To characterize XL01126 and XL01134 and compare them with the top first-generation degrader SD75, a dose dependent degradation assay was carried out in WT and G2019S LRRK2 MEFs (Figure ). SD75 dose-dependently degraded LRRK2 following 24h treatment in WT and G2019S LRRK2 MEFs (Figure ). However, the degradation of LRRK2 was only partial with Dmax reached at 3 µM (Dmax,24h = 51% and 58% for WT and G2019S LRRK2 respectively). Dose-dependent LRRK2 pSer935 and pRab10 dephosphorylation, which account for both LRRK2 inhibition and degradation, were also observed after SD75 treatment, with EC50 = 2270 nM and 379 nM for the dephosphorylation of Rab10 in WT and G2019S LRRK2 MEFs, respectively. XL01134 and XL01126, the top LRRK2 degraders from the second-generation compounds, showed more extensive LRRK2 degradation after a significantly shorter treatment time (4h) when compared to SD75 (Figure and). XL01134 degraded G2019S LRRK2 (DC50, 4h = 7 nM) more potently than WT LRRK2 (DC50, 4h = 32 nM), with maximum LRRK2 degradation reached at 300 nM and Dmax values against WT LRRK2 and G2019S LRRK2 are 59% and 81%, respectively. However, at concentrations above 300 nM, a strong "hook effect" was observed (Figure ). XL01126 also degraded G2019S LRRK2 (DC50, 4h = 14 nM) and WT LRRK2 (DC50, 4h = 32 nM) at nano-molar concentrations, but achieved more complete degradation than XL01134, with Dmax,4h = 82% in WT MEFs and Dmax,4h = 90% in G2019S LRRK2 MEFs, achieved at around 1 µM. Moreover, no "hook-effect" was observed with XL01126 at higher concentrations (Figure ). Due to the potent LRRK2 degradation capabilities, XL01134 and XL01126 resulted in more pronounced pRab10 dephosphorylation (Figure and 5C) than SD75. XL01134, at 4h, showed 30fold more potent pRab10 inhibition than SD75 (at 24h) in both WT MEFs and G2019S LRRK2 MEFs. XL01126 (at 4h) is 40-fold more potent than SD75 (at 24h) in inhibiting Rab10 phosphorylation in WT MEFs, and 25-fold more potent in G2019S LRRK2 MEFs. Figure . Dose dependent LRRK2 degradation, LRRK2 dephosphorylation, and Rab10 dephosphorylation by SD75, XL01134, and XL01126 in WT and G2019S LRRK2 MEFs. Representative Western blots of total LRRK2, LRRK2-pSer935, Rab10-pThr73, total Rab10, and Tubulin levels after treating WT and G2019S LRRK2 MEFs with SD75 (A), XL01134 (B), or XL01126 (C) at the indicated concentrations for indicated time period. The relativeLRRK2 protein and pRab10 levels were obtained by quantifying the ratios of total LRRK2/Tubulin or Rab10-pThr73/total Rab10, respectively, and the ratios were normalized to the DMSO treated samples. The relative total LRRK2 and pRab10 levels were plotted against the compounds concentration and fitted against "non-linear regression, one site-fit LogIC50" in GraphPad to obtain the DC50 and EC50 values. Data were obtained from two to three biological independent experiments.