【作者】 于金梅；

【导师】 郑建全；

【作者基本信息】 中国人民解放军军事医学科学院 ， 药理学， 2011， 硕士

【摘要】 促肾上腺皮质激素释放因子(Corticotropin-releasing Factor,CRF)是由41个氨基酸组成的神经肽。是机体应激反应的关键因子,协调应激相关的自主神经、免疫、生理和行为反应。CRF是应激反应轴下丘脑-垂体-肾上腺轴(HPA轴)的起始因子,通过作用于垂体上的CRF受体1(Corticotropin-releasing Factor receptor,CRFR1),激活HPA轴。CRF受体是典型的G蛋白偶联受体,主要有CRFR1与CRFR2两种亚型。两种受体的信号传导途径相似,以AC-cAMP-PKA信号通路为主介导生物学效应。在长期应激条件下,CRF分泌过多,HPA轴功能亢进,介导了抑郁症等情感类疾病。重症抑郁症患者脑脊液中CRF含量过高,HPA轴功能亢进;crfr1基因敲除小鼠不易表现出焦虑样行为;CRFR1拮抗剂能够拮抗动物的抑郁样行为等证据都表明CRFR1有可能成为新一代抗抑郁症的靶标。尽管CRFR1拮抗剂在动物模型中显现出良好的抗抑郁作用,但在临床试验中表现出严重的毒性反应,使此类药物的研究不得不停止。小肽类药物具有活性高,副反应小等优势,越来越受到人们的重视,目前还没有关于CRFR1小肽类拮抗剂的报道。本课题利用经典的噬菌体展示技术筛选CRFR1肽类拮抗剂,希望筛选到毒副作用小的CRFR1小肽类拮抗剂,用于抑郁症的治疗与研究。1.靶蛋白序列的分析和确定:CRFR1为典型的B1超家族G蛋白偶联受体,七次跨膜,N端位于细胞外,C端位于细胞内。CRFR1 EC1区是CRF及相关肽结合的关键区域,其中包含形成CRFR1结合构象的区域(Q43-N90)和CRFR1的选择性结合区域(R76-A89)。CRFR1与CRFR2具有>70%的同源性,其中EC1区同源性最低(60%)。因此根据重要结构域、同源性低、胞外区的原则,选取CRFR1的EC1区域(M 1-V118)作为靶蛋白。2.靶蛋白的表达和纯化:将EC1的cDNA序列连接到原核表达载体pGEX-4T2上,并将其转化到BL21(DE3)感受态细胞中,用IPTG诱导融合蛋白GST-EC1188的表达。SDS-PAGE分析GST- EC1118蛋白主要以包涵体的形式表达。通过优化诱导条件都不能增加可溶性蛋白的表达。分析EC1的氨基酸序列,EC1的M1-N19区域和N108-V118区域富含疏水性氨基酸和含硫氨基酸,疏水键和二硫键过多容易导致包涵体的形成,影响蛋白的溶解性。因此在保证CRFR1的选择性结合区域完整的基础上,去除M1-N19与N108-V118区域,重新选择EC1(P20-N107)片段作为靶蛋白。重新构建原核表达载体pGEX-4T2-CRFR1 EC1(P20-N107),IPTG低温诱导GST-EC188蛋白的表达。将超声裂解后的上清液用GSTrap FF纯化柱纯化,SDS -PAGE分析鉴定纯化结果,最终获得高纯度的GST-EC188靶蛋白。3.亲和肽的筛选:从Ph.D.-12和Ph.D.-C7C文库中筛选与靶蛋白结合的噬菌体克隆。将靶蛋白GST-EC188与GST固定到固相载体上,Ph.D.-12和Ph.D.-C7C文库先与GST蛋白孵育,将未与GST结合的噬菌体再与靶蛋白结合,洗掉非特异性结合的噬菌体,将特异性结合的噬菌体洗脱下来进行扩增,用于下一轮的筛选,筛选三轮。通过降低靶蛋白的浓度、增加洗脱强度、减少噬菌体与靶蛋白的孵育时间等措施优化筛选条件,筛选到结合力强的噬菌体克隆。用回收率%(回收滴度/投入滴×100)表示噬菌体的富集程度,Ph.D.-12文库第三轮的回收率%(4.2×10-3)是第一轮回收率%(5×10-5)的84倍;Ph.D.-C7C文库第三轮的回收率%(2.5×10-3)是第一轮回收率%(1.5×10-5)的167倍。结果证明经过三轮筛选,与靶蛋白特异性结合的噬菌体克隆得到富集。用ELISA法鉴定噬菌体克隆与靶蛋白的结合力,得到12个阳性克隆。用竞争性ELISA进一步验证亲和力,12个阳性克隆与CRFR1的结合表现为浓度依赖性。分别为Ph.D.-C7C文库1、2、6、7、9、12、14号克隆,Ph.D.-12文库2、5、14、15、16号克隆。分析阳性噬菌体展示肽的氨基酸序列。发现12个噬菌体展示肽均与CRF有一定的同源性,其中Ph.D.-C7C文库P2、P7、P14,Ph.D.-12文库P2、P15、P16与CRF的同源性最高,并且结合力较强。4. HEK293-CRFR1稳定表达细胞株的建立:为鉴定筛选到的小肽的生物活性,构建了HEK293-CRFR1稳定表达细胞株。Western blotting与RT-PCR结果显示CRFR1蛋白表达,以3号克隆表达量高;荧光免疫结果显示CRFR1表达于细胞膜上。CRF刺激HEK293-CRFR1细胞释放cAMP的实验结果说明CRFR1不但稳定表达于HEK293细胞,并且具有功能(EC50 = (8.44±0.68)×10-9 M,n=3)。5.亲和肽功能验证:初步验证12个阳性噬菌体展示肽对CRFR1功能的影响。利用原核表达载体pGEX-4T2,诱导表达与纯化融合表达蛋白GST-小肽,得到高纯度的GST-小肽。用cAMP释放试验验证GST-小肽对CRF刺激HEK293-CRFR1细胞释放cAMP的影响。结果显示,环七肽1、2、6、9、14号小肽,十二肽5、15、16号小肽表现不同程度的抑制作用,其中以十二肽P16抑制作用最强。GST蛋白可能会影响小肽与CRFR1的结合与作用,因此化学合成P16,进一步验证其功能。6. P16拮抗效应及机制研究:6.1验证P16对CRFR1的特异性抑制作用。用CRF刺激HEK293-CRFR1细胞释放cAMP,用P16拮抗此作用。结果显示在CRF(32 nM)存在的条件下,P16抑制CRF刺激HEK293-CRFR1细胞释放cAMP的作用,并且呈现浓度依赖性(IC50 = (1.36±0.60)×10-10 M,n=3)。