CN105859834B - 一种用于靶向治疗的多肽和核酸偶联化合物 - Google Patents
一种用于靶向治疗的多肽和核酸偶联化合物 Download PDFInfo
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Abstract
本发明公布了一种用于靶向治疗的多肽和核酸偶联化合物。在作为效应分子的核酸和作为靶分子的多肽[D‑Lys6]GnRH上分别引入炔烃基团和叠氮基团,然后通过click点击化学反应,合成具有靶向作用的多肽和核酸偶联化合物,可应用于肿瘤的靶向治疗。
Description
技术领域
本发明涉及生物医药领域,具体涉及一种多肽和核酸的偶联化合物及其制备方法,以及该化合物在疾病靶向治疗中的应用。
背景技术
1998年,Fire等发现了RNA干扰(RNA interference,RNAi)能特异性的降解目的基因的mRNA(Fire A,Xu S,Montgomery MK,Kostas SA,Driver SE,Mello CC.Potent andspecific genetic interference by double-stranded RNA in Caenorhabditiselegans.Nature.1998;391:806-11.)。介导该现象的是双链的siRNA。2001年,Elbashir等成功的用体外合成的双链siRNA沉默了哺乳动物细胞内相关基因的表达,为siRNA类基因治疗型药物的发展奠定了基础(Elbashir SM,Harborth J,Lendeckel W,Yalcin A,Weber K,Tuschl T.Duplexes of 21-nucleotide RNAs mediate RNA interference in culturedmammalian cells.Nature.2001;411:494-8.)。但是,由于siRNA相对分子量较大(14000Da左右)且带有大量的负电荷,无法顺利穿越细胞膜到达细胞内,并容易从尿内排出,再外加siRNA自身的不稳定性、易被降解、半衰期短、易引起免疫反应、难以从核内体中逃逸等因素,这些都导致siRNA生物利用度降低,影响其临床应用。如何提高siRNA体内的生物利用度,是siRNA能否广泛利用的关键。利用载体递送siRNA,是一种有效的方法。脂质体和聚合物都可用于siRNA的载体,但都面临像毒性大、靶向性不强甚至脱靶以及微粒大小控制困难等问题。而蛋白类载体可以很好地克服这些问题,其中最常见的有细胞穿透肽和单链抗体。Andaloussi等设计了一种新型的多肽PepFect 6(PF6)(Andaloussi SE,Lehto T,Mager I,Rosenthal-Aizman K,Oprea,II,Simonson OE,et al.Design of a peptide-basedvector,PepFect6,for efficient delivery of siRNA in cell culture andsystemically in vivo.Nucleic Acids Res.2011;39:3972-87.),PF6与HPRT1(hypoxanthine phosphoribosyltransferase1)siRNA通过非共价复合形成PF6/siRNA混合物,该复合物在体外实验展现出很高的递送效率,并且在动物试验中,通过静脉注射PF6/siRNA可以有效地沉默肝、肾和肺部的HPRT1mRNA水平。但因为该多肽和核酸形成的是混合物,导致混合物的形成条件、保存以及标准化都带来非常大的困难。
单链抗体(single chain of variable fragments,scFv)由于具备靶向性好、分子量小、穿透性强等特点而被用作siRNA载体。由于scFv与siRNA结合性较差,常需要一个多肽充当连接器。其中富含碱性氨基酸具备与核酸结合功能的鱼精蛋白截短体(truncatedprotamine,tp)是最常见的连接器。具体方法就是将scFv与tp通过基因重组获得scFv-tp融合蛋白。scFv-tp同时具有scFv的靶向性和tp的siRNA结合性,从而成为稳定的siRNA递送载体。scFv-tp依托scFv的特异性,通过与细胞表面的特异性受体结合进而将siRNA递送至靶细胞内,减少了siRNA的脱靶效应。