【作者】 李翀；

【导师】 陆伟跃； Wuyuan Lu； 魏刚；

【作者基本信息】 复旦大学 ， 药剂学， 2010， 博士

【摘要】 生物体内蛋白质之间的相互作用在生命过程中起着至关重要的作用。据此设计和制备抑制剂的策略,目前已在多种疾病的干预性治疗中得到验证。抑瘤蛋白p53及其负性调节蛋白MDM2之间的相互作用与众多肿瘤发生、发展密切相关。通过抑制p53与MDM2的结合,可恢复p53抑瘤活性而达到抗肿瘤效果。据保守估计,至少25%的肿瘤患者有望经此类策略治疗而受益,因此p53-MDM2抑制剂具有成为今后主要抗肿瘤药物的潜力。p53-MDM2抑制剂主要分为小分子化合物和多肽两大类,其中多肽与小分子化合物相比,具有高亲合力和高选择性。但目前针对p53-MDM2的多肽抑制剂研究仅停留在分子水平,主要原因在于：1)生理环境下多肽稳定性差,易被酶降解而丧失活性；2)靶点位于细胞内,一般多肽不具备入胞能力,无法实现其药效。为此,我们以此作为切入点,设计和制备稳定性好、对p53-MDM2抑制能力强的多肽,并采用主动靶向递药策略,实现肿瘤细胞内的多肽递送,发挥其抗肿瘤作用。本论文分为两大部分介绍。第一部分主要开展了抑制p53与MDM2结合的多肽设计、制备和表征。采用蛋白“嫁接”策略,以能够与MDM2蛋白特异性结合的序列为模拟对象,选用具有三维结构的微型蛋白毒素为模板,通过关键位点的“嫁接”,以期得到稳定性好、对p53-MDM2抑制能力强的多肽。首先,以天然p53序列为模拟对象,以蝎毒毒素BmBKTx1为模板,将p53上结合MDM2的关键位点“嫁接”到蝎毒微型蛋白上,经分子水平表征,设计产物stoppin-1能特异性结合MDM2,具有抑制p53与MDM2结合的能力；借鉴阳离子穿膜肽入胞原理,设计具有穿膜能力的微型蛋白stoppin-2,经细胞水平表征,stoppin-2可通过抑制p53与MDM2结合而选择性杀伤肿瘤细胞；稳定性实验显示,与直链短肽(17-28)p53相比,stoppin-2在近似生理环境下的稳定性大大增强。结果提示,以微型蛋白毒素为模板设计p53-MDM2多肽抑制剂的策略是可行的。其次,开展了p53-MDM2多肽抑制剂的优化设计,选用具有更强MDM2结合能力的PMI序列为模拟对象,以结构更简洁、制备更容易的蜂毒明肽apamin为模板,通过丙氨酸扫描和端基截取技术获取PMI的关键位点和区域信息,进行蛋白“嫁接”优化,获得抑制p53与MDM2结合的微型蛋白stingin。表征结果表明,stingin结合MDM2能力强(Kd=17nM),具有制备容易、产率高、应用前景好的特点。最后,根据镜像噬菌体展示技术筛选结果,制得了全D型的p53-MDM2多肽抑制剂D-PMIα并进行了表征。D-PMIα结合MDM2的能力(Kd=200nM)尽管低于stingin,但因其分子全部由D型氨基酸构成,其稳定性大幅提高,具有深入研究价值。第二部分主要开展了p53-MDM2多肽抑制剂的肿瘤细胞靶向递送和抗肿瘤药效研究。选用能与肿瘤细胞表面整合素ανβ3特异性结合的RGD序列多肽,并修饰于脂质体给药系统表面,将p53-MDM2多肽抑制剂递送至体内外肿瘤细胞内,以期发挥其抗肿瘤效果,提升多肽的应用价值。首先,制备了功能性脂质膜材料c(RGDDYK)-PEG3400-DSPE,采用逆向蒸发法构建了包载stingin-5(L型多肽)和D-PMIα(D型多肽)的c(RGDDYK)-脂质体给药系统。结果表明,c(RGDDYK)-脂质体能有效包载多肽,该脂质体给药系统平均粒径为70-90nm,包封率约30%,可用于后续药物递送研究。其次,以人脑胶质瘤U87为模型,对多肽经递送入胞后的体内外抗肿瘤活性进行评价。细胞水平试验结果表明,stingin-5抑制肿瘤细胞生长活性(IC50≈4.5μM)与阳性对照Nutlin-3 (IC50≈4.0μM)相当,D-PMIα(IC50≈1.9μM)明显强于Nutlin-3,提示c(RGDDYK)-脂质体能有效递送多肽入胞并发挥其抗肿瘤活性,D型多肽D-PMIα综合抑制肿瘤生长效果好于L型。以U87皮下移植瘤和脑部原位肿瘤为模型,选用D型多肽D-PMIα进行体内靶向递送药效评价。结果表明,c(RGDDYK)-脂质体-D-PMIα能显著抑制皮下移植瘤生长、延长脑部原位肿瘤裸小鼠的生存时间。细胞和体内水平均验证了多肽通过抑制p53与MDM2结合、释放p53蛋白而发挥抗肿瘤作用。结果提示,通过主动靶向给药策略,可解决p53-MDM2多肽抑制剂在体内发挥抗肿瘤药效问题。最后,在上述研究工作基础上,以D-PMIα经优化得到的的D-PMIβ为研究对象,在多肽N端修饰脂肪酸以提高其脂溶性,采用薄膜水化法构建了包载该多肽的c(RGDDYK)-脂质体,并进行了体内外药效评价和作用机制探讨。结果表明,多肽脂肪酸化后有利于脂质体包载,且载药量高、稳定性好、制备工艺简单,体内外抗肿瘤药效良好,具有较好的应用前景。综上所述,本文通过多学科的研究手段,设计和制备了抗肿瘤多肽,尤其是D型多肽,解决了多肽药物稳定性问题、提高了其抑制p53与MDM2结合的活性；借助主动靶向递送策略,解决了该类多肽进入细胞的问题,使其在体内外表现出良好的抗肿瘤药效和较理想的治疗指数。高效低毒是抗肿瘤药物研究的长远目标,本研究工作可能为抗肿瘤药物研发带来有益的启示。更多还原

【Abstract】 Protein-protein interactions play a crucial role in biological processes, representing important therapeutic targets for disease intervention. The interaction between the tumor suppressor protein p53 and its negative regulator MDM2 promotes tumorgenesis, thus providing a promising anti-cancer strategy through antagonizing MDM2 to re-activate the p53 pathway. It was estimated that at least 25% of human cancers could be treated by inhibitors of p53-MDM2 binding, which might be the main drugs of tomorrow for cancer therapy therefore. Since protein-protein interfaces