BACE1抑制剂分子水平高通量筛选体系的建立

Establishment of high-throughput screening system for BACE1 inhibitors in vitro

目的建立体外分子水平β-分泌酶(BACE1)抑制剂高通量筛选体系。方法采用均相时间分辨荧光法(HTRF),通过优化反应时间、酶浓度、检测仪器等实验条件,建立BACE1抑制剂高通量筛选体系,根据待测样品对BACE1活性抑制程度筛选其抑制剂。结果采用HTRF法建立了BACE1抑制剂高通量筛选体系,其中反应时间确定为6h、BACE1浓度为670U/L,采用VICTOR3酶标仪测得信噪比为649.6,信背比为44.6,Z′因子为0.91,变异系数小于10%,并筛选到一系列IC50小于10-6mol/L的化合物。结论采用HTRF法建立的BACE1抑制剂筛选体系灵敏度高、特异性和稳定性好,可用于BACE1抑制剂的高通量筛选。

Objective To establish the high-throughput screening system for β-secretase（β-site amyloid precursor protein cleaving enzyme 1,BACE1）inhibitors in vitro.Methods Using the homogeneous time-resolved fluorescence（HTRF） technique to establish a high-throughput screening system for BACE1 inhibitors according to the extent that test compounds inhibited BACE1 activity by optimizing the incubation time,enzyme concentration and the detector.Results A high-throughput screening system for BACE1 inhibitors based on HTRF methods was established.The incubation time was 6 h,the BACE1 concentration is 670 U/L.According to the detector of VICTOR3,the signal-to-noise（S/N） ratio reached 649.6,while the signal-to-background（S/B） ratio and Z＇ factor were 44.6 and 0.91,respectively.The coefficient of variation（CV） was much lower than 10%.Using this method,the inhibitors with IC5010-6mol/L could be screened out.Conclusion The high-throughput screening system based on HTRF Could assure a high sensitivity,specificity and stability,which could be used to high-through put screening for the BACE1 inhibitors.