早在2005年,Song等就用gp160-scFv(F105)与tp的融合蛋白F105-P作为载体将EGFP siRNA递送至小鼠体内被HIV感染的CD4T细胞中,有效地沉默目的基因的表达,抑制了HIV病毒复制,并且在动物模型中,F105-P介导的多种原癌基因siRNA的体内递送也收获巨大的成功(Song E,Zhu P,Lee SK,Chowdhury D,Kussman S,Dykxhoorn DM,et al.Antibody mediated in vivo delivery of small interferingRNAs via cell-surface receptors.Nat Biotechnol.2005;23:709-17.)。这些结果强有力的证明了scFv-tp作为siRNA载体在体内递送的可靠性和高效特异性。之后,Yao等用类似的方法用人类表皮生长因子2(human epidermal growth factor receptor 2,Her2)受体的scFv-tp融合蛋白将PLK1-siRNA成功递送至Her2+的乳腺癌细胞系中,并且在动物模型中也能有效地抑制肿瘤的增殖和转移(Yao YD,Sun TM,Huang SY,Dou S,Lin L,Chen JN,etal.Targeted delivery of PLK1-siRNA by ScFv suppresses Her2+breast cancergrowth and metastasis.Science translational medicine.2012;4:130ra48.)。在这些应用中,都用到了基因技术,包括基因克隆、细菌表达、分离纯化等复杂步骤,过程复杂、技术难度大,对人体和环境还可能带来一些安全问题。
由上可见,多肽或者蛋白可以作为核酸的载体,用于把短的siRNA带入体内,发挥其基因沉默作用,特异性的沉默靶基因的表达从而达到治疗的目的。为了更好地发挥多肽的载体作用,还需要克服一系列的问题。
发明内容
本发明的目的是提供一种多肽和核酸偶联的化合物,其中多肽结构部分赋予其靶向性,引导该化合物定向达到靶分子,核酸作为效应分子,可以沉默目的基因,抑制肿瘤的生长,发挥肿瘤治疗作用。
本发明的多肽和核酸偶联化合物包括共价连接的多肽和核酸两部分,其结构如下式I所示。
式I中,nucleic acid代表核酸,是具有治疗效应的分子,如siRNA、反义核酸、aptamer(核酸适配体)等;多肽部分是[D-Lys6]GnRH(见序列表中SEQ ID No:1),具有靶向作用,通过D-Lys残基上的一个氨基与式I中所示羰基形成酰胺键。
GnRH(Gonadotropin-Releasing Hormone,促性腺激素释放激素)也称作LHRH(Luteinising-hormone releasing hormone,促黄体激素释放激素),是体内自然存在的一种激素,最初是从哺乳类下丘脑分离出的一种十肽激素,其基本生理功能是调节垂体前叶促性腺激素的分泌,在控制和调节哺乳动物生殖功能中发挥重要作用。GnRH发挥其生物学功能必须要通过GnRHR(GnRH–Receptor,GnRH受体)来介导。GnRHR属于类视紫红质G蛋白偶联受体家族(GPCR)中的一员,是具有7次跨膜结构的蛋白受体。近年的研究发现,卵巢、子宫内膜、前列腺、等生殖系统肿瘤细胞表面均有GnRH结合位点,GnRH受体(GnRHR)表达量较高,而高表达的GnRHR是为抗肿瘤治疗提供了潜在靶点。GnRH类似物已经被广泛用于生殖系统疾病的治疗,其中以[D-Lys6]GnRH为基本骨架的类似物最为常见。而siRNA作为一种广泛存在的基因调控机制,理论上可以沉默任何高表达的基因。肿瘤通常都会找到一些肿瘤相关基因的高表达,这些基因的表达对肿瘤的生长、转移起重要作用,如果敲除这些基因,能够有效地抑制肿瘤的生长和转移。在本发明的一些具体实施例中,通过[D-Lys6]GnRH作为靶分子,把效应分子siRNA带到表达GnRH受体的肿瘤细胞,以siRNA特异性的模式沉默肿瘤相关基因,达到靶向治疗肿瘤的目的。
本发明的多肽和核酸偶联化合物是通过如下方法制备的:在作为效应分子的核酸和作为靶分子的多肽上分别引入炔烃基团和叠氮基团,然后通过click点击化学反应,形成均一稳定的化合物。反应式如下所示:
上述click点击化学反应可以以二甲亚砜(DMSO)、叔丁醇(t-BuOH)和水作为溶剂,CuSO4·5H2O-TBTA配合物和抗坏血酸钠作为催化剂,室温下反应2~5小时。
