苯丙素类化合物作用鱼类单殖吸虫靶标研究

Based on the previous studies of the relation between phenylpropanoids and monogenea, this project is focused on finding the targets of the six highly active derivatives against Gyrodactylus kobayashii. Firstly, triethylene glycol and phenylpropanoids were used to synthesize thioctic connective intermediate compound through a series of chemical reactions such as condensation, substitution and esterification. Then gold nanoparticles were synthesized by reaction of gold acid chloride trihydrate and sodium borohydride. Furthermore, it will be electrostatic adsorption generated phenylpropanoids connected gold nanoprobes. Using the nanoprobes, the binded proteins of test compounds were pulled down from the cellular of G. kobayashii proteome through SDS-PAGE separation. The target proteins can be identified by MALDI-TOF/TOY mass spectrometry, then targets of phenylpropanoids will be validated by RNA interference and bioinformatics analysis. Based on above results, spectroscopic methods will be used to study interaction modes between phenylpropanoids and target proteins. All the research work will not only provide theoretical foundation for insecticidal mechanism study, but also be meaningful for acquiring clinical drug candidates with physicochemical property and pharmacological activity highly raised through next constructing computer aided drug design mode and purposeful modify and optimize the structure of phenylpropanoids, which lays the academic foundation important application prospect for creating fishery active pharmaceutical ingredient that possess patent of China.

本项目基于前期苯丙素类杀灭单殖吸虫构效关系研究基础上，以6种杀虫高活性苯丙素类衍生物、小林三代虫模型为材料进行作用靶标研究。首先以三乙二醇为起始，通过与苯丙素类衍生物进行缩合、取代、酯化等一系列化学反应得到硫辛酸类苯丙素化合物。然后将三水合氯金酸和硼氢化钠反应，进一步采用静电吸附将两者共价结合制备含苯丙素类衍生物的金纳米探针，并与小林三代虫裂解液孵育，经SDS-PAGE分离、MALDI-TOF/TOY 质谱进行靶蛋白鉴定、RNA干扰验证；在此基础上，通过光谱学方法研究苯丙素类化合物和靶蛋白的相互作用模式，为构建计算机辅助药物设计模型提供信息。本研究不仅为揭示杀虫机制提供理论依据，而且为有目标的结构改造和优化苯丙素类衍生物，获得理化性质、药理活性等均有大幅提升的临床候选药物，创制具有我国自主知识产权的渔用原料药，有着重要的科学理论意义和应用价值。

本项目基于前期研究基础，围绕如何进一步提高苯丙素类化合物的理化性质和生物活性问题，对该类化合物的分子靶标进行鉴定并对其相互作用开展研究。首先，以牛蒡子苷元为起始原料合成脱氧牛蒡子苷元，再分别经过化学修饰，制备金纳米阳性探针GNP-6与阴性探针GNP-10。通过透射电镜、傅里叶变换红外光谱和高效液相色谱等方法鉴定GNP-6和GNP-10，结果表明两者均成功构建且可用于下一步蛋白下拉试验。随后，通过亲和层析分离、质谱鉴定和秀丽隐杆线虫数据库分析结合蛋白，结果显示与探针结合的蛋白为myosin-2和肌肉M线装配蛋白。转录组学和iTRAQ蛋白定量结果发现1.85 mg/L的牛蒡子苷元在体作用小林三代虫4 h后，参与肌肉收缩的蛋白如肌钙蛋白、肌球蛋白、原肌球蛋白以及肌动蛋白等蛋白表达下调。qRT-PCR发现牛蒡子苷元处理组中myosin-2基因表达量显著下降。组学联合结果证明牛蒡子苷元作用单殖吸虫的靶点是myosin-2。最后，该项目通过三代转录测序得到myosin-2的cDNA全长序列，经昆虫细胞表达得到myosin-2马达区S1蛋白，荧光淬灭试验表明牛蒡子苷元与S1的是相互结合引发荧光淬灭。基于AlphaFlod2构建S1部分的3D结构，利用SYBYL软件进行牛蒡子苷元和myosin-2的分子对接，结果发现牛蒡子苷元的内酯环活性基团与myosin-2的50 kDa附近的疏水口袋结合，从而非竞争性抑制肌球蛋白Ⅱ型水解ATP，导致虫体能量供应不足最终死亡。本项目创新性地将蛋白拉下技术、组学联合分析和分子对接等方法深入研究药物作用单殖吸虫的分子机制，不仅有利于杀灭鱼类单殖吸虫药物的创新与发展，还对其他水产药物的研发提供新方法和新思路。