基于多肽与肿瘤细胞生物亲和的蟾酥大分子活性物质基础研究

Toad venom (chansu) is a common Chinese medicine in the treatment of tumor diseases. The therapeutic effect of toad venom is due to its major active ingredients, not only the bufadienolides but also peptides, secreted by parotid glands. Venom peptides have amphiphilic groups and specific amino acid sequences, which could interact with cancer cells in the modes of electrostatic attraction and receptor-ligand binding, to cause the modification on the surface of cancer cells via peptide self-aggregation. This bio-affinity makes the venom peptides exert anti-tumor and immunostimulatory actions. In this project, the anti-tumor peptides in toad venom (TVP) and their structure-activity relationship would be investigated. Firstly, effects of TVP on the tumor cell proliferation and level of Th1/Th2 cytokines would be examined in vivo and vitro models. Then, TVP would be divided into fractions for the next activity screening. Secondly, the active peptides would be determined by the cell/protein affinity purification-mass spectrometry, and be sequenced using De-novo transcriptomics based database search. Thirdly, the pure anti-tumor peptides would be obtained via bioassay guided isolation/purification, mass identification and chemical synthesis. Finally, the structure-activity relationship of TVP would be identified to reveal the anti-tumor material basis of toad venom. This study would benefit the quality control, refining and new drug development of toad venom.

蟾酥是临床常用抗肿瘤药物，除所含小分子甾烯外，来自腺体分泌的多肽在其中发挥重要作用。由于毒素多肽具水-脂两亲结构和特定氨基酸序列，能通过电荷吸引及受体介导方式与肿瘤细胞直接结合。这种亲和性使蟾酥多肽诱发抗肿瘤/免疫反应效应，构成蟾酥整体取效的一个独特方面。本项目采用药理评价和生化分离技术，对蟾酥抗肿瘤多肽进行鉴定与序列解析及构-效关系研究。工作路径：① 在整体/细胞水平，以抗肿瘤细胞增殖和促淋巴因子释放为指标，评价蟾酥总肽的效应，进一步筛选有效部位。② 建立亲和筛选模型，采用液相-质谱及转录组从头测序技术鉴定有效部位与肿瘤细胞/蛋白直接结合的蟾酥多肽分子。③ 开展目标导向的蟾酥多肽分离/纯化，合成制备及活性验证研究，总结分析序列结构与亲和性及抗肿瘤活性间关系。揭示蟾酥抗肿瘤的大分子物质基础，为药材/制剂质量控制、产品精制和新药开发提供科学依据。

毒肽是两栖动物化学防卫物质。研究表明具抗肿瘤、镇痛、免疫调节活性而备受重视。申请者在10余年对蟾酥研究中发现，除小分子甾烯外，毒肽是其抗肿瘤整体取效的重要部分。本项目基于转录组、肽组学和肿瘤细胞的亲和性筛选，鉴定了蟾酥多肽的序列。1）采用De-novo转录组测序建库、高分辨质谱和经典分子克隆验证技术，分别在mRNA转录、多肽表达和翻译后修饰水平，鉴定了多种类型蟾酥毒肽的序列。在国内外首次报道了蟾酥中高丰度蛋白和毒肽的氨基酸序列。2）完成了蟾酥毒肽组质谱鉴定方法的系统优化。考察不同提取溶剂、修饰处理、不同液相色谱系统、搜索引擎(Peaks和Maxquant)和不同数据库匹配对毒肽检测数目、质谱强度和方法重现性的影响。3）采用肿瘤细胞连续亲和质谱筛选方式，从313条高水平蟾酥毒肽中，鉴定76条肿瘤细胞亲和肽，汇总成18组有代表性毒肽组分，具有抗肿瘤细胞增殖活性，以及对小分子甾烯有促渗和协同增效作用。部分毒肽展现出体内抗肿瘤活性。肽荧光标记实验证明这些肽进入了肿瘤细胞内，分散于细胞质或聚集于细胞核、线粒体等细胞器。4）对毒肽序列同源性比对和分子对接研究，发现作用于肿瘤细胞离子通道TRPM8（冷敏通道）的CL4590来源多肽（在蟾酥毒肽中含量最高）以及蛋白 Histone2A源多肽等具有肿瘤活性作用。相关的研究结果对以揭示蟾酥毒肽抗肿瘤的物质基础，为蟾酥制剂的工艺精制、临床合理用药、质量控制以及创新肽类药物开发提供了科学依据