CN114478707B - 一种构象锁定蜂毒肽衍生物、偶联物及其制备和用途 - Google Patents
一种构象锁定蜂毒肽衍生物、偶联物及其制备和用途 Download PDFInfo
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Abstract
本发明提供了一种构象锁定蜂毒肽Mastoparan衍生物或其药学上可接受的盐,具有以下所示的结构式或其结构类似物:X来源于蜂毒肽MP中的一个氨基酸,所述的蜂毒肽MP序列为INLKALAALAKKIL,三个X为所述的蜂毒肽MP中连续的序列。本发明还提供了一种具有PD‑L1靶向性和肿瘤微环境响应型的构象锁定蜂毒肽MP衍生物‑PEG偶联物的制备及其溶瘤免疫治疗应用方法,本发明的“多功能一体化”多肽‑PEG偶联物不仅能直接裂解结直肠癌细胞CT26,而且与免疫检查点PD‑L1的靶向肽协同,激活机体免疫系统发挥抗肿瘤免疫作用。
Description
技术领域
本发明属于药物化学领域,涉及一种蜂毒肽Mastoparan,具体来说是一种构象锁定蜂毒肽Mastoparan衍生物及其PEG偶联物的制备与溶瘤免疫治疗用途。
背景技术
结直肠癌是全球发病率和死亡率均位居第三的恶性肿瘤。研究表明在新诊断的结直肠癌病例中,20%的患者在就诊时即发现转移,另有25%患者会在随后的治疗中发生转移,而被确诊为转移性结直肠癌的患者五年生存率不足20%。目前,手术切除是结直肠癌的首选方法,放疗、化疗、分子靶向治疗也是结直肠癌的主要治疗手段,但是存在毒副作用和耐药现象。临床研究表明,免疫治疗,主要是免疫检查点抑制剂,如pembrolizumab,在微卫星高度不稳定/错配修复功能缺失的转移性结直肠癌患者中预后良好,但该类患者仅占5%,绝大多数患者为微卫星稳定/错配修复功能正常的结直肠癌,对免疫治疗无响应。因此扩大免疫治疗在结直肠癌患者中的应用具有重要意义。
溶瘤免疫治疗是一种新兴免疫治疗方式,可以通过高效杀伤肿瘤细胞,释放肿瘤相关抗原,从而有效激活机体抗肿瘤免疫反应。溶瘤肽是一种新型的溶瘤药物,具有结构简单、合成简便、潜在风险低的优势。溶瘤肽具有的阳离子和两亲性特性可与肿瘤细胞膜亲电吸附,诱导肿瘤细胞快速裂解,对耐药肿瘤细胞仍然有效,目前已有多个溶瘤肽进入临床试验阶段。
Mastoparan(MP,INLKALAALAKKIL)是从黄蜂Vespula lewisii的毒液中提取分离得到的含有十四个氨基酸残基的阳离子两亲性α-螺旋肽。近年来研究表明,MP具有多种生物活性,如诱导肥大细胞脱颗粒并释放组胺引发炎症,诱导胰岛β细胞使其分泌胰岛素,以及抗耐药菌和抗肿瘤活性。MP的两亲α-螺旋结构和阳离子性对其生物活性至关重要,但是天然来源的多肽存在二级结构不稳定,易被酶解等问题。全碳氢订书肽策略已被证实可以稳定多肽药物的α-螺旋二级结构,增加其代谢稳定性、细胞膜通透性和增强生物活性,有望增强MP的抗肿瘤活性。
由于肿瘤突变负荷较低和肿瘤新生抗原缺乏,大部分结直肠癌患者对免疫治疗并不敏感。溶瘤肽可诱导肿瘤细胞释放大量肿瘤相关抗原,有望成为新型的溶瘤药物。然而,瘤内注射的方式无法实现深部及转移部位肿瘤的给药。本发明聚焦天然蜂毒肽MP,采用多肽-高分子材料偶联物和多价配体技术,设计合成构象锁定蜂毒肽-PEG偶联物,实现构象锁定蜂毒肽的靶向递送,增强结直肠癌的溶瘤免疫治疗效果。
