In ALS-derived lymphoblast cell lines, 5 μM Tideglusib for 24 h reduced elevated TDP-43 phosphorylation and cytosolic accumulation. Western blotting showed a trend toward restoration of nuclear TDP-43, and microscopy supported redistribution. These peripheral cell-line findings do not establish a clinical response or efficacy in motor neurons. [3]
In a separate SH-SY5Y assay, 10 μM Tideglusib for 24 h did not increase nuclear β-catenin, whereas the comparator SB216763 did. Thus, β-catenin responses should be interpreted in the specific model and treatment context. [3]
Preincubation-dependent inhibition of human GSK-3β
Biochemical assay · Human recombinant wild-type GSK-3β; Z′-LYTE peptide phosphorylation assay [1]
A 1-h enzyme–drug preincubation shifted the reported IC50 from 105 to 5 nM. These are assay endpoints and depend on incubation conditions.
Literature-Reported Treatment Conditions
- Treatment concentration
- Serial Tideglusib dilutions; the reported IC50 values are endpoints, not selected treatment doses.
- Exposure duration
- With or without 1 h preincubation; 1 h enzymatic reaction
- Method
- FRET-based Z′-LYTE at 25 °C; enzyme 2–5 nM; ATP 12.5 μM and Ser/Thr-9 peptide 2 μM.
- Vehicle
- 1% DMSO
Methods and source locations
Compound reported in the paper: Tideglusib; Compound label in the paper: Tideglusib
Research evidence for the reported tested material and model; no supplier-lot equivalence or clinical efficacy is established.
Experimental Procedures: Enzymatic Assays; Results Figure 3A and Table 1
Inhibition of wild-type GSK-3β peptide phosphorylation: Table 1: mean of two independent determinations calculated in logarithmic space. Figure 3A separately shows average triplicate determinations ± S.E. — Table 1 (Tideglusib, GSK-3β WT columns); Figure 3A and caption
Persistence of inhibition after removal of unbound Tideglusib
Biochemical assay · Human recombinant wild-type GSK-3β; filtration/dilution reversibility test [1]
Inhibition persisted after removal of unbound drug by 400-fold dilution, whereas the reversible comparator SB-415286 recovered activity. This supports functional irreversibility over the observation window; this experiment alone does not establish covalent bonding.
Literature-Reported Treatment Conditions
- Treatment concentration
- 1 μM Tideglusib; 20 nM GSK-3β during pretreatment; SB-415286 comparator 1 μM
- Exposure duration
- 30 min pretreatment; Results describe 1 h postdilution incubation followed by 1 h enzymatic reaction.
- Method
- Two filtration/dilution cycles (total 400-fold dilution of small molecules); enzymatic activity compared before and after processing.
- Vehicle
- 1% DMSO
Methods and source locations
Compound reported in the paper: Tideglusib; Compound label in the paper: Tideglusib
Research evidence for the reported tested material and model; no supplier-lot equivalence or clinical efficacy is established.
Experimental Procedures: Filtration Experiments and Enzymatic Assays; Results Figure 2 and adjacent paragraph
GSK-3β activity after two filtration/dilution cycles: Figure 2: average of three independent samples measured in duplicate; error bars S.D.; untreated enzyme processed in parallel. — Figure 2 and caption; Results paragraph beginning “Consequently, we decided to test the reversibility”
Independent assay confirms time-dependent potency
Biochemical assay · Full-length human recombinant wild-type GSK-3β; PhosphoSens peptide assay [2]
An independent assay reported IC50 values of 1780 nM without preincubation and 139 nM with 3-h preincubation. These values differ from the earlier Z′-LYTE results and must remain attached to this assay.
Literature-Reported Treatment Conditions
- Treatment concentration
- Serial inhibitor dilutions; IC50 endpoints reported separately.
- Exposure duration
- With or without 3 h preincubation; inhibition quantified after 84 min of reaction.
- Method
- PhosphoSens assay at 30 °C; 0.5 nM GSK-3β, 14 μM ATP, 10 μM peptide substrate.
- Vehicle
- 1% DMSO
Methods and source locations
Compound reported in the paper: Tideglusib; Compound label in the paper: Tideglusib
Research evidence for the reported tested material and model; no supplier-lot equivalence or clinical efficacy is established.
Sections 2.1, 2.2, 2.6; Section 3.4; Figure 5B and Table 1
Inhibition of wild-type GSK-3β: Curve fitting to Eq. 1; Table 1 gives 95% CIs. Figure 5 error bars are SD; n ≥ 2. Asterisk denotes no overlap of the IC50 confidence intervals. — Figure 5B and caption; Table 1 Tideglusib row and footnote; Section 2.6
Reducing agents attenuate inhibition in a single-concentration assay
Biochemical assay · Human recombinant wild-type GSK-3β; PhosphoSens peptide assay [2]
At 10 μM Tideglusib, adding 2 mM DTT, GSH or TCEP markedly reduced measured inhibition. This is a single-concentration comparison, not an IC50 measurement.
