基于SNAP-tag-EGFR细胞膜色谱的北豆根中潜在抗肿瘤活性组分筛选及验证  

Screening and validation of anti-tumor active components from Rhizoma Menispermi based on SNAP-tag-EGFR cell membrane chromatography

作　　者：付佳[1,2] 贾倩倩 韩省力 贺浪冲[1] FU Jia;JIA Qianqian;HAN Shengli;HE Langchong(School of Pharmacy,Xi'an Jiaotong University,Xi'an 710061,China;Institute of Basic&Translational Medicine,Xi'an Medical University,Xi'an 710021,China)

机构地区：[1]西安交通大学药学院,西安710061 [2]西安医学院基础与转化医学研究所,西安710021

出　　处：《西北药学杂志》2023年第1期1-6,共6页Northwest Pharmaceutical Journal

基　　金：国家自然科学基金重点项目(编号:81930096);国家自然科学基金面上项目(编号:81973278)。

摘　　要：目的从北豆根中筛选与表皮生长因子受体(epidermal growth factor receptor,EGFR)作用的活性成分,为抗肿瘤药物研究提供新的先导化合物。方法构建SNAP-tag-EGFR细胞膜色谱模型,将其与高效液相色谱-离子阱-飞行时间质谱构成二维联用系统,靶向筛选北豆根中EGFR的潜在抗肿瘤活性组分。用前沿分析和计算机模拟分子对接研究潜在活性组分与EGFR的相互作用。并用细胞增殖抑制实验初步验证潜在抗肿瘤活性组分的药理作用。结果利用该二维联用系统从北豆根提取物中识别出目标组分蝙蝠葛苏林碱和蝙蝠葛碱。前沿分析测得蝙蝠葛苏林碱、蝙蝠葛碱与EGFR相互作用的平衡解离常数K_(D)值分别为2.48×10^(-6)、3.57×10^(-6)mol·L^(-1),表明蝙蝠葛苏林碱、蝙蝠葛碱与EGFR均存在一定强度的相互作用关系。分子对接实验表明蝙蝠葛碱和蝙蝠葛苏林碱均可结合于EGFR的活性腔,与EGFR的氨基酸残基形成氢键。细胞增殖抑制实验结果显示,蝙蝠葛碱和蝙蝠葛苏林碱在5~100μmol·L^(-1)范围内能够剂量依赖地抑制SNAP-tag-EGFR/人胚肾细胞293(human embryonic kidney 293,HEK293)的增殖,当给药浓度为50μmol·L^(-1)时,蝙蝠葛碱和蝙蝠葛苏林碱对SNAP-tag-EGFR/HEK293细胞生长的抑制率可达到80.59%±3.74%、73.70%±6.93%。结论SNAP-tag-EGFR/CMC模型和高效液相色谱-离子阱-飞行时间质谱联用为在天然产物中快速寻找先导化合物提供了平台,为中药活性组分的作用机制研究奠定了基础。Objective To screen the active components that interact with epidermal growth factor receptor(EGFR)from Rhizoma Menispermi,and to provide new lead compounds for the research of antineoplastic drugs.Methods The SNAP-tag-EGFR/cell membrane chromatography model was constructed,and combined with high performance liquid chromatography-ion trap-time of flight-mass spectrometry(HPLC-IT-TOF-MS)to form a two-dimensional system to screen the potential anti-tumor components in Rhizoma Menispermi.The interactions between potential active components and EGFR were studied by frontier analysis and molecular docking.Finally,the pharmacological effects of potential anti-tumor components were preliminarily verified by cell proliferation inhibition test.Results The two-dimensional system was used to identify the target components of daurisoline and dauricine from the extracts of Rhizoma Menispermi.Frontal analysis showed that the K_(D) values of the interaction of daurisoline and dauricine with EGFR were 2.48×10^(-6)mol·L^(-1)and 3.57×10^(-6)mol·L^(-1),respectively,indicating daurisoline and dauricine could interact with EGFR with a certain binding intensity.Molecular docking show that both daurisoline and dauricine matched the active pocket of EGFR well and form hydrogen bonds with the amino acid residues of EGFR.The results of cell proliferation inhibition test showed that daurisoline and dauricine had a dose-dependent inhibitory activity against SNAP-tag-EGFR/human embryonic kidney 293(HEK293)in the range of 5 to 100μmol·L^(-1).When the concentration was 50μmol·L^(-1),the inhibition rates of dauricine and daurisoline on SNAP-tag-EGFR/HEK293 cell growth were 80.59%±3.74%and 73.70%±6.93%,respectively.Conclusion The combination of SNAP-tag-EGFR/CMC model and HPLC-IT-TOF-MS provides a platform for rapid search for lead compounds in natural products and lays a foundation for the study of the mechanism of action of active components in traditional Chinese medicine.

关 键 词：北豆根 中药 细胞膜色谱 抗EGFR活性 

分 类 号：R917[医药卫生—药物分析学]