因此,本发明采用全碳氢订书肽技术,设计合成十条MP的构象锁定衍生物。通过α-螺旋程度、体外抗肿瘤活性以及溶血副作用等评价,选出治疗指数最优的先导化合物MP9。同时,与高分子材料PEG共价连接得到的蜂毒肽-PEG偶联物协同anti-PD-L1靶向肽,实现蜂毒肽体内高效靶向递送并且增强结直肠癌的溶瘤免疫治疗效果。目前还未见类似的报道。
发明内容
基于以上背景,本发明提供了一种构象锁定蜂毒肽Mastoparan衍生物及其PEG偶联物和其制备与溶瘤免疫治疗用途,所述的这种构象锁定蜂毒肽Mastoparan衍生物及其PEG偶联物和其制备与溶瘤免疫治疗用途要解决现有技术的药物对于治疗结直肠癌的效果不佳的技术问题。
本发明提供了一种构象锁定蜂毒肽衍生物或其药学上可接受的盐,所述衍生物具有以下式(Ⅰ)所示的结构式或其结构类似物:
式(Ⅰ)中,X来源于蜂毒肽MP中的一个氨基酸,所述的蜂毒肽MP的序列为INLKALAALAKKIL,三个X为所述的蜂毒肽MP中连续的序列。
进一步的,其分子式如下所示,
其中,n为0~9的任一整数,m为0~9的任一整数,X、Y、Z来源于蜂毒肽MP中的氨基酸,所述的蜂毒肽MP的序列为INLKALAALAKKIL,三个X为所述的蜂毒肽MP中连续的序列n个Y为所述的蜂毒肽MP中连续的序列;m个Z为所述的蜂毒肽MP中连续的序列。
进一步的,其特征在于,n+m=9。
进一步的,所述的一种构象锁定蜂毒肽衍生物或其药学上可接受的盐为下列各结构式中的任意一种:
本发明还提供了上述的一种构象锁定蜂毒肽衍生物或其药学上可接受的盐的制备方法,包括如下步骤:
a)以蜂毒肽MP为基础,所述的蜂毒肽MP的序列为INLKALAALAKKIL(SEQ ID NO.1所示),将MP序列的i,i+4位点的天然氨基酸替换含有烯烃的氨基酸(2R)-2-N-芴甲氧羰基氨基-2-甲基-6-庚烯酸(Fmoc-S5-OH),i为大于等于1的整数,设计系列MP衍生物MP1-MP10;
b)根据多肽固相合成法,以9-芴基甲氧基羰基保护基的天然氨基酸和Fmoc-S5-OH为原料,Rink Amide氨基树脂为载体,2-肟氰乙酸乙酯/N-甲基吡咯烷酮/N,N-二异丙基碳二亚胺为缩合体系,合成线性多肽;
c)线性多肽合成后,直接进行环合反应,采用Grubbs第一代催化剂进行烯烃复分解反应,得到含有碳氢支架侧链的MP衍生多肽,实现α-螺旋结构的锁定;
d)采用乙酸酐/吡啶体系,对环合后的多肽N-端乙酰化;随后将干燥的树脂从多肽合成管中取出转移至离心管中,用三氟乙酸、水和三异丙基硅烷的混合切割液将环合好的MP衍生多肽从树脂上切下,把含多肽切割液的树脂过滤,得到的过滤液用冰甲基叔丁基醚沉淀,并用氮气吹干沉淀得到多肽粗品,最后将MP衍生多肽粗品通过制备液相纯化,冻干后得到构象锁定MP衍生多肽药学上可接受的盐。
具体的,所述的催化剂为Grubbs第一代催化剂。
本发明还提供了上述的构象锁定蜂毒肽衍生物或其药学上可接受的盐在制备治疗结直肠癌的药物中的应用。
具体的,所述的结直肠癌的相关治疗对应的细胞为CT26细胞。
本发明的构象锁定MP衍生物是在MP的序列中引入非天然氨基酸,在MP肽段侧链形成碳氢支架以稳定多肽的α-螺旋构象,达到构象锁定的目的。首先,基于该策略的构象锁定MP衍生物能够稳定α-螺旋构象且α-螺旋程度有明显提升。其次,体外细胞实验表明该类构象锁定MP衍生物对结直肠癌细胞CT26表现出较强的抑制活性。此外,筛选得到的MP衍生物与MP相比,能够在酶稳定性上得到显著改善,进而提高药物的生物利用度。然而,该类构象锁定MP衍生物的溶血副作用不同程度的增加。
本发明的另一目的是,将候选多肽设计成MMP-2响应型且具有PD-L1靶向性的杂合肽,并与高分子材料PEG偶联得到构象锁定蜂毒肽-PEG偶联物。
