To enable library screening we generated endogenously tagged GSK3β-HiBiT knock-in HEK293 cells (Fig ). Crude compound mixtures, initially made to 10 mM DMSO stock solutions on the assumption of 100% conversion and purity, were taken directly into cell-based high-throughput degradation screens with 24 hours treatment. Dose-dependent degradation profiles and DC50 heatmaps of library plate 1 (Fig ) and library plate 2 (Fig ) show that compounds from both libraries degrade GSK3β to various extents. Importantly, we included in both plates a negative control row, utilising benzyl bromide as a mock-E3 ligase reagent (Fig2C, E-F, row E in both plates). Pleasingly, all wells from negative control compounds showed no effect on GSK3β-HiBiT signal, confirming that the chemistry we used did not interfere with the assay read-out (Fig3A-D). PT-65 is a previously reported GSK3 degrader and was used as a benchmark in our assays . From this single screen structure-activity based relationships were generated, revealing that regardless of the GSK3-binder choice, some linkers (e.g. 2 and 4) as well as E3 ligase binders (16 and 17) showed a trend towards more effective GSK3β degradation. GSK3-binder 20 used in generation of library plate 2 resulted in more potent compounds. In fact, 6 compounds from plate 1 demonstrated >50% Dmax and < 1µM DC50, whereas 35 compounds from plate 2 met the same criteria. Next, we sought to further characterise 6 of the selected compounds from library plate 2. Considering both potency and a desire to further profile compounds with a mix of different linkers and E3 ligase ligands we chose compounds 21, 22, 23, 24, 25 and 26, which originated from wells C7, F2, F12, D2, D7 and D12 respectively. The selected compounds were re-synthesised and purified via preparative HPLC and the degradation profiles of crude vs purified compounds assessed (Fig ). Observed DC50 values were moderately left-shifted (2.5 to 20 fold shift) for all selected pure compounds and Dmax values remained the same between crude and pure compounds. Approximate rank ordering also remained the same amongst the set. We were able to confirm that all compounds, once resynthesised, retained their high levels of degradation potency. All compounds displayed DC50 values ≤20 nM and compound 26 yielded sub-nanomolar potency. PROTAC-mediated degradation of untagged, endogenous GSK3β in HEK293 and HAP1 cells was also confirmed via immunoblotting (Fig and) assuring us that all compounds are bone fide GSK3 degraders. Next, we wanted to investigate degradation rates of the six selected compounds. We used LgBit transfected GSK3β-HiBiT cells to monitor live cell dose dependent degradation over time. Maximal degradation rates (λmax) varied between test compounds (Fig ) from 0.5 to 1.0 hour -1 , with observed maximal degradation rate for 24 (Fig ). Due to the hook effect, maximal degradation rates couldn't be calculated for some of the compounds over the concentration range used, including 26. However, almost complete degradation of GSK3β was observed for 26 after 2 hours with as little as 10 nM compound concentration. All together, these data highlighted that from one orthogonally reactive linker D2B screen, we could identify potent and rapid GSK3 degraders from multiple series. We focussed on assessing impact of GSK3 degradation on total β-catenin and phospho-Collapsin response mediator protein 2 (CRMP2) levels. We chose βcatenin due to the established role of GSK3 in promoting its degradation via phosphorylation and the proposed on-target toxicity risks for GSK3 inhibitors that have been observed to stabilise βcatenin. In addition, CRMP2 is a validated neuronal GSK3 target, which has been found to be hyperphosphorylated in the brains of AD patients and suggested to be an early event in AD pathogenesis . With 24 hours compound treatment the onset of GSK3 degradation was observed at between 0.1 and 10 nM with concurrent reduction in phospho-CRMP2, but no impact on β-catenin stabilisation except at the highest tested dose of 100 nM. 24 also substantially outperformed the nondegrading negative control (27) which would still be capable of equivalent GSK3 inhibition as compared with the degrader.