In Vitro

In vitro activity: Tideglusib irreversibly inhibits GSK-3, reduces tau phosphorylation, and prevents apoptotic death in human neuroblastoma cells and murineprimary neurons. Tideglusib (2.5 μM) inhibits glutamate-induced glial activation as evidenced by decreased TNF-α and COX-2 expression in rat primary astrocyte or microglial cultures. Tideglusib (2.5 μM) also exerts a potent neuroprotective effect on cortical neurons from glutamate-induced excitotoxicity as evidenced by significant reduction in the number of Annexin-V-positive cells in rat primary astrocyte or microglial cultures.

Kinase Assay: [35S]Tideglusib (207 Bq/nmol) at 55 μM is incubated with 5 μM GSK-3β for 1 h at 25°C in 315 μL of 50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl and 0.1 mM EGTA. The incubation is extended for another 30 min after having added 35 μL of the same buffer with or without 100 mM DTE. Samples are then processed in three different ways. First, an aliquot of 125 μL of each sample is mixed with 375 μL of 8 M GdnHCl in H2O and heated at 80°C for 5 min. A second aliquot of 125 μL is diluted up to 500 μL with H2O and left at room temperature for 5 min. In both cases, the free drug is removed afterwards by gel filtration through Sephadex G-25, and the amount of bound drug is determined by liquid scintillation counting on a 1450-MicroBeta TriLux counter. Finally, a third 40 μL aliquot of each original sample is mixed with 10 μL of denaturing electrophoresis sample buffer without reducing agents, and 35 μL of this mixture is loaded onto a 10% polyacrylamide gel and subjected to SDS-PAGE (again in the absence of reducing agents except for the DTE already included in the corresponding sample), followed by fluorography of the dried gel. 

Cell Assay: Tideglusib (NP12) is a small heterocyclic thiadiazolidinone (TDZD) derivative, which is an ATP-non competitive inhibitor of GSK-3β with an IC50value in the micromolar range. Incubation of both astrocyte and microglial cultures with Tideglusib (NP031112) completely abrogates the induction of TNF-α and COX-2 expression after glutamate treatment. These effects of NP031112 are not caused by a loss of cell viability, because the 24 h exposure of astrocyte and microglial cells to this TDZD does not modify cell viability.