本发明还提供了一种肿瘤微环境响应型的PD-L1靶向杂合肽,上述的构象锁定蜂毒肽衍生物与PEG进行偶联。
进一步的,在上述的构象锁定蜂毒肽衍生物的C-端引入一条D型的anti-PD-L1多肽片段NYSKPTDRQYHF(SEQ ID NO.2所示),这两条肽之间以MMP-2响应的短肽PLGLAG(SEQID NO.3所示)连接,得到肿瘤微环境响应型的PD-L1靶向杂合肽,然后在杂合肽的N-端连接一个氨基酸残基Cys,与4-臂-PEG-马来酰亚胺(MW 5KDa)通过迈克尔加成反应得到多肽-PEG偶联物。
本发明还提供了上述的肿瘤微环境响应型的PD-L1靶向杂合肽在制备治疗结直肠癌的药物中的应用。
进一步的,所述的结直肠癌细胞为CT26细胞。
本发明采用全碳氢订书肽技术,将多肽序列的i,i+4位替换成含烯烃的非天然氨基酸S5,通过烯烃复分解反应得到含碳氢支架侧链的MP衍生物,全碳氢订书肽技术可稳定原型肽MP的α-螺旋二级结构,并增加其糜蛋白酶解稳定性。体外药理实验证明MP衍生物MP9具有最佳治疗指数和溶瘤特性,考虑到溶瘤肽类药物在体内易被清除且缺少靶向性,我们在MP9的C末端连接一段MMP-2响应型的anti-PD-L1多肽,并将该杂合肽与高分子材料4-臂PEG偶联得到多肽-聚合偶联物PEG-MP9-aPDL1,以增强偶联物的体内靶向性和长循环,实现溶瘤肽的系统性递送。本发明的“一体化”多肽-PEG偶联物不仅能直接裂解结直肠癌细胞CT26,而且与免疫检查点PD-L1靶向肽协同,激活机体免疫系统发挥抗肿瘤免疫作用。本发明为溶瘤肽类药物的系统性给药、与免疫检查点抑制剂联用增强溶瘤免疫作用等方面提供参考价值。
本发明的构象锁定蜂毒肽-PEG偶联物保留了蜂毒肽的α-螺旋二级结构,且显著降低溶血副作用。其次,细胞实验表明,该偶联物具有诱导免疫原性细胞死亡的特性。最后,体内药理实验表明,该偶联物具有良好的体内靶向性,可在肿瘤部位富集,并且该偶联物对结直肠癌细胞CT26表现出较强的抑制活性,可协同免疫检查点PD-L1靶向肽发挥溶瘤免疫治疗作用。与现有临床试验应用的溶瘤肽药物相比,蜂毒肽-PEG偶联物改变瘤内注射的给药方式,并且联合免疫检查点抑制剂,实现系统性给药与“一体化”治疗,因此可用于制备抗结直肠癌药物。
附图说明
图1为本发明构象锁定蜂毒肽MP9的耐糜蛋白酶稳定性。
图2为本发明具有PD-L1靶向性环境响应型构象锁定杂合蜂毒肽Cys-MP9-MMP-2-aPD-L1的核磁氢谱图。
图3为本发明具有PD-L1靶向性多肽Cys-MMP-2-aPD-L1的核磁氢谱图。
图4为本发明构象锁定蜂毒肽Cys-MP9的核磁氢谱图。
图5为本发明具有PD-L1靶向性构象锁定蜂毒肽-PEG偶联物PEG-MP9-aPDL1的核磁氢谱图。
图6为本发明具有PD-L1靶向性多肽-PEG偶联物PEG-aPDL1的核磁氢谱图。
图7为本发明构象锁定蜂毒肽-PEG偶联物PEG-MP9的核磁氢谱图。
图8为本发明高分子材料4-臂PEG-马来酰亚胺的核磁氢谱图。
图9为本发明构象锁定蜂毒肽-PEG偶联物的溶血副作用。
图10为构象锁定蜂毒肽及其PEG偶联物诱导肿瘤细胞ATP释放
图11为构象锁定蜂毒肽及其PEG偶联物诱导肿瘤细胞HMGB1蛋白外排。
图12为构象锁定蜂毒肽及其PEG偶联物诱导肿瘤细胞CRT蛋白暴露增加。
图13为构象锁定蜂毒肽MP9及其PEG偶联物的生物分布。
图14为构象锁定蜂毒肽-PEG偶联物体内抗结直肠癌作用。
具体实施方式
以下结合具体实施例,对本发明作进一步说明。应理解,以下实施例仅用于说明本发明而非用于限定本发明的范围。
