Fig. 3: Identifying GSK3 degraders from a direct-to-biology screen.

a–d HiBiT lytic assay-based high-throughput screening for GSK3β abundance with 24 h PROTAC treatment in GSK3β-HiBiT HEK293 cells. Dose response curves were generated with non-linear 4 parameter fit correction, and representative graphs are combined results of n = 2 biological replicates, each from n = 2 technical replicates with error bars representing mean and ± SD (a shades of blue correspond to individual compounds from Library Plate 1; b Shades of green correspond to individual compounds from Library Plate 2, with the red curve indicating the positive control PT-65). Heatmaps were generated using DC₅₀ values. Compounds with D_max < 30% were considered as having DC₅₀ > 1 µM. c, d Shading intensity reflects compounds DC₅₀ values, with white indicating >1 µM and deep orange indicating <100 nM. e–j Compounds 21 (orange), 22 (pink), 23 (purple), 24 (green), 25 (teal) and 26 (blue) were selected for resynthesis and further characterisation, which were originated from wells C7, F2, F12, D2, D7 and D12 of library plate 2, with the GSK3 inhibitor 29 (black) included as a negative control for comparison. Potency of these purified compounds were compared with crude mixtures (dashed lines) for GSK3β degradation upon 24 h treatment in GSK3β-HiBiT HEK293 cells (Dose response curves are means of n = 3 biological replicates ± SD, 4-parameter non-linear curve fitting (GraphPad)). k, l Kinetic live cell degradation of GSK3β with selected compounds 24 (green) and 26 (blue), respectively, monitored in LgBit overexpressing GSK3β-HiBiT KI HEK293 cells where shading intensity reflects compound concentration, with darkest shading indicating highest tested concentration (3 µM) and lightest shading indicating lowest tested concentration (0.01 µM) (n = 3 biological replicates, and data representatives of one biological replicate per compound with error bars representing mean of technical replicates and ± SD).