【作者】 张鹏；

【导师】 梁宋平； 刘中华；

【作者基本信息】 湖南师范大学 ， 生物化学与分子生物学， 2020， 博士

【摘要】 癌症是威胁人类健康的主要疾病之一,虽然癌症的临床治疗已取得了巨大进步,但高效专一、低毒且不易产生耐药性的抗癌药物研发依然具有重大临床需求。作为国际抗癌药物研发的重要组成部分,抗癌肽（ACPs）因广谱抗癌性、作用迅速、不易产生耐药性和设计灵活等特点,具备开发成优质抗癌药物的潜能,因而获得科学界和制药界的广泛关注。然而,ACPs靶向性不强、活性和选择性有待提高等局限性限制了它们在临床上的应用。狼蛛抗癌活性肽Lycosin-I是本实验室从新疆穴居狼蛛毒液中鉴定到的具有23个氨基酸残基的双亲性α-螺旋肽,大量的前期研究表明该多肽具有较好的抗癌多肽药物研发潜力,本研究利用糖基化和氨基酸替换的策略对Lycosin-I进行修饰与改构,以期进一步提高其靶向性、活性和选择性,为将其开发成为一个高效低毒的抗癌药物奠定基础。首先,根据葡萄糖转运体在癌细胞膜上高表达的特点,采用糖基化修饰策略,本研究设计了一系列单糖修饰多肽。前期研究表明将Lycosin-I氨基酸序列中的赖氨酸残基替换为精氨酸残基得到的多肽R-lycosin-Ⅰ具有更高的抗癌活性,因此本研究涉及的糖基化修饰以R-lycosin-Ⅰ为模板予以开展。葡萄糖、半乳糖、氨基葡萄糖、阿拉伯糖和甘露糖衍生物通过戊二酸酐分别与R-lycosin-Ⅰ的N端共价偶联,成功制备出五种高纯度的糖肽。多肽糖基化修饰的前阶段工作表明,相比R-lycosin-Ⅰ,仅葡萄糖肽R-Glc同时表现出细胞毒活性和选择性显著提升,因此本研究以R-Glc为对象开展系统性研究。R-Glc能弥散性分布于A549细胞质中,在细胞核内呈现团状聚集,表现出更强的细胞摄取性能。根据设计原理,利用GLUT1的抑制剂与蛋白敲降实验表明,葡萄糖单元更有利于R-Glc和葡萄糖转运体高表达的A549细胞结合,被摄取的效率提高,而与HEK 293T细胞结合较弱,使得R-Glc对癌细胞作用增强,对非癌细胞作用显著减弱。在诱导细胞死亡机制研究中,R-Glc作用细胞后使其形态破碎,胞体干瘪且促使大量LDH释放,表明其具有膜裂解活性;除此之外,R-Glc与线粒体有共定位,能导致线粒体膜电位下降和通透性增加,从而释放细胞色素C,激活Caspase-3诱导细胞凋亡。在对A549肿瘤球体测试中,R-Glc具有更强的肿瘤球体穿透性且诱导球体死亡的能力更强。尾静脉注射FITC-R-Glc后,瘤体中的荧光显著增强,R-Glc肿瘤靶向效能提升。通过瘤体给药的方式,3 mg/kg的R-Glc能显著抑制裸鼠瘤体增长,与R-lycosin-Ⅰ不同,腹腔注射9 mg/kg的R-Glc亦能显著减少裸鼠瘤体体积,诱导大量瘤体细胞凋亡。给药治疗过程对裸鼠的体重无明显影响,对裸鼠主要脏器也无明显毒性。其次,根据肿瘤组织呈现弱酸性微环境（p H=6.5-6.8）的特点,通过对Lycosin-I进行氨基酸替换改构,引入组氨酸,减少带负电荷氨基酸和优化多肽α-螺旋轮结构,从而设计和表征了一个具有选择性作用的酸响应抗癌活性肽-p HTP-1。经培养基优化后,p HTP-1在p H=6.0、6.6和7.4的培养条件下,作用A549细胞的IC₅₀值分别为9、37和大于200μM。对p HTP-1进行丙氨酸扫描分析表明,共有18处氨基酸位点对多肽活性与选择性产生影响,其中突变体I10A在p H=6.6和7.4下,对A549作用的IC₅₀值约为15和大于200μM,将之命名为p HTP-2。由于p HTP-2在近乎肿瘤弱酸性环境（p H=6.6）下细胞毒活性最强,因此以其为对象做系统性生物活性测试,表明其作用癌细胞具有时间与浓度依赖性、广谱性和p H转换性。在p H=6.6下,p HTP-2以直接膜裂解的方式诱导细胞死亡,在细胞质内呈现弥散性分布,在细胞核中也有富集。由于在中性环境下的弱活性,p HTP-2的半溶血浓度在500μM以上,其对分离的乳鼠心肌细胞作用的IC₅₀值也大于500μM。为深入阐明p HTP-2高选择性酸响应机制,我们对其进行了zeta电势、粒径和二级结构表征,将其解释如下:在6.6条件下,序列中的组氨酸发生质子化,增加了多肽的正电荷,多肽间排斥作用增强导致粒径减少且在PBS与SDS中分别呈现无规则卷曲与α-螺旋构象,上述参数有利于多肽与膜结合并插入膜中发挥膜裂解活性。在7.4条件下,多肽组氨酸质子化减弱,正电荷减少,在中性环境下呈现β-折叠构象,分子间的氢键和疏水作用力使得多肽发生自组装聚集,削弱了多肽与膜的结合力与作用力,表现出弱活性。在体内抗癌研究中,尾静脉注射20 mg/kg的p HTP-2对黑色素瘤体具有显著性抑制作用,给药过程对小鼠的体重无明显影响,表明其毒副作用较低。最后,考虑到临床前癌症模型是连接前景药物分子开发与临床治疗的重要桥梁,对药物发掘、药效评判和药理研究具有重要作用,我们尝试建立肿瘤类器官模型。近年来,相对于肿瘤细胞系,越来越多研究表明类器官模型与肿瘤组织具有较好贴合性与真实性。癌症研究模型以细胞系-3D细胞系肿瘤球体-病人肿瘤类器官不断深入。我们在成功构建了小鼠小肠与结肠类器官的基础上,通过组织分离,选择性添加因子和三维培养成功构建了人结肠与结肠癌类器官。人结肠类器官呈囊性致密体结构,能增殖分化出隐窝与绒毛结构域。人结肠癌类器官呈致密无规则球体,具有与肿瘤组织相似的组织学形态,能进行结肠癌类器官生物库的构建,经冻存与复苏后,结肠癌类器官仍生长状态良好。利用结肠癌类器官,我们开展了抗癌肽的初步筛选和探索。结肠癌类器官模型的成功构建为本实验室开展抗癌活性肽的筛选和分子作用机制研究提供了一种新方法,将能极大提升本研究室抗癌活性肽的研究效率和水平。综上所述,糖基化修饰提升了R-lycosin-Ⅰ的选择性与肿瘤靶向性,这种效能的提升是由葡萄糖转运体介导的;氨基酸替换策略赋予Lycosin-I酸响应特性,极大的提升了多肽的选择性且具有显著的体内抗癌活性;系统性类器官模型的构建,丰富了我们对临床前癌症模型的认识,促进了抗癌药物精准性研发。这些修饰与改构策略促进了Lycosin-I的成药性也为类似抗癌肽的优化提供了有力支撑。更多还原

【Abstract】 Cancer is one of the major diseases that threaten human health.Despite substantial progress in the clinical treatment of cancer,the development of high-efficiency,specific and low-toxic anti-cancer drugs still have significant clinical needs.As an important part of anti-cancer drugs development,anticancer peptides（ACPs）have the potential to develop into high-quality anticancer drugs due to their rapid broad-spectrum anticancer activity,low tolerance and flexible design properties.Therefore,they have received widespread attention from the scientific and pharmaceutical communities.However,the clinical application of most ACPs is limited due to the narrow therapeutic index and low tumor targeting.The ACP Lycosin-I is an amphiphilicα-helix