Kinase 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/MgCl2working 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/MgCl2working 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.Cell Assay: MK-2206 is dissolved in DMSO as a stock solution and diluted by culture media before use. Cells (A431, HCC827, NCI-H292, NCI-H358, NCI-H23, NCI-H1299, Calu-6 and NCI-H460 cells) are seeded at a density of 2-3 × 103in 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.MK-2206 is an allosteric inhibitor and is activated by the pleckstrin homology domain. MK-2206 inhibits auto-phosphorylation of both Akt T308 and S473. MK-2206 also prevents Akt-mediated phosphorylation of downstream signaling molecules, including TSC2, PRAS40 and ribosomal S6 proteins. MK-2206 inhibits Ras wild-type (WT) cell lines (A431, HCC827, and NCI-H292) more potently when compared to Ras-mutant cell lines (NCI-H358, NCI-H23, NCI-H1299, and Calu-6). MK-2206 also shows synergistic responses in combination with cytotoxic agents such as erlotinib or lapatinib in lung NCI-H460 or ovarian A2780 tumor cells.MK-2206 or siRNA-mediated Akt inhibition strongly activates autophagy in human glioma cells. However, eukaryotic elongation factor-2 (eEF-2) silencing suppresses MK-2206-induced-autophagy, with a promotion of apoptotic cell death.

MK-2206 shows 60% TGI and inhibits more than 70 % of phospho-Akt1/2 (T308 and S473) in A2780 ovarian cancer xenografts at a dose of 240 mg/kg. MK-2206 exhibits significant antitumor activity in NCI-H292 xenograft in combination with erlotinib or lapatinib.

SK-OV-3, NCI-H292, HCC70, PC-3, and NCI-H460 models in male CD1-nude mice;

:Mol Cancer Ther.2010 Jul;9(7):1956-67;Cancer Res.2011 Apr 1;71(7):2654-63.

C25H21N5O

407.46714

1032349-93-1

MK-2206 dihydrochloride;1032350-13-2;MK-2206;1032349-77-1

C1=CC=C(C=C1)C2=C(C3=CC=C(C=C3)C4(CCC4)N)N=C5C=CN6C(=NN=C6O)C5=C2

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)