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基于SPR技术的表皮生长因子受体与新疆紫草中4种活性成分的相互作用

Interactions Between EGFR and Four Compounds from Arnebia euchroma Based on SPR
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摘要 目的:以人表皮生长因子受体(EGFR)为靶标,从左旋紫草素,乙酰紫草素,β,β-二甲基丙烯酰紫草素,β-乙酰氧基异戊酰紫草素等新疆紫草所含的4种柰醌类活性成分中筛选潜在的EGFR抑制剂。方法:利用表面等离子体共振(SPR)技术,首先通过预吸附实验,优化固定的p H和浓度,选择最佳条件,构筑EGFR生物芯片。在此基础上,以p H 7.4,浓度10 mmol·L-1的磷酸缓冲溶液(含0.005%聚山梨酯-20)作为相互作用时的缓冲溶液,实时动态研究EGFR与4种单体的相互作用。通过软件计算动力学参数,基于结合动力学常数大小,筛选抑制剂。结果:选择p H 4.5,10 mmol·L-1的乙酸缓冲液、质量分数为10 mg·L-1的EGFR为其最佳的固定条件,最终固定量为250 RU。相互作用结果显示4种单体中,β,β-二甲基丙烯酰紫草素与EGFR有明显的结合作用,其他3种单体无明确响应。动力学参数计算结果显示,EGFR与β,β-二甲基丙烯酰紫草素相互作用的结合速率常数ka为1.27×104L·mol(-1)·s(-1),解离速率常数kd为2.92×10-2s-1,解离平衡常数KD为2.31×10-6mol·L-1,卡方值(Chi2)为3.25。KD〈1×10-5mol·L-1,表明他们有较强的亲和力。结论:β,β-二甲基丙烯酰紫草素可能为EGFR的一种抑制剂。 Objective: To screen the potential inhibitor for human epidermal growth factor receptor( EGFR) from four kind compounds including shikonin,acetylshikonin,β-acetoxyisovalerylshikonin and β,β-dimethylacrylshikonin contained in Arnebia euchroma. Method: Through adsorption experiment, the best conditions of the p H and concentration for immobilization of EGFR were determined by using the surface plasmon resonance( SPR) method to construct EGFR biochip. The interactions between EGFR and four compounds were studied on real time after the EGFR immobilization on the chip with PBST( 10 mmol·L-1,p H 7. 4,including0. 005% tween 20) as the interaction buffer. The kinetic parameters were calculated by software,and the inhibitors were then screened based on the combination with kinetic constant. Result: The optimal immobilization conditions were as follows: with 10 mg·L-1 acetate buffer( 10 mmol·L-1,p H 4. 5),and was immobilized with a 250 response unit finally. The interaction results showed that only β,β-dimethylacrylshikonin had good interaction with EGFR among them,and other three compositions had no clear response. The kinetic parameters were calculated further,with binding rate constant ka= 1. 27 × 104 L·mol(-1)·s(-1),dissociation rate constant kd= 2. 92 × 10-2 s-1,dissociation equilibrium constant KD= 2. 31 × 10-6 mol·L-1 and Chi square value( Chi2) of 3. 25. KD 1 × 10-5 mol·L-1,suggesting that they had a strong affinity. Conclusion: It was indicated that the β, β-dimethylacrylshikonin could be a potential EGFR inhibitor.
作者 王嫚 李书文 董旭 申刚义 WANG Man, LI Shu-wen, DONG Xu, SHEN Gang-yi (Key Laboratory of Chinese Minority Traditional Medicine, Minzu University of China, Beijing 100081 , China)
出处 《中国实验方剂学杂志》 CSCD 北大核心 2018年第13期32-36,共5页 China Journal of Experimental Traditional Medical Formulae
基金 国家自然科学基金项目(81573834,81001595) 国家科技支撑计划项目(2014BAC15B04)
关键词 表皮生长因子受体 新疆紫草 表面等离子体共振技术 β β-二甲基丙烯酰紫草素 epidermal growth factor receptor (EGFR) Arnebia euchroma surface plasmon resonance(SPR) β β-dimethylacrylshikonin
作者简介 王嫚,在读硕士,从事药物分析方法研究,Tel:010-68939942,E-mail:18101203672@163.com;[通信作者]申刚义,博士,副研究员,从事药物分析方法研究,Tel:010-68939942,E-mail:shengy@muc.edu.cn
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