在没有CRF作用下,不同浓度P16(5 pM~10 nM)对HEK293 -CRFR1细胞cAMP的基础水平没有影响。说明P16特异性地抑制CRFR1的功能。6.2验证P16对CRFR2的选择性。CRFR2选择性激动剂UCN3刺激瞬时转染HE-K293-CRFR2细胞释放cAMP量效曲线拟合良好(EC50 = (3.65±0.25)×10-8 M , n= 3),说明CRFR2表达于HEK293细胞中,并且具有功能。用UCN3(144 nM)刺激瞬时转染HEK293-CRFR2细胞释放cAMP,同时加入P16测定对HEK293-CRFR2细胞释放cAMP的影响。结果显示不同浓度P16(1 pM~10μM)对UCN3刺激HEK293 -CRFR2细胞释放cAMP没有影响。说明P16对CRFR2的功能没有影响。6.3验证P16对CRF刺激HEK293-CRFR1细胞释放cAMP量效曲线的影响。结果显示没有P16存在时,CRF量效曲线EC50 = (7.22±0.42)×10-8 M(n=3) ;在0.1 nM P16存在时,CRF量效曲线平行右移,EC50 = (1.10±0.25)×10-7 M (n=3) ;在1 nM P16存在时,CRF标准曲线平行右移幅度大于0.1 nM P16组, EC50=(1.50±0.38)×10-7 M(n=3)。说明P16竞争性地抑制CRF刺激HEK293-CRFR1细胞释放cAMP,P16是CRFR1的竞争性抑制剂。综上所述,本研究建立在可溶性表达了CRFR1重要的结合区域EC1(P20-N107)的基础上,以EC1(P20-N107)为靶蛋白,从Ph.D.-12和Ph.D.-C7C文库中筛选到12个与CRFR1结合强的噬菌体克隆,初步验证小肽对CRFR1的作用,其中P16抑制活性最高。通过观察P16对稳定表达CRFR1的HEK293细胞的作用,发现P16以竞争性抑制的方式,选择性拮抗CRFR1功能,而对CRFR2功能没有明显影响。本研究工作利用噬菌体展示技术筛选到CRFR1特异性竞争性拮抗剂P16等小肽,为靶向CRFR1抗抑郁症小肽类药物的研发奠定了基础。更多还原

【Abstract】 Corticotropin-releasing factor (CRF) is a 41-amino acid neuropeptide, originally isolated by Rivier and colleagues. CRF plays a key role in the coordination of neuroendocrine, autonomic, and behavioral responses to stress. In central nervous system, CRF is synthesized and released from parvocellular neurons of the paraventricular nucleus (PVN), bound to CRF receptors (CRFR) locate in anterior pituitary to activate the hypothalamus–pituitary–adrenocortical (HPA) system. CRF and CRF related peptides Urocortin I (UCN1), stresscopin-related peptide (UCN2) and stresscopin (UCN3) act mainly through CRF1 receptors (CRFR1) and CRF2 receptors (CRFR2). The human/rat CRF (h/rCRF) has 15 fold higher affinity for the CRFR1 than the CRFR2. CRFR1 and CRFR2 belong to the class B1 group of the G protein-coupled receptor (GPCR) superfamily. Both receptor subtypes are coupled to the same Gαproteins and signal through similar second messengers, such as the adenylyl cyclase-protein kinase A pathway by coupling with Gs and the phospholipase C-protein kinase C pathway by coupling with Gq.Numerous studies suggest that CRFR1 receptors become the drug target for depression and anxiety. Clinical studies find high levels of CRF in the cerebrospinal fluid of depressed patients. Several selective CRFR1 nonpeptidic antagonists have been demonstrated antidepressant like efficacy in animal models. Mouse mutants lacking CRFR1, showed less stress-induced anxiety-like behavior compared with wild type animals. Since 1991, a large number of small molecule CRFR1 antagonists have been developed. Clinical datas of NBI-30775/RS121919 and NBI-34041 were published. Howere, the