为了方便GnRH的叠氮修饰,在GnRH的第六位通过多肽合成引入D-Lys,D-Lys的一个氨基用于形成氨基酸残基之间的肽键,另个氨基用于引入叠氮基团。[D-Lys6]GnRH与叠氮戊酸发生酰化反应,从而在多肽上引入叠氮基团,反应方程式如下:
对于效应分子,在其5’或3’端偶联上炔烃基团。例如对于一段siRNA双链,为了减少引入的炔烃基团对siRNA干扰效率的影响,采用亚磷酰胺的固相合成法,通过siRNA与3-丁炔-1-醇反应,从而将丁炔基引入核酸正义链的5’末端或3’末端。
通过click反应,将上述经过修饰的多肽和核酸联合到一起,再通过产物的分离纯化,最终获得目标产物。
本发明通过在效应分子核酸上引入炔烃基团,在[D-Lys6]GnRH多肽上引入叠氮基团,通过click反应,得到一个稳定的化合物,该化合物保留了[D-Lys6]GnRH的靶向性,和效应分子核酸的基因沉默作用。作为特异性靶向药物,本发明的多肽和核酸偶联化合物可应用于肿瘤的靶向治疗。
附图说明
图1为实施例1中不同浓度的[D-Lys6(FITC)]GnRH和细胞上受体的结合曲线。
图2为实施例1中确定[D-Lys6(FITC)]GnRH和细胞最佳结合浓度的验证曲线。
图3显示了实施例1中[D-Lys6(FITC)]GnRH与普通GnRH竞争抑制实验结果,横坐标代表8个实验,从第1至第8个实验[D-Lys6(FITC)]GnRH与GnRH浓度比依次为1∶0,1∶1,1∶2,1∶4,1∶8,1∶16,1∶32和0∶0。
图4显示了实施例1中[D-Lys6(FITC)]GnRH和GnRHR抗体封闭抑制试验结果。
图5为实施例2合成化合物的化学反应式。
图6为实施例2合成的[D-Lys6]GnRH-siRNA用UPLC分离纯化的色谱峰。
图7为实施例2合成的化合物[D-Lys6]GnRH-siRNA用UPLC-ESI-MS验证的质谱图。
图8显示了实施例3验证[D-Lys6]GnRH-siRNA具有生物学活性的实验结果,说明该化合物可以敲除目的基因。
图9.显示了实施例3测试[D-Lys6]GnRH-siRNA对靶基因沉默作用的量效关系的结果。
图10显示实施例3中化合物[D-Lys6]GnRH-siRNA对靶基因的沉默作用可以通过封闭细胞表面的受体而阻止,说明其沉默效果是受体依赖性的特异性沉默。
具体实施方式
以下通过实施例对本发明做进一步说明,以便更好地理解本发明,但本发明并不局限于此。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法;下述实施例中所用的试剂、材料等,如无特殊说明,均可从商业途径得到。
实施例1叠氮标记的GnRH多肽具有靶向性和特异性
选取GnRHR高表达的Hela细胞作为实验细胞,来验证Hela细胞对不同浓度梯度的[D-Lys6]GnRH的结合情况。将Hela细胞接种于6孔板中,待细胞汇合度达到70%~80%时,分别加入不同浓度的荧光标记的[D-Lys6(FITC)]GnRH(购自北京中科亚光生物科技有限公司),6h后,用胰酶将细胞消化下来,离心弃上清;再用PBS重悬,离心弃上清(重复三次),去除未结合的[D-Lys6(FITC)]GnRH,最后加入0.5mL PBS,重悬,上流式细胞仪(BD公司)检测,流式结果使用FlowJo 7.6.1软件进行分析,结果见图1。从图1可见,[D-Lys6(FITC)]GnRH和细胞上受体的结合是剂量依赖性的,直到把细胞上所有的受体都结合。紧接着,我们进一步验证了[D-Lys6(FITC)]GnRH饱和结合浓度,如图2所示,当浓度达到300μg/mL时,平均荧光强度达到最大值,随着浓度增加,荧光强度不在相应增加,因此,我们判断300μg/mL为饱和结合浓度。
验证完[D-Lys6]GnRH与Hela细胞结合的最佳浓度后,我们通过竞争性抑制实验进一步验证了[D-Lys6]GnRH与GnRHR的特异性。竞争抑制实验又分为正常GnRH竞争抑制跟GnRHR抗体竞争抑制。在以正常的GnRH作为竞争剂的抑制试验中,先将Hela细胞接种于12孔板中,在细胞汇合度达到70%~80%时,加入终浓度为300μg/mL的[D-Lys6(FITC)]GnRH,再加入不同浓度比例的GnRH([D-Lys6(FITC)]GnRH与GnRH浓度比分别为1∶0,1∶1,1∶2,1∶4,1∶8,1∶16,1∶32以及0∶0)。6h后,收集细胞,用PBS洗三次,和上文一致,上流式细胞仪检测。结果如图3所示,随着GnRH比例的逐渐增加,平均荧光强度不断降低,由此可以看出[D-Lys6]GnRH与GnRH可以竞争性结合GnRHR,也即是说[D-Lys6]GnRH可以特异性结合GnRHR。