tend to be relatively large, potent and selective inhibition necessitates the use of peptide inhibitors rather than low molecular weight compounds. However, most peptide inhibitors of the p53-MDM2 interaction suffer two limitations:1) poor stability in physiological environments and 2) inability to traverse the cell membrane. Our study aims to design stable, high-affinity peptide inhibitors of the p53-MDM2 interaction that are capable of traversing the cell membrane either actively or passively to achieve specific antitumor effects in vitro and in vivo.Our work is divided into two parts. Part one entails the design, preparation, and characterization of three different classes of peptide inhibitors of the p53-MDM2 interaction,which are based on (1) scorpion toxin BmBKTx1 of xx amino acid residues with three disulfides, (2) bee venom toxin apamin of yy amino acid residues with two disulfides, and (3) duodecimal D-peptides, respectively.First, the natural (17-28)p53 sequence was chosen as a donor and scorpion toxin BmBKTx1 as a scaffold. After grafting key residues of p53 to the scorpion toxin, the resultant product, termed stoppin-1, specifically bound MDM2. Inspired by cationic cell-permeable peptides, we re-engineered stoppin-1 by replacing five residues near its C terminus with arginines, creating a second-generation inhibitor capable of permeabilizing the cell membrane (stoppin-2). Stoppin-2 showed p53-dependent tumor-killing activity with enhanced proteolytic stability in cell media. These results illustrated the feasibility of using peptide toxins as scaffold in general to design miniature protein inhibitors of the p53-MDM2 interaction with antitumor activity.Second, we further optimized the miniprotein design approach by using a phage-selected, high-affinity MDM2-binding peptide termed PMI as a donor and apamin as a template with improved properties. A systematic mutational analysis of PMI was also performed to elucidate the molecular basis of peptide inhibition of the p53-MDM2 interaction. Grafting xx critical residues of PMI to apamin resulted in several potent miniprotein inhibitors, termed stingins, that directly competed with p53 for MDM2 binding at low nanomolar affinities as verified by X-ray crystallography and biochemical and biophysical studies.Third, a duodecimal D-peptide inhibitor of the p53-MDM2 interaction, termed D-PMIa, was designed based on mirror image phage screening. Structural and functional studies validated D-PMIa as a competitive inhibitor of p53 binding to MDM2 with a binding affinity for MDM2 of 200 nM. D-PMIa is a promising candidate for therapeutic development as it is fully resistant to proteolytic degradation.The second part of the thesis describes targeted delivery and functional evaluation of peptide inhibitors of the p53-MDM2 interaction in vitro and