Akt1 (IC50 = 8 nM); Akt2 (IC50 = 12 nM); Akt3 (IC50 = 65 nM) Akt1 (IC50 = 8 nM); Akt2 (IC50 = 12 nM); Akt3 (IC50 = 65 nM)

In a dose- and time-dependent manner, MK-2206 (0-10 μM; 72 and 96 hours) suppresses the growth of the nasopharyngeal carcinoma (NPC) cell lines CNE-1, CNE-2, HONE-1, and SUNE-1[3]. For CNE-2 and HONE-1 cells, MK-2206 (0-10 μM; 24 and 48 hours) causes a dose-dependent rise in the proportion of G0/G1 phase cells and a corresponding decrease in S phase cell numbers[3]. The phosphorylation levels of S6 and PRAS40 are attenuated in a dose-dependent manner by MK-2206 (0-10 μM; 24 hours). GSKα/β and AKT phosphorylation is unaffected by MK-2206.[/3]. In CNE-2 cells, MK -2206 (0-10 μM; 24 hours) dose-dependently promotes the appearance of LC3-II. Autophagy requires the crucial protein microtubule-associated protein 1 (LC3)[3].

Human CNE-2 xenograft development in nude mice can be inhibited by MK-2206 at both oral gavage doses (480 mg/kg once a week and 240 mg/kg three times a week; for two weeks). In mice, no further apparent harm is noted[3]. When given orally, MK-2206 (120 mg/kg on alternate days) dramatically reduces the formation of tumors in 3–5 week old athymic nude mice harboring GEO colon cancer cells[4].

Akt kinases are assayed by a GSK-derived biotinylated peptide substrate. By combining a lanthanide chelate (Lance)-coupled monoclonal antibody that is specific for the phosphopeptide with a streptavidin-linked allophycocyanin (SA-APC) fluorophore that will bind to the peptide's biotin moiety, homogeneous time-resolved fluorescence (HTRF) can be used to determine the degree of phosphorylation. When the Lance and APC are close together, the Lance transfers non-radiative energy to the APC, and the APC then emits light at a wavelength of 655 nm. Protease inhibitor cocktail (PIC) 100X: Benzamidine 1 mg/mL, Pepstatin 0.5 mg/mL, Leupeptin 0.5 mg/mL, Aprotinin 0.5 mg/mL; 10X assay reagent: 20 mM 9-glycerol phosphate, 50 mM HEPES, pH 7.3, 16.6 mM EDTA, 0.1% BSA, 0.1% Triton X-100, 0.17 nM labeled monoclonal antibody, and 0.0067 mg/mL SA-APC make up the quench buffer. Working solution for the ATP/MgCl2 assay: 1X Assay buffer, 1 mM DTT, 1X PIC, 5% glycerol, active Akt; Peptide working solution: 2 TM GSK biotinylated peptide, 1X Assay buffer, 1 mM DTT, 1X PIC, and 5% glycerol. 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.

Cell Proliferation Assay[3]
Cell Types: The NPC cell lines CNE-1, CNE-2, HONE-1, and SUNE-1
Tested Concentrations: 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10 μM
Incubation Duration: 72 and 96 hrs (hours)
Experimental Results: At 72 and 96 hrs (hours), the IC50 values in CNE-1, CNE-2, and HONE-1 cell lines were 3-5 μM, and in SUNE-1, they were less than 1 μM.

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Cell Cycle Analysis[3]
Cell Types: CNE-2 and HONE-1 cells
Tested Concentrations: 0.625, 1.25, 2.5, 5, 10 μM
Incubation Duration: 24 or 48 hrs (hours)
Experimental Results: Induced cell cycle arrest at G1 in a dose-dependent manner.

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Western Blot Analysis[3]
Cell Types: SUNE-1 and CNE-2 cells
Tested Concentrations: 0.625, 1.25, 2.5, 5, 10 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Inhibited phosphorylation of AKT downstream targets. Cell Autophagy Assay[3]
Cell Types: CNE-2 cells
Tested Concentrations: 0.625, 1.25, 2.5, 5, 10 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Induced autophagy.

Animal/Disease Models: Four - to 6weeks old male BALB/c nude mice with CNE-2 xenografts[3]
Doses: 240 mg/kg and 480 mg/kg
Route of Administration: po (oral gavage); 240 mg/kg for three times a week; 480 mg/ kg for once a week; for 2 weeks
Experimental Results: Both doses inhibited the growth of human CNE-2 xenografts in nude mice.

[1]. Abstract #DDT01-1: MK-2206: A potent oral allosteric AKT inhibitor. 2009.

