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Protocol
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Kinase Assay
[4]
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| Akt kinases assay |
Akt kinases are assayed by a GSK-derived biotinylated peptide substrate. The extent of peptide phosphorylation is determined by Homogeneous Time Resolved Fluorescence (HTRF) using a lanthanide chelate (Lance)-coupled monoclonal antibody specific for the phosphopeptide in combination with a streptavidin-linked allophycocyanin (SA-APC) fluorophore which will bind to the biotin moiety on the peptide. When the Lance and APC are in proximity, a non-radiative energy transfer takes place from the Lance to the APC, followed by emission of light from APC at 655 nm. Working Solution: 100X protease inhibitor cocktail (PIC): 1mg/mL benzamidine, 0.5 mg/mL pepstatin, 0.5 mg/mL leupeptin, 0.5 mg/mL aprotinin; 10X assay buffer: 500 mM HEPES, pH7.5, 1% PEG, 16.6 mM EDTA, 1 mM EGTA, 1% BSA, 20 mM 9-glycerol phosphate; Quench buffer 50 mM HEPES pH 7.3, 16.6 mM EDTA, 0.1% BSA, 0.1% Triton X-100, 0.17 nM labeled monoclonal antibody, 0.0067 mg/mL SA-APC; ATP/MgCl2 working solution: 1X Assay buffer, 1 mM DTT, 1X PIC, 5% glycerol, active Akt; Peptide working solution: 1X Assay buffer, 1 mM DTT, 1X PIC, 5% glycerol, 2 TM GSK biotinylated peptide. The reaction is assembled by adding 16 μL of ATP/MgCl2 working solution to the appropriate wells. MK-2206 or vehicle (1.0 μL) is added followed by 10 μL of peptide working solution. The reaction is started by adding 13 μL of the enzyme working solution and mixing. The reaction is allowed to proceed for 50 min and then stopped by the addition of 60 μL HTRF quench buffer. The stopped reactions are incubated at room temperature for at least 30 min and then read in the instrument. |
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Cell Assay
[2]
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| Cell Lines |
A431, HCC827, NCI-H292, NCI-H358, NCI-H23, NCI-H1299, Calu-6 and NCI-H460 cells |
| Concentrations |
0, 0.3, 1 and 3 μM |
| Incubation Time |
72 or 96 hours |
| Methods |
MK-2206 is dissolved in DMSO as a stock solution and diluted by culture media before use. Cells are seeded at a density of 2-3 × 103 in 96-well plates and incubated for 24 hours. Then MK-2206 (0, 0.3, 1 and 3 μM) is added to the cells. Cell proliferation is determined after 72 or 96 hours. |
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Animal Study
[2]
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| Animal Models |
SK-OV-3, NCI-H292, HCC70, PC-3, and NCI-H460 models in male CD1-nude mice |
| Formulation |
Formulated in 30% Captisol |
| Doses |
120 mg/kg |
| Administration |
Orally administered |
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References |
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[1] Yan L, AACR Annual Meeting 2009: Abstract Number: DDT01-1.
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[2] Hirai H, et al. Mol Cancer Therapy, 2010, 9(7), 1956-1967.
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[3] Cheng Y, et al, Cancer Res, 2011, 71(7), 2654-2663.
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[4] WO2008070016 (A2)
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