实施例1一类构象锁定MP衍生物MP1-10的制备
表1本发明构象锁定MP衍生物MP1-10的氨基酸序列
下面以制备构象锁定MP衍生物MP1为例进行具体描述,其余构象锁定MP衍生物的制备操作相同。
合成路线:
合成步骤:
称取735mg氨基树脂(取代度0.34mmol/g)放入多肽合成管中,于室温加入5mL二氯甲烷(DCM)溶胀20分钟,然后用水泵抽干溶剂。将7mL含有20%哌啶的N,N’-二甲基甲酰胺(DMF)溶液加入合成管中与树脂共孵育,35℃反应5分钟后抽干液体,随后再加入7mL含有20%哌啶的DMF溶液,重复操作1次。脱除保护基后,树脂用DCM洗涤3遍,再用DMF洗涤3遍。
称取Fmoc-Leu-OH(353mg,1mmol),2-肟氰乙酸乙酯(Oxyma pure,142mg,1mmol)至10mL离心管中,充分溶于6mL N-甲基吡咯烷酮,再加入155μL N,N-二异丙基碳二亚胺(N,N’-diisopropylcarbodiimide,DIC,1mmol)活化氨基酸。随后将该混合液加入合成管中,于60℃、60rpm反应20分钟。随后抽干液体,用DMF洗涤3遍,DCM洗涤3遍,再用DMF洗涤3遍,除去未反应的氨基酸。然后脱去第一个氨基酸Leu的Fmoc保护基,方法同上。我们用上述方法依次接入Fmoc-Ile-OH,Fmoc-Lys(Boc)-OH,Fmoc-Lys(Boc)-OH,Fmoc-Ala-OH,Fmoc-Lys(Boc)-OH,Fmoc-Ala-OH,Fmoc-Ala-OH,Fmoc-Leu-OH,Fmoc-S5-OH,Fmoc-Lys(Boc)-OH,Fmoc-Leu-OH,Fmoc-Asn(Trt)-OH,Fmoc-S5-OH,脱Fmoc保护。值得注意的是,含有α-甲基、α-烯基的非天然氨基酸Fmoc-S5-OH的缩合时间延长至2小时。得到接好线性多肽的树脂之后,先用10mL 1,2-二氯乙烷(dichloroethane,DCE)冲洗树脂3次,然后加入6mL含Grubbs第一代催化剂(10mmol/L)的DCE,于35℃、60rpm反应2小时。随后抽干反应液,重复反应1次。反应结束后,树脂用DCM洗涤3遍,再用DMF洗涤3遍。向多肽合成管中加入10mL混合液(乙酸酐:吡啶:DMF=1:1:8)将N-端氨基乙酰化,于35℃晃动反应15分钟后洗涤树脂。先用10mL甲基叔丁基醚(methyl tert-butyl ether,MTBE)冲洗树脂3次,水泵抽干。将干燥的树脂从多肽合成管中取出转移至50mL离心管中,加入15mL cocktail TFA,即95%三氟乙酸,2.5%水,2.5%三异丙基硅烷混合切割液,室温振荡2小时。将含多肽切割液的树脂过滤至50mL离心管中,每5mL过滤液加40mL冰MTBE,4000rpm离心3分钟,倾倒上清后得到沉淀,随后再加入冰MTBE重复沉淀1次,用氮气吹干沉淀得到多肽粗品。最后经制备液相纯化后冻干得到构象锁定MP衍生物MP1。
实施例中所用试剂均为市售分析纯,制备液相所用溶剂为色谱纯。
实施例2本发明构象锁定MP衍生物的质谱和α-螺旋程度数据
本发明构象锁定MP衍生物MP1-10的质谱和α-螺旋程度数据见表2
表2本发明构象锁定MP衍生物MP1-10的质谱和α-螺旋程度数据表
由上表可见,本发明化合物与原性肽MP相比,α-螺旋程度均增加。
实施例3本发明构象锁定MP衍生物体外抗肿瘤实验