peptide which identified from the venom of Lycosa singoriensis.Our previous study indicated Lycosin-I have potential for the drug development.In this study,glycosylation and amino acid substitution strategies were performed to improve the anticancer activity,selectivity and tumor targeting of Lycosin-I,which lays the foundation for its development into a highly effective and low toxicity anticancer drug.Firstly,based on the high expression of glucose transporters in cancer cells,and using glycosylation modification strategies,a series of glycopeptides were designed.Previous studies have shown that R-lycosin-Ⅰ which was obtained by replacing the lysine in the sequence of Lycosin-I with arginine has higher anticancer performance.Therefore,we choose the R-lycosin-Ⅰ as a template in glycosylation modification study.Glucose,galactose,glucosamine,arabinose,and mannose derivatives were covalently coupled to the N-terminus of R-lycosin-Ⅰ through glutaric anhydride,which yield 5 high-purity glycopeptides.Only the glucose modified peptide（R-Glc）showed significant improvement in cytotoxicity and selectivity.R-Glc can be diffusely distributed in the A549 cytoplasm,presenting clusters in the nucleus and showing stronger cell uptake ability.According to design principle,the GLUT1 inhibitors and protein knockdown experiments were performed which showed that GLUT1 with high expression in A549 cells was approved to be involved in the cytotoxicity and selectivity enhancement of R-Glc.In the mechanism,R-Glc can disrupt the morphology of cells and promote the release of LDH,indicating that it has membrane lytic activity;R-Glc also can co-localization with mitochondria,decrease mitochondrial membrane potential,increase mitochondrial membrane permeability and release cytochrome C,which caused caspase-3 activation and then promoted cell apoptosis.In the A549 tumor spheroids,R-Glc demonstrated stronger tumor spheroid penetrability and cytotoxicity than that of R-lycosin-Ⅰ.Compared to R-lycosin-Ⅰ,the lung tumor xenograft model of nude mice injected with FITC-R-Glc showed strong fluorescent signals around tumor.By the way of intratumoral injection,3 mg/kg of R-Glc could significantly inhibit nude mice tumor growth.Unlike R-lycosin-Ⅰ,intraperitoneal injection of 9 mg/kg of R-Glc can also significantly reduce the tumor volume and induce numerous tumor cell apoptosis.The administration process had no significant effect on body weight and had no obvious toxicity to the main organs of nude mice.Secondly,according to the p H characteristics of the tumor microenvironment（p H=6.5-6.8）,amino acid substitution strategy was performed to reconstructe the Lycosin-I,which yeild a p H-triggered peptide p HTP-1.Under the culture conditions of p H=6.0,6.6 and 7.4,the IC₅₀values of p HTP-1 against A549 cells were 9,37 and>200μM,respectively.Alanine scanning analysis of p HTP-1 demonstrated that the I10A mutant had the highest cytotoxicity at p H=6.6 and named it p HTP-2.At p H=6.0 and 7.4,the IC₅₀values of p HTP-2 against A549 cells were 15and>200μM,respectively.Due to the best performance,p HTP-2 was choose as a research object in the following experiment.Systematic biological activity tests indicated that p HTP-2 had broad-spectrum,time-concentration dependent and p H conversion in treating cancer cells.In addition,p HTP-2 induced cell death by direct membrane