Fig 3. IC₅₀ shift of inhibitors of TbGSK3β as a function of preincubation time with drug and kinase (10 nM) at 10 μM GSM peptide substrate and 10 μM ATP.

Preincubated reactions were preincubated with inhibitor, TbGSK3β, GSM peptide, and buffer at room temperature; after 30 minutes reactions were initiated with the addition of ATP. Reactions with 0 min preincubation were initiated with the addition of TbGSK3β. Reactions were run for 5 min at room temperature, and stopped heat at 80°C, followed by ice bath, and ADP product formation was measured by ADP-Glo kit. All concentrations of tideglusib and controls with and without TbGSK3β were kept at 1% DMSO. Black = 30 minute preincubation Grey = 0 minute preincubation. (A) tideglusib IC₅₀s 180 nM and 43 nM (B) GW8510, IC₅₀s 14.5 nM and 15.1 nM