[2]. Phase II trial of AKT inhibitor MK-2206 in patients with advanced breast cancer who have tumors with PIK3CA or AKT mutations, and/or PTEN loss/PTEN mutation. Breast Cancer Res. 2019 Jul 5;21(1):78.

[3]. Effects of an oral allosteric AKT inhibitor (MK-2206) on human nasopharyngeal cancer in vitro and in vivo. Drug Des Devel Ther. 2014 Oct 10;8:1827-37.

[4]. Akt inhibitor MK-2206 promotes anti-tumor activity and cell death by modulation of AIF and Ezrin in colorectal cancer. BMC Cancer. 2014 Mar 1;14:145.

The PI3K pathway plays an important role in regulating cancer cell proliferation, growth, survival and metabolism. The serine/threonine kinase Akt, a central node of the PI3K pathway, is frequently activated in a significant proportion of human solid tumors, making Akt an attractive target for therapeutic intervention. Akt inhibitors, which target the signaling pathway downstream of some of the most important growth factors and their tyrosine kinase receptors such as HER2, IGF1R, EGFR, and c-MET, should have broad utility against a wide array of human tumors. Utilizing a traditional compound screening approach, we have identified Akt inhibitors that block both the activation and kinase activity of the enzyme. Medicinal chemistry exploration of the structure activity relationships of these leads resulted in potent and selective Akt compounds including MK-2206. MK-2206 inhibits Akt isozymes 1, 2, and 3 with in vitro IC50 values of 8, 12, and 65 nM, respectively. MK-2206 is an allosteric inhibitor requiring the presence of the Pleckstrin homology domain for activity, and therefore is highly selective against Akt exhibiting no inhibitory activities against over 250 protein kinases when tested at 1\#956;M. In multiple cancer cell lines, MK-2206 inhibited Akt1 kinase activity (IC50 \#8776; 20 nM), and blocked Akt2 and Akt3 activities with 2- to 6-fold less potency. MK-2206 inhibited auto-phosphorylation of both Akt T308 and S473, as well as prevented Akt-mediated phosphorylation of down-stream signaling molecules, including TSC2, PRAS40 and ribosomal S6 proteins. MK-2206 exhibited potent anti-proliferative activity against a number of cancer cell lines harboring one or more of the following genetic defects: 1) gene amplification resulting in constitutive activation of upstream receptor tyrosine kinases such as HER2, 2) a PI3KCA activating mutation, 3) inactivation of tumor suppressor PTEN, and 4) amplification and mutation of Akt itself. In addition, activation of the Ras pathway tended to predict non-response to MK-2206.[1]

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MK-2206 is currently being studies in two Phase I trials, one in healthy volunteers (HV) and one in cancer patients. In the first-in-human HV trial, twenty-four healthy, male subjects participated in this Phase I randomized, double-blind, placebo-controlled, sequential-panel, multiple-period, rising single oral dose study. Eight (8) subjects were assigned to each of 3 panels (Panels A, B, and C) where in each treatment period in a panel the same 6 subjects received MK-2206 and 2 subjects received placebo after an overnight fast. The volunteers were administered single doses from 0.25 to 100 mg and blood samples were collected predose and at prespecified postdose time points for pharmacokinetic and pharmacodynamic (whole blood inhibition of phospho Akt) assays. Single doses of MK-2206, up to 100 mg, were found to be generally well tolerated. No serious clinical or laboratory adverse experience was reported. The most commonly reported adverse experiences were headache, common cold, and diarrhea. One subject was discontinued from the study due to the clinical adverse experience of blurry vision which resolved. There were no clinically meaningful changes in laboratory safety tests or ECG evaluations. No clinically significant hyperglycemia or hyperinsulinemia was seen in these subjects. Preliminary pharmacokinetic results found that orally administered MK-2206 was readily absorbed with a median Tmax of 6 to 8 hours. The median half-life was 55 to 78 hours. AUC0-\#8734; and Cmax displayed dose proportional behavior from 2-mg to 100-mg. Preliminary pharmacodynamic results found that single doses of 40-, 80- and 100-mg MK-2206 inhibited Akt in whole blood to a greater extent than placebo. Maximum Akt inhibition occurred at 6 hours postdose for both the 80- and 100-mg doses with mean plasma concentrations of >65 nM. There was evidence of Akt inhibition from 2 through 24 hours. In conclusion, MK-2206 was generally well tolerated following single dose administration to healthy subjects. MK-2206 displays dose proportional pharmacokinetics with clear evidence of Akt inhibition. Clinical development of MK-2206 in cancer patients is ongoing with a focus on tumors harboring PI3K pathway activation events.[1]

