The HEK293 cells were transfected with LRRK2-NanoLuc fusion vector (Promega, NV3401) following Promega's protocol and seeded into white 384-well plate (Corning3570) at a density of 6000 cells/well. The cells were then treated with 50 µg/ml of digitonin, 125 nM of LRRK2 tracer, testing compounds at decreasing concentrations (11 concentrations with 2-fold serial dilution starting from 10 µM) or testing compounds and VCB mix (11 concentrations with 2-fold serial dilution starting from 10 µM compound for the compound. The first 6 concentrations of VCB start from 32 µM with 2-fold dilution, the last 5 concentrations of VCB keep at 1 µM), and NanoBRET NanoGlo Substrate (Promega) at concentration recommended by the manufacturer's protocol. In the maximum signal control samples (DMSO control), DMSO was added instead of testing compounds. In the minimum signal control samples (no tracer control), DMSO and LRRK2 tracer dilution buffer were used to replace testing compounds and LRRK2 tracer separately. The filtered luminescence was measured within 10 min following addition of the substrate on a PHERAstar (BMG LABTECH) plate reader equipped with a 450-nm bandpass filter (donor) and a 600-nm long pass filter (acceptor). The fractional occupancy was calculated according to the equation: fractional occupancy = (mBRETtesting compound -mBRETno tracer control)/(mBRETDMSO control -mBRETno tracer control).