CN114409808B - 基于核酸适配体的靶向嵌合体及其对tau蛋白的降解 - Google Patents
基于核酸适配体的靶向嵌合体及其对tau蛋白的降解 Download PDFInfo
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Abstract
本发明公开了一种基于核酸适配体的靶向嵌合体。该嵌合体分子包括核酸适配体,连接单元,E3连接酶配体。该嵌合体与tau蛋白之间具有高的亲和力,可特异性地靶向tau蛋白,且具有良好的血清稳定性。该嵌合体通过招募泛素使得tau蛋白发生多聚泛素化并进入泛素‑蛋白酶体系统,从而实现人神经母细胞瘤等细胞中对tau蛋白的高效降解,为治疗tau蛋白相关的神经性疾病提供了新的治疗方案。相比于小分子及多肽的靶向tau蛋白水解嵌合体,本发明中的嵌合体降解效率显著提高。另外,考虑到核酸适配体筛选技术日益成熟,本发明中的嵌合体不限于靶向tau蛋白的降解,有望降解其它核酸适配体可靶向的致病蛋白,是一种适用性广泛的治疗手段。
Description
技术领域
本发明属于生物医药领域,涉及一系列基于核酸适配体的靶向嵌合体,该嵌合体可用于降解重要的致病蛋白。
背景技术
蛋白质的异常表达与许多疾病的产生和发展密切相关,多年来抑制剂策略是调控蛋白活性的主要方式。然而,由于抑制剂分子采用占据目标蛋白活性位点的作用方式,其通常需要高的给药浓度维持活性,并且仍有许多蛋白(如转录因子、支架蛋白)难以靶向。近年来,蛋白水解靶向嵌合体(PROTACs)的发展为我们提供了新的治疗及研究方案。PROTAC是一种高效的蛋白降解嵌合体分子,它由靶蛋白配体、linker及E3连接酶配体三部分组成,其作为一种“桥梁”使得靶蛋白与E3酶相互靠近,形成靶蛋白-PROTAC-E3连接酶的三元复合物,使得靶蛋白被泛素化标记并进入细胞内的泛素-蛋白酶体系统,实现靶蛋白的降解。PROTAC具有高选择性、高降解效率及催化特性,已经被用于降解多种致病蛋白,如雄性激素受体、雌性激素受体及各种激酶蛋白。此外已有一系列的PROTAC药物进入临床实验阶段。
核酸适配体是一段通过人工筛选获得的短链核酸分子,它可以通过特定的三维结构与靶标分子相结合。核酸适配体又被称为“化学家的抗体”,与抗体相比,核酸适配体具有易合成与修饰、免疫原性低、组织渗透性高等优点,已被广泛的应用于疾病诊断、治疗等生物医药领域。此外,与化学小分子相比,通常情况下核酸适配体对靶蛋白的亲和力更强,筛选手段更加丰富。因此我们构建基于核酸适配体的靶向嵌合体,其由核酸适配体、linker和E3连接酶配体三部分组成,通过核酸适配体和E3连接酶配体分别对靶蛋白和E3连接酶的招募,形成靶蛋白-嵌合体分子-E3连接酶的三元复合物,由于靶蛋白与E3连接酶距离上的靠近,并在E1、E2酶的共同作用下,使得靶蛋白被泛素化标记,最终进入蛋白酶体被降解。Tau蛋白作为我们的降解靶标之一,它是众多神经相关性疾病中的重要致病蛋白,如阿尔兹海默症、帕金森症、皮克氏症等等。虽然已有许多针对tau相关疾病的方案,但特异性降解tau蛋白被认为是一种极具吸引力的策略。目前仅有少量文献报道小分子PROTAC及多肽PROTAC分子用于降解tau蛋白,且降解浓度范围在μM以上。然而,本发明所提出的基于核酸适配体的嵌合体降解tau浓度范围在nM级别,显著提升了降解效率。此外,在本发明所提出的策略适用性广泛,基于成熟的适配体筛选技术,有望通过替换核酸适配体去靶向降解其他致病蛋白。
发明内容
本发明要解决的技术问题在于设计与合成基于核酸适配体的靶向嵌合体,并有效降解相关致病蛋白。其特征在于:
