Table 2.
In vitro data for different classes of GSK-3 inhibitors including effects on enzyme activity, p-tau reduction and neuroprotection in LUHMES cells
| Inhibitor | Recombinant GSK-3β assay IC50 [nM], Reference | Recombinant GSK-3β assay IC50 [nM], Selenica et al | Cell-based GSK-3β assay IC50 [nM] | Max brain concentration [nM] | % inhibition p-tau at 2 μM, LUHMES | % inhibition LY294002 toxicity at 2 μM, LUHMES |
|---|---|---|---|---|---|---|
| AR-A014418 | 104 | |||||
| Bhat et al. (2003) | 300 | 16200 | 2000 | 0 | 31 | |
| SB216763 | ||||||
| 34 | 9.2 | 150 | 15000 | 41 | 46 | |
| Coghlan et al. (2000) | ||||||
| TDZD-8 | ||||||
| 2000 | >10000 | 9727 | ND | 0 | 3 | |
| Martinez et al. (2002) | ||||||
| Indirubin-3′- monoxime | ||||||
| 22 | 26 | 236 | 13000 | 54 | 68 | |
| Leclerc et al. (2001) | ||||||
| Alsterpaullone | 4 | 15 | 62 | 350 | 62 | 51 |
| Leost et al. 2000 | ||||||
| CHIR98014 | 0.63 | 3.7 | 70 | 7000 | 93 | 52 |
| Ring et al. (2003) |
Abbreviations: DMSO, dimethylsulphoxide; GS, glycogen synthase; GSK-3, glycogen synthase kinase-3; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
The IC50 for each compound was determined by the incorporation of 33P into a GS peptide by human recombinant GSK-3β. The incorporated radioactivity was measured via scintillation and IC50 values calculated by nonlinear regression. Data are shown in [nM]. For the cell-based GSK-3β activity assay, LUHMES cells were treated with GSK-3 inhibitors for 6 h and GSK-3β activity measured, as described in Methods section. Densitometry analysis was performed on autoradiographic film and IC50s calculated using sigmoidal dose-response curve fit analysis. Data are shown in [nM]. For determination of tau phosphorylation, LUHMES cells were treated for 6 h with 2 μM inhibitors, lysed, and human, endogenous Ser396 p-tau levels measured using an ELISA kit from Biosource. Data are shown as percentage inhibition of Ser396 p-tau compared to DMSO-treated cells. For neuroprotection experiments, LUHMES cells were co-treated with 2 μM inhibitors and 50 μM of the PI3 kinase inhibitor, LY294002, for 72 h and cell viability measured using the MTT reduction assay. Data are shown as % inhibition of LY294002-induced toxicity compared to DMSO-treated controls.