We began our efforts by designing and synthesizing a small set of PROTACs aiming to maximize sampling of chemical space and target-PROTAC-E3 ternary complex pairing. HG-10-102-01(Figure ), a BBB penetrant type 1 LRRK2 inhibitor, was chosen as the LRRK2 ligand, on the basis of its small molecular size and favorable physicochemical properties . According to homology modeling of HG-10-102-01 with LRRK2, the morpholine ring is pointing towards solvent , suggesting of a suitable exiting vector for PROTAC linkage. We converted the morpholine ring to piperazine to facilitate linker attachment. For the E3 ubiquitin ligases, we decided to recruit Cereblon (CRBN), Cellular Inhibitor of Apoptosis (cIAP), and VHL, which have readily available ligands with known "PROTACable" sites (Figure ). After converting both the warhead and E3 ligase ligands into "PROTACable" intermediates, they were tethered together through linkers and a small library of first-generation compounds containing 12 LRRK2 PROTACs (Figure ) were generated (Schemes S3, S4, and S5). Figure . Screening of the first-generation PROTACs in WT and G2019S LRRK2 MEFs. (A) Representative Western blots monitoring total LRRK2, LRRK2-pSer935, Rab10-pThr73, total Rab10, and Tubulin levels following the treatment of WT and G2019S MEFs with the indicated compounds at 33 nM, 1 µM, or DMSO for 4h. (B) Quantitative analysis of the relative LRRK2 protein and Rab10-pThr73 levels, which are presented as ratios of total LRRK2/Tubulin or Rab10-pThr73/total Rab10, normalized to the DMSO treated sample. Data were obtained from two biological independent experiments. These PROTACs were then biologically evaluated in mouse embryonic fibroblasts (MEFs) by Western blotting. Briefly, MEFs were treated with compounds at 33 nM and 1 µM for 4 h (Figure ) and 24 h (Figure ) separately, and the intracellular level of LRRK2, phosphorylated LRRK2 at Ser935, and phosphorylated Rab10 (pRab10) at Thr73 were determined. Rab10 is one of the bona fide substrates of LRRK2, whose phosphorylation status is directly affected by LRRK2 kinase activity and protein level. Phosphorylation of LRRK2 at Ser935 is a well-studied biomarker site used to assess the efficacy of type 1 LRRK2 inhibitors . HG-10-102-01 based PROTACs can potentially dephosphorylate LRRK2 at Ser935 through both LRRK2 degradation and inhibition. Among the first-generation PROTACs, compounds SD75, SD82, and SD100 (Figure ) degraded 30-70% of G2019S LRRK2 at 1 µM/4h (Figure ) and achieved 70-85% G2019S LRRK2 degradation after 1 µM/24h treatment (Figure ). These three compounds also showed substantial dephosphorylation of LRRK2 and Rab10, with 75-90% pRab10 dephosphorylated after 1 µM/24h treatment in G2019S LRRK2 MEFs (Figure ). A fourth compound, SD13, also looked promising as it degraded 60% G2019S LRRK2 at 33 nM/4h treatment (Figure ) and 68% G2019S LRRK2 at 33 nM/24h (Figure ). However, less G2019S LRRK2 was degraded upon 1µM treatment by SD13, compared to the 33 nM treatments, suggestive of the "hook effect" . Although SD75, SD82, and SD100 showed only moderate LRRK2 degradation, they did not show any sign of the "hook effect" at 1µM concentration. Notably, all three compounds share the same E3 ligase and ligand (VHL, VH101) and exit vector out of the tert-leucine group, suggesting a potential hot-spot of ternary complex formation between VHL and LRRK2. We therefore decided to focus further medicinal chemistry optimization on this chemical series with the goal to further improve the compounds' fitness as LRRK2 degraders.