In the NanoBRET-based ternary binding affinity assay, LRRK2-NanoLuc was transiently expressed in HEK293 cells as the BRET donor and LRRK2 tracer which is prepared by conjugating HG-10-102-01 with a fluorophore (BODIPY 576/589 ) (Figure and Scheme S11) was introduced as the acceptor. Titration of PROTAC degraders to the lysed cells and LRRK2 tracer in the presence or absence of recombinant VCB protein (VHL complexed with elongin B-elongin C) gives ternary and binary binding affinities of PROTACs against LRRK2 respectively. Similarly, the ternary complex formation assay also used LRRK2-NanoLuc transiently expressed in HEK293 as the BRET donor, but the acceptor was recombinant VCB protein labeled with BODIPY 576/589 via NHS ester-activated crosslinking reaction. PROTACs that can bridge LRRK2 and VCB together will produce BRET signal (Figure ). In line with the degradation potency, XL01126 induced the most cooperative ternary complex as indicated by its positive cooperativity (α = 5.7) (Figure ) and the highest maximal level of ternary complex formation (Figure ). In contrast, XL01134 induced significantly lower cooperativity (α = 1.4) and SD75 has a negative cooperativity with VHL and LRRK2 (Figure ) Figure . Binding affinities to VHL and LRRK2. (A) Binding affinity of the tested compounds to VHL using FP assay. The indicated compounds were titrated to a solution of VCB protein (10 nM) and JC9 (5 nM) (a FAM labeled probe that binds to VCB) to displace JC9 and the percentage of displacement was plotted against the compounds' concentration and fitted into the "non-linear regression, one site-LogIC50" to obtain the IC50 values, which were used to back calculate the Ki values. NanoBRET target engagement assays of tested compounds to VHL (B) and LRRK2 (C) in permeabilized and live cell modes. The indicated compounds were titrated into HEK293 cells transfected with VHL-NanoLuc (B) or LRRK2-NanoLuc fusion (C) in the presence of VHL tracer (B) or LRRK2 tracer (C). 0.25 µM and 0.5 µM VHL tracer were used for the permeabilized and live mode VHL engagement assay separately. 0.125 µM and 0.5 µM of LRRK2 tracer were used for the permeabilized and live mode LRRK2 engagement assay separately. The Fractional occupancy of the tracers are plotted against the tested compounds' concentrations and fitted into "non-linear regression, one site-LogIC50" to obtain the IC50 values of each compound against both permeabilized and live cells, separately. Data points are presented as mean ± SEM from three independent experiments. (D) The IC50 ratios between permeabilized and live mode target engagements of each compound were used to compare their permeabilities.