SB415286

SB 415286 inhibits GSK3α in an ATP competitive manner with K_i of 31 nM and shows similar potency against GSK3β. SB 415286 has little or no activity against 24 other protein kinases with IC50 > 10 μ M. SB 415286 stimulates glycogen synthesis in the Chang human liver cell line with EC50 of 2.9 μM, and induces expression of a β-catenin-LEF/TCF regulated reporter gene in HEK293 cells. ^[1] SB 415286 protects both central and peripheral nervous system neurones in culture from death induced by reduced PI3-kinase pathway activity in a concentration-dependent manner, which is correlated with inhibition of GSK-3 activity and modulation of GSK-3 substrates tau and β-catenin. ^[2] In L6 myotubes, SB 415286 induces a much greater activation of GS (6.8-fold) compared to that elicited by insulin (4.2-fold) or Li (4-fold). ^[3] SB 415286 (10 μM) inhibits rapamycin-induced down-regulation of cyclin D1, and blocks rapamycin and -induced apoptosis, suggesting a critical role for GSK3β in rapamycin-mediated -sensitization. ^[4] SB 415286 prevents coxsackievirus-induced cell death in a dose-dependent manner via stabilization of β-catenin. ^[5] SB 415286 exerts a protective effect on hydrogen peroxide-induced cell death in B65 rat neuroblastoma cells and neurons, while lithium does not attenuate the toxic effects of hydrogen peroxide. ^[7] SB 415286 treatment potentiates TRAIL- and CH-11-induced apoptosis in HepG2 cells. ^[8] Inhibition of GSK-3 by SB 415286 causes multiple myeloma (MM) cell growth arrest and apoptosis through the activation of the intrinsic pathway. ^[9] SB 415286 decreases the viability of Neuro-2A cells, and induces the accumulation of cells in the G2/M phase of the cell cycle and subsequent apoptosis. ^[10]

Administration of SB 415286 (~10 mg/kg twice daily) reduces the extent and degree of the trinitrobenzene sulphonic acid (TNBS)-provoked colonic inflammation in the rat, and reduces the fall in body weight, which is related to downregulation of NF-κB activity, involved in the generation of proinflammatory mediators. ^[6] SB 415286 treatment at 1 mg/kg significantly delays the growth of Neuro-2A cells in vivo in nude mice. ^[10]

• GSK-3 activity assay

  GSK-3 kinase activity is measured, in the presence of various concentrations of SB 415286, in a reaction mixture containing final concentrations of: 1 nM human GSK3α or rabbit GSK3α; 50 mM MOPS pH 7.0; 0.2 mM EDTA; 10 mM Mg-acetate; 7.5 mM L-mercaptoethanol; 5% (w/v) glycerol; 0.01% (w/v) Tween-20; 10% (v/v) DMSO; 28 μM GS-2 peptide substrate. The GS-2 peptide sequence corresponds to a region of glycogen synthase that is phosphorylated by GSK-3. The assay is initiated by the addition of 0.34 μCi [³³P]γ-ATP (IC50 determinations) or 2.7 μCi [³³P]γ-ATP (Ki determinations). The total ATP concentration is 10 μM (IC50 determinations) or ranged from 0 to 45 μM (Ki determinations). Following 30 minutes incubation at room temperature the assay is stopped by the addition of one third assay volume of 2.5% (v/v) H₃PO₄ containing 21 mM ATP. Samples are spotted onto P30 phosphocellulose mats and these are washed six times in 0.5% (v/v) H₃PO₄. The filter mats are sealed into sample bags containing Wallac betaplate scintillation fluid. 33P incorporation into the substrate peptide is determined by counting the mats in a Wallac microbeta scintillation counter.

• Cell lines

  OPM-2, RPMI-8226, U-266, and INA-6

• Concentrations

  Dissolved in DMSO, final concentrations ~10 μM

• Incubation Time

  48, or 72 hours

• Method

  Cells are exposed to different concentrations of SB 415286 for 48 or 72 hours in 96-flat well plates. After 48 or 72 hours, [³H]thymidine is added to the cultures (10 μCi/well) for the last 12 hours. The [³H]thymidine incorporation is evaluated by scintillation counting by using a top count β-counter. Apoptosis is assessed by annexin V/Propidium Iodide staining or by detection of mitochondrial membrane potential. Cell death is evaluated by the analysis of Forward/Side scatter fluorescence changes. Fluorescence Activated Cell Sorting (FACS) analysis is performed using a FACS-Calibur Cell Cytometer.

• Animal Models

  Male Wistar rats with acute colitis provoked by trinitrobenzene sulphonic acid (TNBS)

• Dosages

  ~1 mg/kg

• Administration

  Administered subcutaneously twice daily