and KH2 (26) (Fig ), as has been described previously . Equilibrium dialysis suggests an unbound fraction of 0.5% for KH1 (24) in mouse plasma, whereas the serum shift degradation assay showed a shift in DC50 of 8.2x with 10% Mouse serum (Fig ), therefore predicting a shift of ~80x in whole blood and equating to a free fraction of ~1.2% in mouse plasma. Using a 2-hour total plasma concentration of 16 nM at a dose of 0.37 mg/kg (Supplementary Tables 5) and using the predicted free fractions, an unbound total plasma concentration at this time point of between 0.4 nM and 0.9 nM was calculated. In addition, we measured brain binding, predicting a free concentration in brain of 20 pM based on the measured free fraction of 0.12%. This equates to a low Kpu,u of 0.05. Finally, to understand if KH1 (24) could reasonably be expected to degrade GSK3 in vivo and at what dose, we measured the ability of KH1 (24) to degrade mouse GSK3β in mouse embryonic fibroblasts (Fig ), suggesting a DC50 in the region of 0.1-1 nM. We therefore reasoned that although the Kpu,u was low, the high potency of KH1 (24) and its measured brain concentration/penetration and total plasma levels could reasonably mean it may reach required free concentrations to observe effects on GSK3 abundance in vivo at a ~10-fold elevated i.v. dose. Given this understanding we dosed mice at 5mg/kg i.v. and measured KH1 (24) levels in plasma and brain at 4 hours and GSK3β as well as downstream substrate levels in liver and brain at the same time point (FigS4C-D). Pleasingly, we observed almost complete removal of GSK3β in liver as well as significant degradation of GSK3β in mouse brain (Fig ) supporting KH1 (24) as a GSK3 PROTAC suitable for in vivo use.