BACE1 Enzyme Inhibition Assay

The ability of bromophenols to inhibit the BACE1 enzyme was evaluated in vitro using human recombinant β-secretase and a BACE1 fluorescence resonance energy transfer assay kit (Life Technologies, Carlsbad, CA). All of the experimental conditions and procedures were similar to those in our previous report.⁴⁸ In brief, 10 μL of assay buffer (pH 4.5), 10 μL of BACE1 (1.0 U/mL), 10 μL of the BACE1 substrate (750 nM), and 10 μL of different bromophenols/quercetin concentrations were mixed in 384-well back plates and incubated for 60 min at RT in the dark. After incubation, mixtures in wells were irradiated at 545 nm and emission intensity at 585 nm was recorded using the SpectraMax Gemini XPS microplate spectrofluorometer (Molecular Devices, Sunnyvale, CA). The percentage (%) inhibition of BACE1 activity was calculated from the following equation: % inhibition = [1 – (S₆₀ – S₀)/(C₆₀ – C₀)] × 100%, where S₀ and C₀ are the initial fluorescence of the test samples and control groups, and S₆₀ and C₆₀ are the final (after 60 min) fluorescence of the test samples and control groups, respectively, and are expressed as IC₅₀ values calculated from the log dose–inhibition curve.