3.1实验方法:采用CCK-8细胞增殖毒性检测试剂盒测试构象锁定MP衍生物对结直肠癌细胞CT26的抗增殖活性。在96孔板中配置100μL的CT26细胞悬液(5000个/孔),将培养板在37℃、5%CO2培养箱预培养24小时。吸弃培养基,向培养板加入100μL不同浓度的构象锁定MP衍生物的基础培养基,并在培养箱孵育24小时。吸弃培养基,向每孔加入100μL含10%CCK-8溶液的基础培养基。将培养板在培养箱内孵育2小时后用酶标仪测定96孔板在450nm处的吸光度。按如下公式计算药物对CT26细胞的抑制率:抑制率=[(As-Ac)]/[(Ab-Ac)]×100%,As:实验孔(含有细胞的培养基、CCK-8、待测药物),Ab:对照孔(含有细胞的培养基、CCK-8、没有待测药物),Ac:空白孔(不含细胞和待测药物的培养基、CCK-8)。
3.2实验结果:体外抗肿瘤实验见表3。
表3本发明的化合物MP1-10体外抗肿瘤实验结果
由上表可见,本发明化合物均对CT26细胞有较强的抑制活性,不同程度地提升了MP的抗肿瘤活性。
实施例4本发明的化合物MP1-10的溶血副作用
本发明的化合物MP1-10的溶血实验结果见表4。
表4本发明的化合物MP1-10的溶血实验
由上表可见,本发明MP衍生物均对红细胞的溶血毒性均有增加,其中,MP9的溶血副作用相对较小。
实施例5本发明构象锁定MP衍生物的耐糜蛋白酶稳定性
优选上述实验α-螺旋程度和体外抗肿瘤活性均较高的MP9进行耐糜蛋白酶稳定性的考察。称取适量的药物配制成浓度为1mM的肽储存液。称取一定量的糜蛋白酶溶于含有2mM CaCl2的磷酸盐缓冲液(50mM,pH 7.4)至糜蛋白的浓度为0.5ng/μL。在2mL离心管中分别加入1000μL含糜蛋白酶的磷酸盐缓冲液和100μL的药物储存液进行酶消化反应。分别取药物各个时间点的100μL反应液加入20μL 1M的盐酸淬灭糜蛋白酶活性。最后使用高效液相色谱仪分析不同时间点多肽的百分残余量并作图。
图1为MP9对糜蛋白酶稳定性结果,原型肽MP迅速降解,而经过构象锁定能够显著提高MP9的稳定性,24小时后才降解完全。
实施例6本发明具有PD-L1靶向性的肿瘤响应型构象锁定杂合蜂毒肽的设计合成
本发明将MP9设计为一条PD-L1靶向性的MMP-2响应杂合肽及相关多肽的氨基酸序列的质谱和α-螺旋程度数据见表5
表5本发明PD-L1靶向性的MMP-2响应杂合肽及相关多肽的氨基酸序列
合成方法:
以多肽Cys-MP9-MMP-2-aPD-L1为例,首先采用多肽固相合成法合成线性多肽,构象锁定订书肽MP9部分通过烯烃复分解反应进行环合,并对N-端进行乙酰化,合成步骤同实施例1。
由于N-末端连有氨基酸残基Cys,切割液替换为15mL cocktail B TFA切割试剂(88%三氟乙酸,5%水,5%苯酚,2%三异丙基硅烷),室温振荡2小时。将含多肽切割液的树脂过滤至50mL离心管中,每5mL过滤液加40mL冰MTBE,4000rpm离心3min,倾倒上清后得到沉淀,随后再加入冰MTBE重复沉淀1次,用氮气吹干沉淀得到多肽粗品。最后经制备液相纯化后冻干得到PD-L1靶向性的MMP-2响应杂合肽及相关多肽。
表6本发明PD-L1靶向性的MMP-2响应杂合肽及相关多肽的质谱数据表
实施例7本发明构象锁定蜂毒肽-PEG偶联物的合成
以构象锁定蜂毒肽-PEG偶联物PEG-MP9-aPDL1为例
