Given the modular nature of PROTAC molecules, the structural modification of the second generation of LRRK2 PROTAC degraders focused on modifying the LRRK2 ligand, the linker, and the VHL ligand (Figure and Figure ), separately. To best assess which structural modification would confer the most significant activity improvement, we designed molecular match pairs of SD75, SD82, and SD100 by changing one structural moiety at a time. XL01078B, XL01072, and XL01070B were designed (Figure ) and synthesized (Scheme S3) where the 5-chlorine substitution on the aminopyrimidine ring of HG-10-102-01 was replaced with -CF3 substitution which was reported to improve binding affinity to LRRK2 . XL01119, XL01118 and XL01120 (Figure ) were molecular match pairs of SD75, SD82, and SD100 respectively, by harboring an extra methyl group on the benzylic position of VHL ligand, which was introduced to increase binding affinity to VHL E3 ligase . Fluorine substitution was introduced on the phenyl group of the VHL ligand of XL01123, XL01122, and XL01121 (Figure ) attempting to fine-tune physicochemical properties at a permissible site . The linker length, composition, and rigidity, which can significantly affect the physicochemical and pharmacokinetic properties of PROTACs, as well as their ternary complex formation and activity , were explored as represented by compounds XL01131, XL01140, XL01111, XL01126, XL01134, and XL01076 (Figure ). In an attempt to improve the drug-like properties and reduce molecular size, we designed XL01145, XL01149 and XL01168 (Figure ). These compounds are derived from truncated HG-10-102-01 with the morpholinoamide moiety removed as its absence retains binary binding affinity to LRRK2 . These 18 new compounds were synthesized as outlined in Schemes 1, S1, S2, S3, S6, S7, S8 and S9 and were also screened via Western blotting (Figure and Figure ). Quantitative analysis of the Western blots (Figure and Figure ) revealed that at 33 nM/4h treatment, XL01126 and XL01134 were the most effective optimized compounds that degraded 20-30% of WT LRRK2 and 50-60% of G2019S LRRK2 (Figure ). Accordingly, these two compounds were also the most potent in decreasing pRab10 in both WT and G2019S LRRK2 MEFs, with > 60% pRab10 inhibited in G2019S LRRK2 MEFs at 33 nM/4h (Figure ). In contrast, the first-generation degraders SD75, SD82, and SD100 induced little to no degradation of LRRK2 at 33 nM/4h treatment (Figure ) and showed weak (<40%) degradation at 33 nM/24h treatment (Figure ), at which XL01126 and XL01134 degraded 50-60% of WT LRRK2 and 70-80% of LRRK2 G2019S (Figure ). Most of the compounds exhibited substantial WT LRRK2 and G2019S LRRK2 degradation (30-80%) at 1 µM/4h or 1 µM/24h treatment (Figure and Figure ), leading to potent and almost complete pRab10 inhibition in WT MEFs and G2019S LRRK2 MEFs, respectively. Multiple new compounds, including XL01078B, XL01119, XL01123, XL01131, XL01126 and XL01134, surpassed SD75, SD82 and SD100 in degrading WT LRRK2 and G2019S LRRK2 at 1 µM/4h and 1 µM/24h treatment, suggesting modifications at the warhead (XL01078B), the E3 ligase ligand (XL01119 and XL01123), and the linkers (XL01131, XL01126, and XL01134) can all improve the degraders' fitness to some extent. Nonetheless, the significant improvement exhibited by XL01126 and XL01134, which are isomers of each other, encouraged us to characterize them further.