clinic research has been ended due to adverse side effects such as hepatotoxicity. There is no CRFR1 antagonist is on market so far. Small peptide drugs present a lot of advantages, such as mild side effects, high activity with low dose, generally non-immunogenic. Peptide drug development has gained more and more attention. The aim of this paper is to identify novel CRFR1 peptide antagonists from phage display library.CRF1 receptor’s EC1 domain has no receptor activation, but it plays a key role in peptide ligand binding. The Gln43-Asn90 domain constituted the CRF1 receptor binding pocket, Arg76-Ala89 domain formed the CRF1 receptor ligand binding selectivity domain. So the eighty eight amino acids (Pro20–Asn107) of CRFR1 were selected for panning small peptides from Ph.D.-12 and Ph.D.-C7C. After three rounds panning, the binding positive phages were enriched. The coefficient of recovery of the Ph.D.-12 and Ph.D.-C7C increased approximately 84-fold and 167-fold respectively. Twelve phages were shown binding positive in dose dependent manner by ELISA. Twelve polypeptides share sequence homology with CRF, No2, No7, No14 of Ph.D.-C7C and No2, No15, No16 of Ph.D.-12 shared higher homology with CRF. The affinity of six phages for CRFR1 was higher than other phages, which shared lower homology with CRF.We successfully constructed two cell strains which stably expressed pcDNA3.1-hCRFR1 and pcDNA3.1 in HEK293 cells respectively. Twelve polypeptide fragments were expressed and purified with the pronucleus expression vector pGEX-4T2. Twelve coexpression polypeptides could attenuate cAMP accumulation of HEK293-CRFR1 cells not HEK293-pcDNA3.1 cells induced by CRF (32 nM) in different degree. P16 was chemically synthesized due to higher inhibition. P16 shown strong inhibition of the cAMP formation of HEK293-hCRFR1 cells concentration -dependently induced by CRF (32 nM), but had no influence in the cAMP formation of HEK293-hCRFR1 at the absence of CRF. P16 could not inhibit the cAMP accumulation while HEK293-hCRFR2 cells were exposed to UCN3, the CRFR2 selective agonist. The cAMP accumulation concentration-responsive curve of CRF shifted right at the presence of P16 (0.1nM, 1nM), which suggested that P16 was the competitive antagonist of CRFR1. It is implied that P16 specifically bound to CRFR1, but not CRFR2, and competitively inhibited CRFR1 activation. We conclude that P16 is a selective and competitive antagonist of CRFR1. It has the potential to become a new and high activity antidepressant and antianxiety drug with little side effects.更多还原