首先,提出一种基于核酸适配体的靶向嵌合体,其结构通式如下式(Ⅰ):A-L-Y
A为靶向可筛选的蛋白的核酸适配体,所述蛋白是胞内蛋白;
Y为靶向E3连接酶的配体,为来那度胺或VHL配体;
L为核酸适配体与E3连接酶配体之间的连接基团;
n为连接基团L中乙基基团的重复个数;
为了合成所述的基于核酸适配体的靶向嵌合体,本发明采取的技术方案为:
(a):利用DNA合成仪合成一系列末端氨基的核酸适配体。
(b):通过有机反应得到末端羧基修饰的E3连接酶配体。
(c):将氨基修饰的核酸适配体与羧基修饰的E3连接酶配体进行偶联。
(d):通过脱除核酸固相、HPLC等分离纯化手段得到基于核酸适配体的蛋白水解靶向嵌合体。
具体的,本发明所述的基于核酸适配体的靶向嵌合体可特异性靶向tau蛋白,其中靶向tau蛋白的序列如:0301:5’-GCT TGG TCC TCC CGG GGT TCT GGA AAA GC-3’和0222:5’-GCG GAG CGT GGC AGG-3’。为了实现高效降解tau蛋白,本发明还采用了核酸链上不同位置和数量的2’-OMe或P-S修饰,所构建的一系列靶向嵌合体不会显著改变适配体本身的结构,与tau蛋白之间具有高的亲和力,此外还具有良好的血清稳定性。
进一步的,本发明将一系列的基于核酸适配体的靶向嵌合体通过脂质体包载给药至表达tau蛋白的细胞,其中包括人神经母细胞瘤细胞和人肾上皮细胞,以及其他外源tau蛋白表达的细胞。通过western blot检测发现嵌合体可以有效降解细胞中的tau蛋白。
本发明的有益效果是:
1.所涉及的基于核酸适配体的靶向嵌合体具有高的蛋白降解效率,其中30nM的嵌合体分子可以有效降解tau蛋白,该降解效率是现有PROTAC分子的几十倍左右;
2.基于核酸适配体筛选手段不断提升,有望将这种技术应用至其他蛋白的靶向水解;
3.为治疗相关疾病提供可靠的新方法;
附图说明
下面结合附图和具体实施方式对本发明作进一步详细的说明。
图1是一系列嵌合体分子结构图。
图2是0222的质谱结果图。
图3是0228-1-f的质谱结果图。
图4是0312-f的质谱结果图。
图5是0320S的质谱结果图。
图6是0301的质谱结果图。
图7是0228-2-f的质谱结果图。
图8是1105-1的质谱结果图。
图9是1105-2的质谱结果图。
图10是1106-1的质谱结果图。
图11是1106-2的质谱结果图。
图12是0311-f的质谱结果图。
图13是0320SS-f的质谱结果图。
图14是嵌合体分子0228-2-f与适配体0301的圆二色光谱对比图。
图15是嵌合体分子0228-2-f与适配体0301的紫外吸收随温度变化的曲线对比图。
图16是嵌合体分子1106-2与tau蛋白之间的结合/解离曲线图。
图17是嵌合体分子0311-f在血清中孵育时间变化的聚丙烯酰胺凝胶电泳图。
图18是嵌合体分子0228-2-f在人神经母细胞瘤细胞中tau降解的Western blot电泳图。
具体实施方式
实施例中所用主要试剂:
实施例1:本发明中嵌合体分子的合成
(1)首先用乙腈分别溶解dA、dT、dC、dG,2’-OMe修饰的dA、dT、dC、dG的亚磷酰胺单体,以及C6氨基的亚磷酰胺单体,所有亚磷酰胺单体的浓度均为0.1g/mL。然后将所要用的单体、碘液、盖帽试剂、TCA Deblok试剂、乙腈装至DNA合成仪。然后开启合成仪,将核酸合成柱装至合成仪上,最后通过仪器合成末端氨基修饰的核酸适配体;(2)取3.4mg的末端羧基修饰的E3配体溶于无水DMF中,并加入2.2mg HATU,2.5μL的DIPEA,活化5min,然后加入20mg负载于固相载体的核酸适配体,室温反应24h;(3)反应完成后,离心弃上清液,并分别用无水DMF和乙腈洗涤固相载体3次。