ABSTRACT
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 IC50<106mol/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.