将多肽Cys-MP9-MMP-2-aPD-L1与4-臂PEG-马来酰亚胺(MW 5KDa)分别溶于PBS(pH7.4)溶液中,浓度均为2mg/mL。然后将溶于PBS中的多肽与4-臂PEG-马来酰亚胺以摩尔比8:1混合均匀,水浴超声10分钟除氧,随后室温搅拌反应10小时。将反应混合物移至超滤管(10KDa)中,用蒸馏水置换PBS,4000g离心15min,共离心5次,除去未反应完全的游离多肽、PEG和盐离子。将超滤产物冷冻干燥得到白色粉末,即为多肽-PEG偶联物PEG-MP9-aPDL1,用核磁共振1H谱对反应物及产物进行表征,并通过圆二色谱测定偶联物的α-螺旋程度。按照上述方法,多肽Cys-MMP-2-aPD-L1、Cys-MP9分别与4-臂PEG-马来酰亚胺反应得到多肽-PEG偶联物PEG-aPDL1和PEG-MP9。
表7本发明的构象锁定蜂毒肽-PEG偶联物的α-螺旋程度
由上表可知,本发明的构象锁定蜂毒肽-PEG偶联物PEG-MP9和PEG-MP9-aPDL1保留了蜂毒肽的α-螺旋二级结构,而只含靶向肽的偶联物PEG-aPDL1无α-螺旋二级结构。
图2-8为不同多肽及其PEG偶联物的核磁氢谱。图9为偶联物的溶血副作用结果,与游离构象锁定蜂毒肽MP9相比,与PEG连接的构象锁定蜂毒肽-PEG偶联物的溶血副作用得到改善。
实施例8本发明构象锁定蜂毒肽及其PEG偶联物诱导免疫原性细胞死亡
(1)ATP释放实验
根据增强型ATP检测试剂盒操作说明书,检测游离肽MP9、多肽-PEG偶联物PEG-MP9、PEG-MP9-aPDL1裂解细胞膜后释放的ATP含量。CT26细胞按5×103个/孔接种于96孔板中,于37℃、5%CO2条件孵育过夜。弃去原有培养基,加入含有不同药物(IC50浓度)的基础培养基作用6h,每组设置3个复孔,阴性对照组只加基础培养基,阳性对照组则在给药作用结束前5分钟加入含20μL裂解液的基础培养基。随后将96孔板于4℃、800g离心5分钟。另取黑色96孔板中预先加入100μL ATP检测工作液,室温放置3-5分钟消耗底物,然后从离心好的96孔板中每孔取20μL上清加入到含有ATP检测工作液的孔中,立即采用多功能酶标仪检测样品的相对化学发光强度(Relative Luminescence Units,RLU)。
(2)高迁移率族蛋白(HMGB1)的免疫荧光染色
将CT26细胞以5×104个/孔的密度接种于共聚焦皿,培养过夜。给药游离肽MP9、偶联物PEG-MP9及PEG-MP9-aPDL1(IC50浓度)作用6小时,吸弃培养基,每孔加入1mL预冷PBS轻轻冲洗细胞并吸弃。随后吸弃上清,4%多聚甲醛室温固定20分钟,再用冰PBS洗涤2次。加入1%Triton X-100破膜5分钟,用冷的PBS洗涤2次。洗涤后将肿瘤细胞置于1%BSA中室温封闭1小时。封闭结束,冰PBS洗涤3次。加入anti-HMGB1 anitbody,4℃孵育过夜;吸弃上清,加入荧光二抗Alexa Fluor 488-conjugated secondary antibody,室温孵育2小时。再加入Hoechst 33342(5μM)标记细胞核,继续染色10分钟。冷PBS洗涤3次,使用GE DeltaVisionOMX SR超高分辨显微镜观察HMGB1的释放情况。
(3)钙网蛋白(Calreticulin,CRT)的免疫荧光染色
将CT26细胞以5×104个/孔的密度接种于共聚焦皿,培养过夜。给药游离肽MP9、偶联物PEG-MP9及PEG-MP9-aPDL1(IC50浓度)作用6小时,吸弃培养基,每孔加入1mL预冷PBS轻轻冲洗细胞并吸弃。随后吸弃上清,4%多聚甲醛室温固定20分钟,再用冰PBS洗涤2次。洗涤后将肿瘤细胞置于1%BSA中室温封闭1小时。封闭结束,冰PBS洗涤3次。加入anti-Calreticulin-Alexa Fluor 647antibody孵育1小时;吸弃上清,再加入Hoechst 33342(5μM)标记细胞核,继续染色10分钟;冷PBS洗涤3次,使用GE DeltaVision OMX SR超高分辨显微镜观察CRT的暴露情况。
