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

摘要

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.%目的 建立一种基于荧光共振能量转移(FRET)法筛选β分泌酶(BACE1)抑制剂的适用性评价方法.方法 选取2种基于FRET方法建立的商品化BACE1活性测定体系,按照标准流程检验表没食子儿茶素-3-没食子酸酯(EGCG)和Compound 1对于BACE1的抑制活性,计算抑制剂在2个测定体系中的抑制率和催化曲线斜率,同时定量计算化合物加入对测定体系荧光强度的影响,并进行对比评价.结果 曾报道的BACE1抑制剂EGCG加入基于FRET方法的测定体系后,导致体系荧光强度降低,且荧光强度降低得到的直线斜率(10.8±2.6)与抑制曲线斜率(10.2±3.4)数据一致,证明EGCG对BACE1的抑制活性为假阳性;而Compound 1加入对于体系荧光强度无影响,证实了其对BACE1的抑制活性.在基于FRET方法的测定体系中初筛显示阳性的化合物,其对测定体系荧光强度的影响与未加入BACE1的空白对照进行校正,评价待测化合物在所选测定体系中的适用性,避免出现假阳性结果.结论 采用此化合物空白荧光校正方法可定量测定被测化合物对于体系荧光强度的影响,从而评价BACE1抑制剂在FRET体系中的适用性.