For VHL and LRRK2 target engagement experiments in live and permeabilized cells, the HEK293 cells were transfected with VHL-NanoLuc fusion vector (Promega, N275A) or LRRK2-NanoLuc fusion vector (Promega, NV3401) following Promega's protocol and seeded into white 384well plate (Corning3570) at a density of 6000 cells/well. To measure NanoBRET in permeabilized cells, the cells were treated with 50 µg/ ml digitonin (Sigma, D141), 125 nM VHL tracer/125 nM LRRK2 tracer, testing compounds at decreasing concentrations (12 concentrations with 2-fold serial dilution starting from 33 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 tracer dilution buffer were used to replace testing compounds and tracer separately. The filtered luminescence was measured within 10 min following addition of the substrate on a GloMax Discover microplate reader (Promega) or a PHERAstar (BMG LABTECH) plate reader equipped with a 450-nm bandpass filter (donor) and a 600-nm long pass filter (acceptor). To measure NanoBRET in live cells, the cells were treated with 250 nM VHL tracer/500 nM LRRK2 tracer, testing compounds at testing compounds at decreasing concentrations (12 concentrations with 2-fold serial dilution starting from 33 µM) and incubated at 37 °C in an incubator for 2 h. The plates were then cooled down and added with NanoBRET NanoGlo Substrate and Extracellular NanoLuc Inhibitor (Promega, N2160) before performing the same NanoBRET reading as the permeabilized mode on plate readers. NanoBRET ratio of each well was expressed in milliBRET according to the equation: mBRET = [(signal at 610 nM/signal at 450 nM) -(signal at 610 nMno tracer control/signal at 450 nMno tracer control)]×1000. The fractional occupancy was calculated according to the equation: fractional occupancy = (mBRETtesting compound -mBRETno tracer control)/(mBRETDMSO control -mBRETno tracer control).