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The serine/threonine kinase Akt lies at a critical signaling node downstream of phosphatidylinositol-3-kinase and is important in promoting cell survival and inhibiting apoptosis. An Akt inhibitor may be particularly useful for cancers in which increased Akt signaling is associated with reduced sensitivity to cytotoxic agents or receptor tyrosine kinase inhibitors. We evaluated the effect of a novel allosteric Akt inhibitor, MK-2206, in combination with several anticancer agents. In vitro, MK-2206 synergistically inhibited cell proliferation of human cancer cell lines in combination with molecular targeted agents such as erlotinib (an epidermal growth factor receptor inhibitor) or lapatinib (a dual epidermal growth factor receptor/human epidermal growth factor receptor 2 inhibitor). Complementary inhibition of erlotinib-insensitive Akt phosphorylation by MK-2206 was one mechanism of synergism, and a synergistic effect was found even in erlotinib-insensitive cell lines. MK-2206 also showed synergistic responses in combination with cytotoxic agents such as topoisomerase inhibitors (doxorubicin, camptothecin), antimetabolites (gemcitabine, 5-fluorouracil), anti-microtubule agents (docetaxel), and DNA cross-linkers (carboplatin) in lung NCI-H460 or ovarian A2780 tumor cells. The synergy with docetaxel depended on the treatment sequence; a schedule of MK-2206 dosed before docetaxel was not effective. MK-2206 suppressed the Akt phosphorylation that is induced by carboplatin and gemcitabine. In vivo, MK-2206 in combination with these agents exerted significantly more potent tumor inhibitory activities than each agent in the monotherapy setting. These findings suggest that Akt inhibition may augment the efficacy of existing cancer therapeutics; thus, MK-2206 is a promising agent to treat cancer patients who receive these cytotoxic and/or molecular targeted agents.[2]

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Inhibition of the survival kinase Akt can trigger apoptosis, and also has been found to activate autophagy, which may confound tumor attack. In this study, we investigated regulatory mechanisms through which apoptosis and autophagy were modulated in tumor cells subjected to Akt inhibition by MK-2206, the first allosteric small molecule inhibitor of Akt to enter clinical development. In human glioma cells, Akt inhibition by MK-2206 or siRNA-mediated attenuation strongly activated autophagy, whereas silencing of eukaryotic elongation factor-2 (eEF-2) kinase, a protein synthesis regulator, blunted this autophagic response. Suppression of MK-2206-induced autophagy by eEF-2 silencing was accompanied by a promotion of apoptotic cell death. Similarly, siRNA-mediated inhibition of eEF-2 kinase potentiated the efficacy of MK-2206 against glioma cells. Together, these results showed that blunting autophagy and augmenting apoptosis by inhibition of eEF-2 kinase could modulate the sensitivity of glioma cells to Akt inhibition. Our findings suggest that targeting eEF-2 kinase may reinforce the antitumor efficacy of Akt inhibitors such as MK-2206.[3]

C₂₅H₂₂CLN₅O

443.93

443.151

C, 67.64; H, 5.00; Cl, 7.99; N, 15.78; O, 3.60

1032349-77-1

MK-2206 dihydrochloride;1032350-13-2;MK-2206 free base;1032349-93-1

67254077

Solid powder

6.157

3

4

3

32

760

0

O=C1NN=C2C3C=C(C4C=CC=CC=4)C(C4C=CC(=CC=4)C4(CCC4)N)=NC=3C=CN21

LFYOZCBFOSSLNJ-UHFFFAOYSA-N

InChI=1S/C25H21N5O.ClH/c26-25(12-4-13-25)18-9-7-17(8-10-18)22-19(16-5-2-1-3-6-16)15-20-21(27-22)11-14-30-23(20)28-29-24(30)31;/h1-3,5-11,14-15H,4,12-13,26H2,(H,29,31);1H

8-(4-(1-aminocyclobutyl)phenyl)-9-phenyl-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3(2H)-one hydrochloride

MK2206; MK-2206 hydrochloride; 1032349-77-1; MK-2206 Monohydrochloride; UNII-4HA45S22ZZ; 4HA45S22ZZ; 1,2,4-Triazolo(3,4-f)(1,6)naphthyridin-3(2H)-one, 8-(4-(1-aminocyclobutyl)phenyl)-9-phenyl-, hydrochloride (1:1); 8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-2H-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3-one;hydrochloride; MK2206; MK 2206

2934.99.9001

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)

Typically soluble in DMSO (e.g. > 10 mM)

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.)