A Four-Point Screening Method for Assessing Molecular Mechanism of Action (MMOA) Identifies Tideglusib as a Time-Dependent Inhibitor of Trypanosoma brucei GSK3β
Time dependent inhibition of TbGSK3β by tideglusib and GW8510 at 100 μM ATP and 80 μM GSM. A/B: TbGSK3β incubated with 0.2 μM (green circles), 0.4 μM (blue squares) or 0.6 μM (red triangles) tideglusib at 5, 10, 20, and 30 minute time intervals. C/D: TbGSK3β incubated with 0.6 μM (purple squares) GW8510 at 5, 10, 20, and 30 minute time intervals. All reactions were made up of 100 μM ATP, 80 μM GSM and buffer, mixed with inhibitor or DMSO. TbGSK3β [5 nM] was added to start reaction and incubated at room temperature at different time intervals. Reactions were stopped at 80°C and worked up as described in Materials and Methods. Data in A best fit the progress curve for irreversible inhibition (P = vst + (v0 –vs) (1-e-kt)/k) where vs equals zero. In C the data fit a straight line as expected for reversible inhibition, however the y-intercept was above zero. Values are mean +/- SE. All experiments run in duplicates. A. ADP formed with time (N = 6). B. Replot of data in A expressed as percent inhibition by tideglusib with time. C. ADP formed with time (N = 6). D. Replot of data in A expressed as percent inhibition by GW8510 with time.