在抗体抑制试验中,将细胞平均分成三组,分别为control组,anti-GnRHR(+)组和anti-GnRHR(-)组。在细胞汇合度达到70%~80%时,Control组不做任何处理;anti-GnRHR(-)组加入终浓度为300μg/mL的[D-Lys6(FITC)]GnRH;anti-GnRHR(+)组提前用5%BSA在37℃条件下封闭30分钟后用过量的GnRHR单克隆抗体再预处理2小时,再加入终浓度为300μg/mL的[D-Lys6(FITC)]GnRH。6h后,收集细胞,用PBS洗三次,上流式细胞仪检测,结果如图4所示。从图4可以看出,[D-Lys6(FITC)]GnRH多肽和受体的结合可以被抗体阻止。
实施例2[D-Lys6]GnRH-siRNA的合成、分离、纯化和验证
本实施例通过click反应合成[D-Lys6]GnRH-siRNA,然后通过UPLC对产物进行分离纯化,并通过UPLC-MS对产物进行验证。
实验材料及试剂:[D-Lys6(azidopentanonicacid)]GnRH(由北京中科亚光生物科技有限公司合成),正义链5’端丁炔基修饰的PLK1siRNA(正义链:5’-CH≡C-CH2-CH2(mU)GAAGAAGA(mU)(mC)A(mC)(mC)(mC)(mU)(mC)(mC)(mU)(mU)A dTdT-3’(序列中“m”代表2’位甲氧基修饰即2’-OMe,下同,参见SEQ ID No:2),反义链:5’-UAAGGAGGGUGAUCUUCUUCAdTdT-3’(SEQ ID No:3),购自广州锐博生物科技有限公司),二甲亚砜(DMSO),CuSO4·5H2O,叔丁醇(t-BuOH),TBTA(tris(benzyltriazolylmethyl)amine),UPLC,ESI-Orbitrap MS。
色谱柱:Jupiter 300A 5u C18(150×4.6mm)
合成步骤如下:吸取50μL,10mmol/L的[D-Lys6(azidopentanonicacid)]GnRH(溶剂为DMSO/t-BuOH:3/1,体积比)加入至100μL 0.5mmol/L的siRNA-CH2-CH2-C≡CH水溶液中,再分别加入4μL 0.1M的CuSO4·5H2O-TBTA配合物(体积比1/1)(配合物溶剂为H2O/DMSO/t-BuOH:13.5/3.5/3,体积比)和4μL 0.8M的抗坏血酸钠水溶液,室温下搅拌三个小时。化学反应方程式见图5。
反应结束后,使用3KD的超滤管(购自德国millipore)处理反应体系,收集截留物。再使用UPLC进行分离纯化,分离条件如下:流动相A=0.02M TEAA(三乙胺醋酸盐,pH=7.4)的H2O/CH3CN(体积比95/5)溶液;流动相B=MeOH;洗脱梯度为100%A 1min,100/0~20/80(A/B)30min,100%B 6min;流速:1mL/min;进样量:10μL;分离纯化的结果见色谱图6。纯化完后冻干,加水溶解后再使用UPLC-ESI-MS验证纯化产物的分子量,结果见图7,正义链测量值8912.68(m/z,z=4,2227.17;标准值8911.57),反义链测量值7322.82(m/z,z=4,1829.45;标准值7322.50),总链测量值16234.49(m/z,z=6,2704.75;标准值16234.07)。
实施例3CLICK反应后的[D-Lys6]GnRH-siRNA活性验证
为了验证click反应后的产物[D-Lys6]GnRH-siRNA的生物学活性,我们通过将[D-Lys6]GnRH-siRNA转染Hela细胞后通过qPCR来检测plk1的表达水平来验证。Hela细胞在转染前一天接种至24孔板中,每孔细胞数目为5×104,转染时要求细胞密度能达到30%~50%。转染时将细胞平均分为四组,分别为空白组(Blank)、阴性对照组(NC)、PLK1组(P+)和click产物组(Click+)。