基于G3BP蛋白的靶向抗肿瘤多肽药物研究

发布时间：2019-06-25 12:59

【摘要】：G3BP1(Ras GAP SH3-binding protein)是Ras活性的负反馈调节因子GTP酶激活蛋白(Ras GTPase activating protein,Ras GAP)SH3结构域的特异结合蛋白。由466个氨基酸组成,位于胞质,是Ras信号通路中重要的调控分子及下游靶分子。G3BP1具有核酸内切酶、DNA解旋酶活性,能诱导应急颗粒形成,参与多种细胞生长、分化、凋亡和RNA代谢的信号传递。因此G3BP1可能是肿瘤生长的关键蛋白。本文研究目的是在前期Ras GAP与G3BP1的NTF2样结构域结合模型研究的基础上,设计合成了全新多肽药物GAP162,在分子和细胞水平上研究多肽药物的抗肿瘤作用及可能的作用机制。对以G3BP1为作用靶点的的多肽药物从体内抗肿瘤药效学和药动学方面进行成药性初步研究。第一部分:基于G3BP靶点的多肽药物设计及体外抗肿瘤作用机理研究首先本课题与中科院大学合作在计算机模拟Ras GAP与G3BP1 NTF2样结构域的结合模型的基础上,通过氨基酸残基突变设计了理论上比天然Ras GAP多肽片段具有更强亲和力的多肽片段P160,在此基础上引入穿膜肽设计了多肽药物GAP161和GAP162。首先利用ATP法研究GAP162对HCT116和A549细胞的体外抗肿瘤作用。结果表明GAP162对HCT116和A549的增殖均具有显著的抑制作用,并且这种增殖抑制作用优于GAP161,且不依赖于穿膜肽片段P167。为了探究GAP162的体外抑瘤作用机理,采用流式细胞术和Annxin V/PI双染检测给药后HCT116的细胞凋亡情况;采用Western blot分析HCT116凋亡通路上的关键蛋白Caspase-3;用SPR技术测定GAP162与G3BP1亲和力;免疫共沉淀法检测GAP162对Ras GAP与G3BP1的结合的影响;免疫荧光法检测GAP162对SG颗粒形成的影响;Western blot检测GAP162对G3BP1蛋白总量,G3BP1蛋白Ser-149位磷酸化水平和C-myc蛋白表达的影响。实验结果表明GAP162能够显著引起HCT116细胞凋亡,并且证明这种凋亡是通过提高caspase-3的剪切活性来实现的。Biacore测定结果表明GAP162能够特异地与G3BP1蛋白结合,并且亲和力大于GAP161。在HCT116细胞经GAP162处理后,分别用G3BP1和Ras GAP抗体进行互逆免疫共沉淀后发现GAP162能够减少Ras GAP与G3BP1的结合。这与我们基于G3BP蛋白与Ras GAP结合设计多肽药物一致。用Western blot检测不同浓度GAP162对HCT116细胞G3BP1总蛋白及Ser-149位磷酸化水平的影响,结果发现GAP162对G3BP1的总蛋白没有影响,但能够抑制Ser-149位去磷酸化水平。免疫荧光实验结果发现在A549细胞中,GAP162能够显著抑制SG颗粒的形成,并且呈现浓度依赖性。最后在HCT116细胞中,GAP162能够显著降低C-myc蛋白的表达量。因此推测GAP162的肿瘤抑制作用可能的机制是:GAP162能够与G3BP1特异结合,抑制G3BP1的Ser-149去磷酸化,进而抑制SG颗粒的形成和C-myc蛋白表达,引起细胞凋亡,起到对肿瘤细胞的增殖的抑制作用。第二部分:基于G3BP靶点的多肽药物体内药效及药动学初步研究GAP161是第一个合成和进行体内外药效研究的以G3BP1为靶点的多肽药物。因此本文首先对GAP161进行药动学研究。建立了HPLC-MS/MS法测定大鼠血浆中GAP161浓度。由于多肽药物的特殊性,方法的选择优化包括:1)选择EP管而非玻璃管作为样品处理的容器,工作液配制的过程中加入了0.5%大鼠空白血浆来解决非特异性吸附的问题;2)非酶切进行整体多肽分析;3)加入0.5%的DMSO作为电荷聚集试剂;4)使用离子交换固相萃取96孔微板Oasis MAX进行前处理;5)选择孔径较大的C4色谱柱进行分离。本文建立的生物分析方法,线性范围为5~2000 ng·m L-1,线性良好;最低定量下限为5ng·m L-1,选择性良好;GAP161和内标GAP120的保留时间分别为1.51 and 1.50 min,样品的进样时间3 min以内;样品处理用96孔板进行,提高了样品前处理的速度,样品回收率大于57%;日间和日内精密度,准确度满足研究需求;在大鼠血浆中-80℃冻融2次、冰上放置12 h、样品处理后在自动进样器上放置24 h均不影响其稳定性,但是在常温下放置4 h,稳定性会下降。因此整个实验处理过程在冰上进行。方法学验证表明大鼠血浆中GAP161测定的LC-MS/MS方法满足药物代谢动力学研究的要求。Sprague-Dawley大鼠单次静脉注射5 mg·kg-1 GAP161后测定不同时间的血浆药物浓度,并用非房室模型进行参数计算。末端相半衰期T1/2为1.84±0.14 h,而MRT为0.258±0.053 h,表观分布容积为35894±1621 m L·kg-1。结果表明GAP161半衰期较短、在体内较易降解、稳定性差。部分地解释GAP161体内肿瘤抑制作用不强。在GAP161生物分析方法的基础上,建立了HPLC-MS/MS测定大鼠血浆中GAP162浓度的生物分析方法,并对GAP162在大鼠体内的药动学进行研究。Sprague-Dawley大鼠单次静脉注射5 mg·kg-1 GAP162后测定不同时间的血浆药物浓度,用非房室模型进行参数计算。末端相半衰期T1/2为1.43±0.34 h,而MRT为0.649±0.053 h,表观分布容积为7872±1061 m L·kg-1。结果表明GAP162与GAP161有相似的半衰期。本文采用Iodogen标记法结合三氯醋酸沉淀法和分子筛排阻HPLC来研究GAP162在HCT116肿瘤裸鼠体内重要器官及肿瘤组织的分布及其随时间的变化。采用总放射性进行定量。HCT116肿瘤裸鼠静脉注射125I-GAP162后总放射性分布的AUC排序显示GAP162在肺中的暴露水平最高,脑浓度最低,不经血脑屏障。由数据可看出心脏,血清和肿瘤均在给药后2 min达到组织的Cmax,并且在肿瘤组织当中消除慢。为了评价GAP162的体内抗肿瘤药效,本课题用小鼠结肠癌C26细胞构建小鼠移植肿瘤模型。接种后24 h,采用两种给药方式进行剂量范围的腹腔和皮下连续给药GAP162(每天一次),或者剂量范围的GAP162与顺铂剂量1 mg·kg-1联合用药后,比较动物体重、荷瘤变化,计算瘤重抑制率指标。结果显示单独腹腔和皮下给药后,GAP162对C26肿瘤肿瘤模型具有显著的抑制作用。腹腔给药的有效剂量为40 mg·kg-1,皮下给药的有效剂量为160 mg·kg-1。此外,GAP162与其他抗肿瘤药物联合给药治疗将会提高抗肿瘤效果。与GAP161相比,相同剂量下,抑瘤率提高了最少2倍。
