Fig 1.
Kinetic scheme for slow binding kinetics.
One-step binding: the inhibitor [I] and substrate [S] are in equilibrium; binding is competitive and mutually exclusive. Increasing [S] will shift the equilibrium to product formation and reduce the amount of enzyme in bound to inhibitor (EI); for two-step binding the formation of the E’I complex is time dependent and not in rapid equilibrium with ES; increasing [S] will have a smaller effect on reducing the fraction of enzyme in the E’I complex. This behavior looks noncompetitive and is termed insurmountable.
Fig 2.
Substrate kinetics for TBGSK3β.
Panel A. Time course of TbGSK3β, 1.5 nM, run in kinase buffer (15 mM HEPES, pH 7.4, 10 mM MgCl2, 1 mM EGTA, 0.02% Tween 20) with 10 μM ATP and 10 μM GSM. Reactions were initiated with the addition of enzyme to a total volume of 20 μL. Microtubes were mixed and sealed, and reactions were terminated by placement of the tube in a heat block for 5 min. Reaction mixtures were transferred to a 384-well plate, and ADP-Glo (Promega) detection was used according to the manufacturer’s instructions. Panels B and C. Michaelis Menten plots of reaction velocity with increasing concentrations of (B) GSM peptide and (C) ATP. They velocities are calculated from the nmol of ADP formed/nmol of enzyme (TbGSK3β)/ time.
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
Fig 4.
Four point MMOA screen for tideglusib and GW8510.
Time dependent inhibition was evaluated by preincubation of TbGSK3β with 60 nM tideglusib and 6 nM GW-8510 with 10μM and 100μM ATP. (A). Tideglusib [60 nM] in 10μM ATP. (B). GW8510 [60 nM] in 10μM ATP. (C.) Tideglusib [60 nM] at 100μM ATP. (D.) GW8510 [60 nM] at 100μM ATP. All reactions preincubated or not preincubated with TbGSK3β for 30 min at room temperature. Experiments run with 10μM GSM peptide, 10μM ATP, and buffer. Minute preincubation (30 min) was preincubated with inhibitor, TbGSK3β, GSM peptide, and buffer. ATP was mixed to initiate reaction. No preincubation contained inhibitor, GSM peptide, ATP, and buffer. The reaction was initiated with TbGSK3β. Reactions were run at room temperature for 5 min and stopped at 80°C. ADP formed was measured by ADP-Glo kit. Values are mean +/- standard error. N = 3 for each experiment and experiments were run in duplicates. Control reactions contained DMSO and background was determined using a zero time incubation and subtracted from all reactions. Black = 30 min preincubation Grey = No preincubation.
Fig 5.
Progress curves for tideglusib and GW8510.
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.
Fig 6.
Tideglusib retains activity following a 100-fold dilution of TbGSK3β.
Tideglusib [100 nM, GW8510 [100 nM] and DMSO were preincubated with 50 nM TbGSK3 for 30 min. After a 100 fold dilution activity was measured and compared to DMSO containing reaction. Data expressed percent inhibition of DMSO activity. Data represent the average of two separate experiments for GW8510 and three for tideglusib. All points in each experiment were run in duplicate.
Table 1.
Evaluation of time dependent inhibition withTbGSK3β inhibitors.
Tideglusib, AZ960, CT99021, LY278544, sorafenib, and TWS119 were evaluated for time-dependent activity. The procedures are described in Fig 4. Data are the mean of duplicate experiments with N = 2 for each experiment. Data shown are percent inhibition of DMSO control. Ratio is % inhibition at 30 min preincubation/ % inhibition at 0 min preincubation at 10 μM ATP. Concentrations were selected based on preliminary screening IC50 measurements with no preincubation. IC50 values ± standard deviation +n = 3) were determined with 10 μM ATP and 10 μM GSM.
Fig 7.
Two point ATP competition analysis for TbGSK3β inhibitors.
Tideglusib, AZ960, CT99021, LY2784544, sorafenib, and TWS119 at 10μM GSM and 10 μM or 100 μM ATP. GSM mixed with 1x buffer and 10μM or 100μM ATP. TbGSK3β [5 nM] was added to start reaction and incubated at RT for 5 minutes. Reactions were stopped at 80°C and worked up as described in Materials and Methods. Experiments ran in duplicate, N = 2.
Fig 8.
Tideglusib inhibits growth of T. brucei.
Bloodstream trypanosomes (3 x 103 cells/mL) were treated for 48 hours at varying concentrations of tideglusib (in DMSO solvent, final concentration of DMSO was 0.1%). Percent growth was determined by comparison to trypanosomes treated with equivalent volume of solvent (0.1% DMSO). Non-linear regression fit of the dose-response curve was determined using GraphPad Prism 6 software.
Fig 9.
Trypanosome viability after short term exposure with tideglusib.
Trypanosomes (5 x 105/mL in HMI-9 medium) were treated with pentamidine (or water solvent control) or tideglusib (5 μM, 10 μM, or DMSO solvent control) for 6 hours (37°C, 5% CO2) and then cultured in drug-free treatment as detailed in panel (A). Trypanosome densities were determined after 6 hour treatment (B), and the drugs washed from the cells by centrifugation and resuspension in fresh medium (to a density of 0.5 x 105/mL). Trypanosome proliferation in the fresh medium was monitored for 44 hours (37°C, 5% CO2) (C). Error between four separate cultures are shown. Cultures in which no cells were observed is denoted by an asterisk.