空白组不经过任何处理,阴性对照组使用lipofectamine 2000(以下简写为lip2000)转染稳定的无关序列的siRNA(正义链5’-UUCUCCGAACGUGUCACGUdTdT-3’(SEQ ID No:4);反义链:5’-ACGUGACACGUUCGGAGAAdTdT-3’(SEQ ID No:5)),P+组用lip2000转染正常的PLK1siRNA(正义链5’-(mU)GAAGAAGA(mU)(mC)A(mC)(mC)(mC)(mU)(mC)(mC)(mU)(mU)A dTdT-3’(SEQ ID No:2);反义链:5’-UAAGGAGGGUGAUCUUCUUCAdTdT-3’(SEQID No:3)),Click+组使用lip2000转染click产物[D-Lys6]GnRH-siRNA,各组siRNA的终浓度为90nM。转染后4h换液,30h进行细胞总RNA提取,进而再用qPCR来分析各组的PLK1mRNA的表达含量。
如图8所示,P+组跟click+组相比于NC组PLK1mRNA水平显著性降低(p<0.01),且P+与click+组无显著性差异,实验结果表明,click产物[D-Lys6]GnRH-siRNA具有完整的PLK1siRNA生物学活性的基础。
GnRH-siRNA特异性验证
本实验主要目的是验证click产物[D-Lys6]GnRH-siRNA是否保留了GnRH作为载体的特异性。
同上,我们也是运用了qPCR的方法来验证click产物[D-Lys6]GnRH-siRNA是否可以借助自身的[D-Lys6]GnRH的特异性而穿透细胞膜进入细胞内起到特异性的沉默作用。首先,我们探索了[D-Lys6]GnRH-siRNA的特异性沉默PLK1的量效关系,将Hela细胞接种于24孔板,当细胞汇合度到达50%时,直接加入不同终浓度的[D-Lys6]GnRH-siRNA(终浓度分别为30nM,60nM,90nM,120nM,150nM),30h后,进行总RNA提取以及qPCR分析,量效关系结果如图9所示,当[D-Lys6]GnRH-siRNA浓度达到90nM时,PLK1沉默效果最佳。
细胞平均分为四组,分别为Blank组,Plk1组,Clickpro组以及Click+anti组。Blank组不做任何处理;Plk1组将正常的Plk1siRNA在不加载体的情况下直接加入培养基中(终浓度为90nM);Clickpro组是将click产物[D-Lys6]GnRH-siRNA(终浓度为90nM)直接加入培养基中;Click+anti组将细胞提前用5%BSA在37℃条件下封闭30分钟后,用过量的GnRHR单克隆抗体再预处理2小时提,移除抗体,加入新鲜培养基,再加入[D-Lys6]GnRH-siRNA(终浓度为90nM)。24h后,提取总RNA,继而进行qPCR实验和分析。结果见图10,首先,Clickpro组相比于Plk1组,PLK1mRNA水平显著性降低,说明[D-Lys6]GnRH-siRNA可以不借助载体亦能沉默目的基因的表达,其次,Clickpro组相比于Click+anti组,PLK1mRNA水平也显著性降低,也就是说[D-Lys6]GnRH-siRNA对靶基因的沉默作用可以通过封闭细胞表面的受体而阻止,说明其沉默效果是受体依赖性的特异性沉默。
Claims (6)
1.一种多肽和核酸偶联化合物,包括共价连接的多肽和核酸两部分,其结构式I所示:
式I中,nucleic acid代表核酸,是PLK1 siRNA,连接位置在其正义链5’端;多肽部分是[D-Lys6]GnRH,其D-Lys残基上的一个氨基与式I中所示羰基形成酰胺键。
2.权利要求1所述多肽和核酸偶联化合物的制备方法,在核酸和多肽上分别引入炔烃基团和叠氮基团,然后通过click点击化学反应形成所述化合物,反应式如下:
3.如权利要求2所述的制备方法,其特征在于,所述click点击化学反应以二甲亚砜、叔丁醇和水作为溶剂,CuSO4·5H2O-TBTA配合物和抗坏血酸钠作为催化剂,室温下反应2~5小时。
4.如权利要求2所述的制备方法,其特征在于,首先合成[D-Lys6]GnRH多肽,然后将其与叠氮戊酸发生酰化反应,从而在多肽上引入叠氮基团,反应式如下:
5.如权利要求2所述的制备方法,其特征在于,所述核酸是PLK1 siRNA,在核酸上引入炔烃基团的方式是采用亚磷酰胺的固相合成法,通过PLK1 siRNA与3-丁炔-1-醇反应,从而将丁炔基引入PLK1 siRNA正义链的5’末端。
6.权利要求1所述的多肽和核酸偶联化合物在制备特异性靶向药物中的应用,所述特异性靶向药物为生殖系统肿瘤治疗药物。
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