[Abstract]:G3BP1 (Ras GAP SH3-binding protein) is a specific binding protein of the Ras GTPase activating protein (Ras GAP) SH3 domain. It is composed of 466 amino acids and is located in the cytoplasm. It is an important regulatory molecule and downstream target molecule in the Ras signal pathway. G3BP1 has endonuclease and DNase activity, can induce the formation of emergency particles, and participates in the signal transmission of various cell growth, differentiation, apoptosis and RNA metabolism. Therefore, G3BP1 may be the key protein of tumor growth. The purpose of this paper is to study the anti-tumor effect and possible mechanism of the polypeptide drug on the molecular and cellular level. In order to inhibit the formation of SG particles and the expression of C-myc protein, the effect of the expression of C-myc protein on the proliferation of the tumor cells was studied. The second part: The drug effect and the pharmacokinetics of the polypeptide drug based on the G3BP target were the first to synthesize and carry out the in-vivo drug-effect study. The GAP161 was the target of G3BP1. In this paper, the pharmacokinetic study of GAP161 was carried out in this paper. In this paper, the pharmacokinetics of GAP161 was studied in this paper. In this paper, the concentration of GAP161 in the rat plasma was determined by HPLC-MS/ MS. The selection of the method includes:1) selecting EP tube instead of glass tube as the container for sample treatment, adding 0.5% of blank plasma to solve the problem of non-specific adsorption in the process of preparation of working fluid;2) performing whole polypeptide analysis by non-enzyme digestion;3) adding 0.5% DMSO as the charge aggregation reagent;4) extracting 96-hole microplate Oasis MAX with ion exchange solid-phase extraction for pretreatment; and 5) selecting a C4 chromatographic column with a larger pore diameter to separate. In this paper, a bioanalytical method for determining the concentration of GAP162 in plasma of rat plasma by HPLC-MS/ MS was established based on the bioanalytical method of GAP161, and the pharmacokinetics of GAP162 in rats were studied. In Sprague-Dawley rats, a single intravenous injection of 5 mg 路 kg-1 GAP162 was used to determine the plasma drug concentration in different time, and the parameters were calculated with non-compartmental model. The half-life of the end phase was 1.43-0.34 h, and the MRT was 0.649-0.053 h. The apparent distribution volume was 7872-1061 m...
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R96

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