中国药理学与毒理学杂志2018，Vol.32Issue(5)：371-376,6.DOI:10.3867/j.issn.1000-3002.2018.05.004
基于荧光共振能量转移法建立筛选β分泌酶抑制剂的适用性评价方法
Evaluation method for compatibility of compounds in fluorescence resonance energy transfer model to screen β-secretase inhibitors
摘要
Abstract
OBJECTIVE To develop a method to evaluate the compatibility of compounds in the fluorescence resonance energy transfer (FRET) model for β-secretase (BACE1) inhibitor screening.METHODS Two commercially available BACE1 inhibitor screening systems based on FRET were selected to evaluate the BACE1 inhibitory activities of (-)-epigallocatechin-3-gallate (EGCG) and Compound 1 according to the supplier's protocol.The inhibitory rates and slopes of the catalytic curves of the inhibitors were calculated.The effect of inhibitors on the fluorescence intensity of the systems were quantitatively calculated and the comparatively evaluated.RESULTS EGCG,a reported non-competitive inhibitor of BACE1,directly induced the reduction of fluorescence intensity of one of the systems.The slope of the line with the addition of EGCG (10.8±2.6) conformed to that of the line of EGCG inhibition (10.2±3.4),which indicated that EGCG was a pseudo-positive inhibitor of BACE1.Compound 1 had little effect on the fluorescence intensity of the systems,so the inhibitory activity of Compound 1 was confirmed.The compounds which showed inhibitory activity in preliminary screening should be checked in the blank control without BACE1 to calibrate the effect of compound on the system fluorescence intensity.The applicability of the tested compounds in the screening system could thus be evaluated to prevent pseudo-positive results.CONCLUSION This fluorescence calibration method with compound control can be universally used for assays based on FRET theory to evaluate the applicability of tested BACE1 inhibitors.关键词
荧光共振能量转移/β分泌酶/荧光强度/表没食子儿茶素-3-没食子酸酯Key words
fluorescence resonance energy transfer/β-secretase/fluorescence intensity/(-)-epigallocatechin-3-gallate分类
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赵颖,张家淮..基于荧光共振能量转移法建立筛选β分泌酶抑制剂的适用性评价方法[J].中国药理学与毒理学杂志,2018,32(5):371-376,6.基金项目
高等学校博士学科点专项科研基金(20070023078)The project supported by Research Fund for Doctoral Program of Higher Education of China (20070023078) （20070023078）