Literature-Reported Treatment Conditions
- Treatment concentration
- 10 μM Tideglusib; 2 mM DTT, GSH or TCEP in the corresponding assay conditions
- Exposure duration
- 84 min reaction endpoint
- Method
- PhosphoSens peptide phosphorylation; compare each reducing-agent condition with no reducing agent. AR-A014418 was included as an assay comparator.
- Vehicle
- 1% DMSO
- Co-treatment
- Assay modifier: DTT, GSH or TCEP; no therapeutic combination.
- Reported limits
- Figure 6 caption reports n=2 without identifying these as independent biological experiments.
Methods and source locations
Compound reported in the paper: Tideglusib; Compound label in the paper: Tideglusib
Research evidence for the reported tested material and model; no supplier-lot equivalence or clinical efficacy is established.
Section 2.2 (Figure 6 exception), Section 3.5; Figure 6 and caption
GSK-3β inhibition with versus without reducing agents: Figure 6: 10 μM inhibitor; 2 mM reducing agent; SD error bars; n=2. — Figure 6 and caption; Sections 2.2 and 3.5
TDP-43 phosphorylation and localization in ALS-derived lymphoblasts
Cell-based study · Immortalized lymphoblast cell lines from sporadic ALS patients and control individuals [3]
At 5 μM for 24 h, Tideglusib reduced elevated pTDP-43 and cytosolic TDP-43 in ALS-derived lymphoblasts. The nuclear Western blot response was described as a trend toward restoration; microscopy provided additional localization evidence. These are cell-line findings, not treatment responses in patients.
Literature-Reported Treatment Conditions
- Treatment concentration
- 5 μM
- Exposure duration
- 24 h
- Method
- Western blotting of total and subcellular fractions; confocal TDP-43/DAPI microscopy; treated and untreated lines from both donor groups.
- Reported limits
- Solvent, final vehicle concentration and supplier/lot of the tested Tideglusib are not specified in the retrieved main text. No vehicle-matched solvent control is asserted.
Methods and source locations
Compound reported in the paper: Tideglusib; Compound label in the paper: TDG
Research evidence for the reported tested material and model; no supplier-lot equivalence or clinical efficacy is established.
Results 2.2; Figures 2–3 and captions; Methods 4.1, 4.3–4.5 and statistical analysis
pTDP-43/total TDP-43 and subcellular TDP-43 distribution: Figure 2: mean ± SEM, observations from five cell lines per group. pTDP-43 normalized to total TDP-43; cytosol and nucleus assessed with α-tubulin and Lamin B1 loading/purity controls. — Figure 2a–b and caption; Results 2.2; Methods 4.3
Cytosolic/nuclear TDP-43 fluorescence distribution: Figure 3 caption: mean ± SEM, two cell lines per donor group and at least 45 cells per individual. Methods 4.4 instead describes approximately 45 cells per group in n=2 independent experiments. The exact sampling hierarchy remains unresolved; cells are not independent donors. — Figure 3 and caption; Methods 4.4 (different sampling description retained)
No nuclear β-catenin accumulation in the tested neuroblastoma condition
Cell-based study · Human SH-SY5Y neuroblastoma cells [3]
Tideglusib at 10 μM for 24 h did not produce nuclear β-catenin accumulation in the reported assay, whereas the ATP-competitive comparator SB216763 did. This limits a general assumption of Wnt/β-catenin activation across cell contexts.
Literature-Reported Treatment Conditions
- Treatment concentration
- 10 μM Tideglusib; separate comparator SB216763 at 10 μM
- Exposure duration
- 24 h
- Method
- Western blots of nuclear and cytosolic fractions, with Lamin B1 and α-tubulin loading/purity controls; untreated cells included.
- Reported limits
- Solvent, final vehicle concentration and compound lot are not specified in the retrieved main text.
Methods and source locations
Compound reported in the paper: Tideglusib; Compound label in the paper: TDG
Research evidence for the reported tested material and model; no supplier-lot equivalence or clinical efficacy is established.
Results 2.4; Figure 5 and caption; Methods 4.1 and 4.3; statistical analysis
Nuclear and cytosolic β-catenin: Figure 5: mean ± SEM of six different experiments; nuclear β-catenin normalized to Lamin B1, cytosolic signal to α-tubulin. — Figure 5 and caption; Results 2.4; Methods 4.3