50℃干燥固相载体,然后加入浓氨水0.8mL,并于50℃孵育8h;(4)孵育完毕后,离心,取上清液转移至新的EP管中,50℃浓缩得到嵌合体粗产物;(5)取粗产物进行HPLC分离纯化,洗脱剂为TEAA和乙腈;(6)将HPLC纯化完成后的样品50℃浓缩仪浓缩得到固体;(7)用超纯水中溶解上步骤所得的固体,然后用微量核酸定量仪进行定量;(8)进行嵌合体分子的质谱测试,附图1所示为一系列嵌合体分子的结构。附图2至附图13所示一系列分子合成的质谱结果。
实施例2:本发明中两个实施例0228-2-f和0301的结构和热稳定性能测试配制浓度为1.33μM的0228-2-f及0301溶液450μL,以含5mM镁离子的PBS溶液作为空白对照,然后进行圆二色光谱测试,结果如附图14所示,表示VHL配体的缀合并没有显著改变适配体本身的结构。此外,将0228-2-f及0301溶于5mM镁离子的PBS溶液,使其紫外吸收值在0.2-0.8之间,利用紫外可见光谱的附件加热设备给样品加热,同时检测嵌合体分子0228-2-f及0301在260nm处紫外吸收值随温度上升的变化,测试完毕后绘制吸收变化曲线图,结果如附图15所示,表示二者的热稳定性能相差较小。
实施例3:本发明中一个实施例1106-2的亲和力性能测试
首先配制浓度梯度为0.25μM、0.5μM、1μM、2μM、4μM的1106-2溶液,然后将链霉素传感器浸泡于PBS缓冲液(含5mM镁离子)中。然后按摩尔比1:4混合生物素与tau蛋白,室温下反应1h,之后通过除盐柱除去未反应的生物素,得到生物素标记的tau蛋白溶液。分别在样品板中加入每个浓度的样品溶液200μL,以及蛋白溶液和PBS缓冲溶液(含5mM镁离子)200μL,将tau蛋白通过生物素-链霉素之间的作用力固定于传感器上,利用生物膜干涉仪测试一系列浓度的1106-2嵌合体分子与tau蛋白之间的亲和力。结果如附图16所示,1106-2与tau二者之间结合力为79.9nM。
实施例4:本发明中一个实施例0311-f的血清稳定性测试
取98ng的0311-f溶于含10%血清的PBS中,将其孵育于37℃,并于不同时间点取出5μL样品,并加入5uL上样液,先用液氮将其快速冷冻,之后冻存于-80℃。然后进行20%变性聚丙烯酰胺凝胶电泳,取50mL EP管,加入8.4g尿素,2mL 10×TBE,10mL 40%的原胶,补水至20mL。然后取上步骤溶液12mL,加入10%APS溶液100μL,加入TEMED10μL,然后将该溶液加入至提前装好的两片玻璃板中,并插入梳子,等待约20min。取下已经制好的胶,以1×TBE为电泳液,将之前冻存的样品溶解,然后上样10μL。电压为150V,电泳约1h,然后用sybr金核酸凝胶染料染色,最后置于Bio-RAD仪器成像,结果如附图17所示,0311-f具有良好的血清稳定性。
实施例5:本发明中一个实施例0228-2-f的tau蛋白降解性能测试
配制浓度梯度为30nM、100nM、300nM的0228-2-f溶液,分别通过脂质体包载给药至人神经母细胞瘤细胞,孵育24h,提取蛋白通过Western blot检测靶蛋白水平的变化。具体实验步骤如下:
1.弃去细胞培养基,十二孔板每孔用1ml预冷的PBS洗两遍,并按照每孔80μl的体积加入RIPA裂解液(含终浓度为1mM的Cocktail蛋白酶抑制剂);
2.收集细胞,转移细胞悬液至1.5ml离心管,冰上裂解20min,每隔10min涡旋振荡30s使其充分裂解,4℃、15000rpm离心15min,弃去沉淀,转移上清液至新的1.5ml离心管。