图10-12为构象锁定蜂毒肽及其PEG偶联物可诱导免疫原性细胞死亡,主要是ATP释放增加、细胞核内HMGB1蛋白减少和CRT蛋白暴露增加。
实施例9本发明构象锁定蜂毒肽-PEG偶联物的生物分布
(1)ICG标记的蜂毒肽-PEG偶联物合成
精密称取多肽Cys-Lys(N3)-MP9-MMP-2-aPD-L1与Cys-Lys(N3)-MP9溶于PBS中,浓度均为2mg/mL,分别与4-臂PEG-马来酰亚胺发生迈克尔加成反应,超滤冻干得到多肽-PEG偶联物PEG-K(N3)-MP9-aPDL1和PEG-K(N3)-MP9,反合成步骤同实施例7。随后,精密称取一定量的ICG-DBCO粉末溶于DMSO,浓度为5mg/mL。ICG-DBCO分别与溶于PBS中的偶联物PEG-K(N3)-MP9-aPDL1和PEG-K(N3)-MP9以摩尔比8:1在PBS(pH 7.4)溶液中混合均匀,30℃条件搅拌反应12小时,通过点击化学反应在偶联物上标记荧光ICG。将反应产物移至超滤管中,4000g离心15分钟,除去未反应完全的,共离心5次,将超滤产物冻干得到ICG标记的蜂毒肽肽-PEG偶联物ICG-PEG-MP9和ICG-PEG-MP9-aPDL1。多肽MP9亦通过点击化学反应标记ICG,得到游离多肽ICG-MP9。
(2)活体成像
1.造模:5周龄雌性BALB/c小鼠背部右侧大腿外侧异位接种CT26细胞,数量1×106细胞/只。待肿瘤平均体积达到200mm3时,将荷瘤小鼠随机分成3组,具体如下:①ICG-MP9组;②ICG-PEG-MP9组;③ICG-PEG-MP9-aPDL1组。
2.给药:每组小鼠尾静脉注射不同药物,每只以0.01mg ICG计,PBS溶剂体积100μL。
3.监测:分别在给药1h、2h、4h、8h、12h、24h后,麻醉小鼠,使用VISQUE小动物活体成像仪进行拍摄,观察ICG体内荧光分布情况。体内成像拍摄结束后,处死小鼠,剖取小鼠的肿瘤、心脏、肝脏、脾脏、肺和肾脏等组织进行离体荧光成像拍摄。荧光结果通过VISQUEClevue软件分析。
图13为构象锁定蜂毒肽MP9及其PEG偶联物的生物分布结果,PEG-MP9-aPDL1组在肿瘤部位的荧光强度最强,表明PEG-MP9-aPDL1可以系统靶向递送多肽药物到达肿瘤部位并蓄积。(A)给药1h、2h、4h、8h、12h、24h后,小鼠体内荧光分布图;(B)离体组织心、肝、脾、肺、肾中荧光分布图;(C)离体肿瘤组织的荧光分布图;(D)离体组织肿瘤、心、肝、脾、肺、肾的荧光统计图。
实施例10本发明构象锁定蜂毒肽-PEG偶联物的体内抗肿瘤作用
(1)动物模型建立
雌性BALB/c小鼠,左腹第二对乳房垫接种CT26细胞,数量8×105细胞/只,约3天形成肿瘤。
(2)评价构象锁定蜂毒肽-PEG偶联物的体内抗肿瘤作用
实验分组:①空白组(PBS);②PEG-aPDL1组;③PEG-MP9组;④PEG-MP9-aPDL1组。给药方式:采用尾静脉注射的方式,每组以3mg/kg MP9多肽计算给药量,溶液体积100μL,每隔两天给一次药共给6次。评价指标为:①以瘤体积1000mm3为终点,解剖出小鼠肿瘤,比较各组肿瘤大小;②跟踪记录实验过程中小鼠一般健康状况,饮食、饮水和体重变化,比较副作用。
