizol提取乳腺癌细胞SK-BR-3的总RNA，之后用PrimeScript RT reagent Kitwith gDNA Eraser(Takara)试剂盒反转录出第一条链cDNA，用PCR合成出Grb2N-SH3片段(SEQID NO.1)并用琼脂糖凝胶电泳验证，之后测序确定。经测序确定后，片段用T4连接酶(Takara)连接原核表达质粒pGEX-6P-3(NdeI和XhoI之间)然后酶切验证。
1.2 E.Coli原核表达并纯化蛋白Grb2N-SH3
经验证正确的pGEX-6P-3-Grb2N-SH3质粒转化于BL-21感受态细胞中，挑克隆验证正确后，加在灭菌的5mL LB培养基中37℃摇过夜。之后取4mL加于400mL LB中37℃摇到OD600约04-0.6，加IPTG诱导，然后16℃摇18小时。之后离心机去掉菌液上清，菌体用超声破碎。利用GST标签纯化蛋白，并用PreScission酶(GE Healthcare Life Sciences)切除GST标签，得到蛋白Grb2N-SH3并用SDS-PAGE验证。
1.3检测RP与Grb2N-SH3蛋白共价结合
RP与Grb2N-SH3蛋白按5:1的量混合反应30分钟。取反应的混合物加热变性脱盐后，用MALDI-TOF MS精确验证RP与Grb2N-SH3蛋白是否共价结合。
2结果
RP与Grb2N-SH3蛋白共价结合的质谱鉴定结果见图3，结果表明RP与Grb2N-SH3蛋白发生了共价结合。
三、乳腺癌细胞系中检测RP抗肿瘤效果
1方法
1.1RP对乳腺癌细胞生长活性的影响检测
乳腺癌细胞SK-BR-3铺于96孔板，每孔3000个细胞。加带有穿膜肽(YGRKKRRQRRR，SEQ ID NO.2)的RP和带有穿膜肽非共价对照多肽(VPPPVPPRRR，SEQ ID NO.3)，浓度为40μM。之后培养24小时、48小时和72小时三个时间点，吸出培养液，加MTT溶液，细胞培养箱培养2小时后用酶标仪检测。
1.2 RP对乳腺癌细胞迁移的影响检测
乳腺癌细胞SK-BR-3铺于24孔板，每孔1×105个细胞。加都带有上述穿膜肽的RP和非共价对照多肽，浓度为40μM，8小时后。细胞用胰酶消化下，并加于Transwell上孔，上孔用无血清培养液，下孔用10％血清培养液，培养18小时后，吸出培养液。用PBS洗两遍后，用结晶紫染色细胞30分钟，之后用PBS洗净，在显微镜下计数被染色细胞个数。
1.3 RP对乳腺癌细胞凋亡的影响检测
乳腺癌细胞SK-BR-3铺于24孔板，每孔1×105个细胞。加都带有上述穿膜肽的RP和非共价对照多肽，浓度为40μM，之后培养24小时、48小时和72小时三个时间点，吸出培养液，用胰酶消化下细胞后，用Annexin V/FITC凋亡试剂盒(Sigma-Aldrich)染色，之后用流式细胞仪检测。
2结果
2.1 RP对乳腺癌细胞生长活性的影响
见图4，结果表明RP可显著抑制乳腺癌细胞的生长活性。
2.2 RP对乳腺癌细胞迁移的影响
见图5，结果表明RP可显著抑制乳腺癌细胞的迁移。
2.3 RP对乳腺癌细胞凋亡的影响
见图6，结果表明RP可显著促进乳腺癌细胞的凋亡。
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，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
SEQUENCE LISTING
<110> 上海市第一妇婴保健院
<120> 靶向Grb2蛋白的抗肿瘤共价多肽抑制剂及其制备方法和应用
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Claims (6)
1.一种靶向Grb2蛋白的抗肿瘤的共价多肽抑制剂，其特征在于，所述的共价多肽抑制剂的氨基酸序列为：VPPPVPPRRX，其中X为(2S)-2-氨基-3-[(2-氯乙酰)氨基]丙酸。
2.权利要求1所述的共价多肽抑制剂的制备方法，其特征在于，所述的制备方法为Fmoc固相多肽合成法。
3.根据权利要求2所述的制备方法，其特征在于，X的原料为Fmoc-Dap(Mtt)-OH和氯乙酸。
4.权利要求1所述的共价多肽抑制剂在制备治疗Grb2蛋白介导的疾病的药物中的应用。
5.根据权利要求4所述的应用，其特征在于，所述的Grb2蛋白介导的疾病为肿瘤。
6.根据权利要求5所述的应用，其特征在于，所述的肿瘤为乳腺癌。
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| CN110669146A (zh) * | 2019-10-24 | 2020-01-10 | 常州大学 | 一种可特异性阻断衔接蛋白Nck的结合位点用于预防肠道EPEC感染的活性肽 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108640974A (zh) * | 2018-05-19 | 2018-10-12 | 上海市第妇婴保健院 | 一种靶向作用Syntenin蛋白PDZ结构域的多肽及其二聚体 |
| CN108640974B (zh) * | 2018-05-19 | 2021-07-06 | 上海市第一妇婴保健院 | 一种靶向作用Syntenin蛋白PDZ结构域的多肽及其二聚体 |
| CN110669146A (zh) * | 2019-10-24 | 2020-01-10 | 常州大学 | 一种可特异性阻断衔接蛋白Nck的结合位点用于预防肠道EPEC感染的活性肽 |
| CN110669146B (zh) * | 2019-10-24 | 2023-05-23 | 常州大学 | 一种可特异性阻断衔接蛋白Nck的结合位点用于预防肠道EPEC感染的活性肽 |
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