3.BCA蛋白浓度测,取浓度为2mg/ml的BCA标准品溶液倍比稀释,得到2,1.5,1,0.75,0.5,0.375,0.25,0.125mg/ml梯度浓度的标准溶液;配制BCA工作液:将BCA Reagent与Cu Reagent按50:1的比例混合均匀。吸取4μl待测蛋白液,用ddH2O稀释5倍;将配制好的BCA工作液按照每孔200μl的量加入专用的酶标板,37℃孵育30min。用酶标仪测上述混合物在562nm处的吸光度值,根据标准曲线计算每个样品的蛋白浓度。
4.剩余上清液加入5×的SDS loading buffer混合均匀成1×,于100℃金属浴中变性10min,即可进行后续实验,多余蛋白可置于-20℃保存。
5.Western blot检测Tau蛋白水平
①蛋白变性后按照每组蛋白样品40μg的上样量,进行SDS-聚丙烯酰胺凝胶电泳,浓缩胶浓度为4%,分离胶浓度为10%;80V恒压待蛋白样品跑至浓缩胶和分离胶界面时将电压调至120V,待loading buffer跑至凝胶底部即终止电泳。
②转膜,封闭及一抗孵育:电泳结束后,取下凝胶并按照Bio-Rad湿转装置组装“三明治”结构,转膜采用0.45μm的PVDF膜,并且转膜前已用甲醇激活,转膜条件为100mA恒流,90min,转膜结束后可在膜上看见预染蛋白Marker,将膜取出用TBST漂洗3次,每次10min,置于5%的脱脂牛奶(TBST配制)中室温封闭1h。弃去封闭液,TBST漂洗3次,每次10min,将PVDF膜置于一抗稀释液中4℃孵育过夜,抗体与一抗稀释液按相应比例稀释。
③二抗孵育,显影:一抗孵育结束后,取出PVDF膜,用TBST漂洗三次,每次10min。HRP标记的二抗按照1:5000的比例用TBST配制的5%的脱脂牛奶稀释,将PVDF膜置于二抗稀释液中室温孵育1h。结束后,PVDF膜用TBST漂洗三次,每次10min。按照A液和B液1:1的比例配制ECL显影液,将显影液均匀覆盖在PVDF膜表面,即可置于Tanon化学发光成像系统显影检测。结果如附图18所示,浓度为30nM的0228-2-f能够有效降解tau蛋白。
上面结合附图对本发明优选的具体实施方式和实施例作了详细的说明,但是本发明并不限于上述实施方式和实施例,在本领域技术人员所具备的知识范围内,还可以在不脱离本发明构思的前提下作出各种变化。
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Claims (5)
2.如权利要求1所述的一种基于核酸适配体的靶向嵌合体,其特征在于:所述基于核酸适配体的靶向嵌合体特异性结合tau蛋白。
3.如权利要求1所述的一种基于核酸适配体的靶向嵌合体的制备方法,其特征在于包括以下步骤,(a):利用DNA合成仪合成末端氨基的核酸适配体;(b):通过有机反应得到末端羧基的E3连接酶配体;(c):将末端氨基的核酸适配体与末端羧基的E3连接酶配体进行偶联;(d):脱除核酸固相、HPLC分离纯化得到基于核酸适配体的靶向嵌合体。
4.非诊断和治疗目的的测试tau蛋白降解的方法,所述方法包括:通过脂质体将权利要求1或2所述的基于核酸适配体的靶向嵌合体给药至表达tau蛋白的细胞,24h后通过western blot检测tau蛋白含量变化。
5.如权利要求4所述的测试tau蛋白降解的方法,其特征在于,所述表达tau蛋白的细胞选自人神经母细胞瘤细胞和人肾上皮细胞。
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