图14为构象锁定多肽MP9与靶向肽anti-PD-L1的杂合肽的PEG偶联物PEG-MP9-aPDL1显著抑制BALB/c小鼠皮下CT26结直肠癌的生长。(A)动物给药示意图;(B)CT26结直肠肿瘤实物图;(C)小鼠体重曲线;(D)CT26结直肠肿瘤体积大小;(E)CT26结直肠肿瘤体重大小。与空白组和PEG-aPDL1组相比,PEG-MP9和PEG-MP9-aPDL1明显抑制肿瘤生长,其中蜂毒肽与免疫检查点抑制抑制多肽联用的PEG-MP9-aPDL1组抗肿瘤效果最佳。
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。
序列表
<110> 上海中医药大学
<120> 一种构象锁定蜂毒肽衍生物、偶联物及其制备和用途
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Claims (9)
1.一种构象锁定蜂毒肽衍生物或其药学上可接受的盐,其特征在于,为下列各式中的任意一种:
。
2.权利要求1所述的一种构象锁定蜂毒肽衍生物或其药学上可接受的盐的制备方法,其特征在于包括如下步骤:
a)以蜂毒肽MP为基础,所述的蜂毒肽MP的序列为INLKALAALAKKIL,将MP序列的i,i+4位点的天然氨基酸替换为含有α-甲基,α-烯烃的氨基酸(2R)-2-N-芴甲氧羰基氨基-2-甲基-6-庚烯酸,i为大于等于1的整数;设计系列MP衍生物MP1-MP10;
b根据多肽固相合成法,以9-芴基甲氧基羰基保护基的天然氨基酸和Fmoc-S5-OH为原料,Rink Amide氨基树脂为载体,2-肟氰乙酸乙酯/N-甲基吡咯烷酮/N, N-二异丙基碳二亚胺为缩合体系,合成线性多肽;
c)线性多肽合成后,直接进行环合反应,采用Grubbs第一代催化剂进行烯烃复分解反应,得到含有碳氢支架侧链的MP衍生多肽,实现α-螺旋结构的锁定;
d)采用乙酸酐/吡啶体系,对环合后的多肽N-端氨基乙酰化;随后将干燥的树脂从多肽合成管中取出转移至离心管中,用三氟乙酸、水和三异丙基硅烷的混合切割液将环合好的MP衍生多肽从树脂上切下,把含多肽切割液的树脂过滤,得到的过滤液用冰甲基叔丁基醚沉淀,并用氮气吹干沉淀得到多肽粗品,最后将MP衍生多肽粗品通过制备液相纯化,冻干后得到构象锁定MP衍生多肽药学上可接受的盐。
3.根据权利要求2所述的一种构象锁定蜂毒肽衍生物或其药学上可接受的盐的制备方法,其特征在于:所述的催化剂为Grubbs第一代催化剂。
4.权利要求1所述的一种构象锁定蜂毒肽衍生物或其药学上可接受的盐在制备治疗结直肠癌的药物中的应用。
5.根据权利要求4所述的应用,其特征在于,所述的结直肠癌细胞为CT26细胞。
6.一种肿瘤微环境响应型的PD-L1靶向杂合肽,其特征在于,权利要求1所述的构象锁定蜂毒肽衍生物与PEG进行偶联。
7.根据权利要求6所述的一种肿瘤微环境响应型的PD-L1靶向杂合肽,其特征在于,在权利要求1所述的构象锁定蜂毒肽衍生物的C-端偶联一条D型的anti-PD-L1多肽片段NYSKPTDRQYHF,这两条肽之间以MMP-2响应短肽肽PLGLAG连接,得到肿瘤微环境响应型的PD-L1靶向杂合肽,然后在杂合肽的N-端连接一个氨基酸残基Cys,与4臂PEG-马来酰亚胺通过迈克尔加成反应得到多肽-PEG偶联物。
8.权利要求7所述的肿瘤微环境响应型的PD-L1靶向杂合肽在制备治疗结直肠癌的药物中的应用。
9.根据权利要求8所述的应用,其特征在于,所述的结直肠癌细胞为CT26细胞。
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