in vivo. An integrin-binding RGD peptide ligand was used as a tumor-targeting moiety; RGD-decorated liposomes were constructed to encapsulate peptide inhibitors to achieve their tumor-specific intracellular delivery.First, functionalized membrane materials c(RGDDYK)-PEG34oo-DSPE were synthesized, followed by the preparation of peptide-loaded liposomes through the reverse evaporation method. Two liposomal delivery systems were constructed, encapsulating stingin-5 (L-peptide inhibitor) and D-PMIα(D-peptide inhibitor), respectively. The mean particle sizes of peptide-loaded liposomes were 70-90 nm with a-30% encapsulation efficiency, indicative of an efficient encapsulation of the peptide inhibitors.Second, the antiumor efficacy of liposomal peptides, after intracellular delivery, were evaluated using human glioblastoma U87 cell line. The L-peptide (stingin-5) killed tumor cells with an IC50 value of 4.5μM, simialr in potency to the small-molecule positive control Nutlin-3 (IC50≈4.0μM). By contrast, D-PMIa was more active, displaying an IC50 value of 1.9μM. Further, Western blot analysis indicated that the peptide inhibitors induced tumor cell death by activating the p53 pathway. Collectively, these results demonstrate the efficiency of intracellular delivery of peptide inhibitors using the RGD-coated liposomal vehicle.Then the antitumor activity of c(RGDDYK)-liposomal D-PMIαwas evaluated in vivo. Administered intravenously, this formulation showed strong inhibitory activity against tumor growth in a subcutaneous U87 glioblastoma-bearing nude mice model, and significantly prolonged the survival of mice with intracranial glioblastoma. Our work demonstrates antitumor therapeutic efficacy of D-PMIa in combination with a targeted delivery vehicle, thus validating D-peptide activation of the p53 pathway as a promising therapeutic paradigm for the treatment of malignant neoplasms.Finally, the D-peptide inhibitor D-PMIβwas N-terminally palmitylated and then encapsulated by c(RGDDYK)-PEG-liposome through thin film hydration method. This improved formulation showed high encapsulation efficiency and significant antitumor efficacy both in vitro and in vivo with low toxicity, thus might be a potential therapeutic strategy for treating according tumors.In conclusion, we have combined contemporary synthetic protein chemistry, phage display, structural biology, cancer biology, drug delivery, and various biochemical and biophysical techniques, and showcased a powerful, innovative, and integrated approach to drug discovery of a novel class of p53 activators for cancer therapy. This work may ultimately lead to the addition of new weapons to the existing anticancer arsenal, and will broadly impact the development of D-peptide therapeutics for targeted molecular therapy of a great variety of human diseases.更多还原