lysis.Due to its weak cytotoxicity at 7.4,the HC₅₀value against erythrocyte and IC₅₀value against cardiomyocytes of p HTP-2 were all above 500μM.In order to expound the highly selective mechanism of p HTP-2,we performed a series of physicochemical parameters tests including zeta potential,particle size and secondary structure and parsed the mechanism as follows:At 6.6,histidines in the p HTP-2 sequence were protonated,which increased the positive charge of the peptide,strengthened the peptides repulsion and decreased the peptide particle size.In PBS and SDS,p HTP-2 presented random coil andα-helix conformations,respectively.Thus,the increased positive charge andα-helix conformation of peptide enhanced its membrane binding and insertion ability,which exerted membrane lysis.At 7.4,p HTP-2 demonstrated weakly protonation andβ-sheet conformation in PBS,which caused peptides to aggregate though intermolecular hydrogen bonding and hydrophobic interaction.The state of aggregation decreased the binding and interaction ability between peptide and cell membrane,showing low cytotoxicity.In the in vivo anticancer study,tail vein injection of 20mg/kg of p HTP-2 showed a significant inhibitory effect on melanoma,and the administration process had no significant effect on the body weight of mice,indicating that p HTP-2 had low side effects.Finally,considering that the preclinical cancer model is an important bridge connecting prospective drug development and clinical treatment,and has important effects on drug discovery,pharmacodynamic evaluation and pharmacological research,we try to establish a tumor organoid model.In recent years,compared to cell lines,more and more studies have shown that the authenticity between organoid models and tumor tissues.Current cancer research models develop in the order of cell lines-3D cell line spheroids-organoids.Based on the successful construction of mouse small intestine and colon organoids,we successfully constructed human colon and colon cancer organoids through tissue separation,selective addition of factors and three-dimensional culture.Human colonic organoids demonstrated a cystic structure that can proliferate and differentiate into crypt and villous domains.Human colon cancer organoids showed dense and irregular spheroids and the histological morphology was similar to that of tumor tissues.After cryopreservation and then resuscitation,colon cancer organoids still grow well.Using colon cancer organoids,we performed a preliminary screening and exploration of ACPs in animal venom.The successful construction of a colon cancer organoid model provides a new method for our laboratory,which will greatly improve the efficiency and level of ACPs research.In summary,the glycosylation modification enhanced the selectivity,cytotoxicity and tumor targeting of R-lycosin-Ⅰ and this improvement was mediated by glucose transporter;the amino acid substitution strategy endowed Lycosin-I with p H triggered characteristic,which greatly enhanced the peptide selectivity and also had significant anticancer activity in vivo.The systemic estabalishment of organoid model has enriched our understanding of preclinical cancer models and promoted the precise development of anticancer drugs.Those peptide design strategies promote the druggability of Lycosin-I and also provide support for the ACPs optimization.更多还原