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 a crucial role in regulating cancer cell proliferation, growth, survival, and metabolism. Serine/threonine kinase Akt is a core node in the PI3K pathway and is frequently activated in a significant number of human solid tumors, making Akt a highly attractive therapeutic target. Akt inhibitors target signaling pathways downstream of several important growth factors and their tyrosine kinase receptors (such as HER2, IGF1R, EGFR, and c-MET), thus holding promise for a wide range of treatments for various human tumors. Using conventional compound screening methods, we identified inhibitors that simultaneously block Akt enzyme activation and kinase activity. Structure-activity relationship (SAR) medicinal chemistry studies of these lead compounds yielded highly efficient and selective Akt compounds, including MK-2206. MK-2206 inhibits Akt isoenzymes 1, 2, and 3, with in vitro IC50 values of 8, 12, and 65 nM, respectively. MK-2206 is an allosteric inhibitor whose activity requires the presence of the Pleckstrin homologous domain, thus exhibiting high selectivity for Akt. At a concentration of 1956 M, it showed no inhibitory activity against more than 250 protein kinases. In various cancer cell lines, MK-2206 inhibited Akt1 kinase activity (IC50 of 20 nM), but its inhibitory potency against Akt2 and Akt3 activity was 2 to 6 times lower. MK-2206 inhibited the autophosphorylation of Akt T308 and S473 and prevented Akt-mediated phosphorylation of downstream signaling molecules, including TSC2, PRAS40, and ribosomal S6 proteins. MK-2206 exhibits potent antiproliferative activity against a variety of cancer cell lines carrying one or more of the following gene defects: 1) gene amplification leading to constitutive activation of upstream receptor tyrosine kinases (such as HER2); 2) PI3KCA activating mutations; 3) inactivation of the tumor suppressor PTEN; and 4) amplification and mutation of Akt itself. In addition, activation of the Ras pathway often indicates no response to MK-2206. [1] MK-2206 is currently undergoing two Phase I clinical trials, one in healthy volunteers (HV) and the other in cancer patients. In the first human healthy volunteer trial, 24 healthy male subjects participated in this Phase I randomized, double-blind, placebo-controlled, sequential, multi-cycle, escalating single oral dose study. Eight subjects were randomized to three groups (Group A, Group B and Group C), and in each treatment cycle, the same six subjects in each group received MK-2206 after an overnight fast, and two subjects received placebo. Volunteers received single doses ranging from 0.25 to 100 mg, and blood samples were collected before and at pre-defined post-dose time points for pharmacokinetic and pharmacodynamic (whole blood phosphorylated Akt inhibition) analysis. Results showed that MK-2206 was generally well tolerated with single doses up to 100 mg. No serious clinical or laboratory adverse events were reported. The most common adverse events were headache, common cold, and diarrhea. One subject withdrew from the study due to a clinical adverse event of blurred vision, but the symptom resolved. No clinically significant changes were found in laboratory safety tests or electrocardiographic assessments. None of these subjects developed clinically significant hyperglycemia or hyperinsulinemia. Preliminary pharmacokinetic results showed rapid absorption of orally administered MK-2206, with a median time to peak concentration (Tmax) of 6 to 8 hours and a median half-life of 55 to 78 hours. AUC_0-t and C_max were dose-proportional in the dose range of 2 mg to 100 mg. Preliminary pharmacodynamic results showed that single doses of 40 mg, 80 mg, and 100 mg MK-2206 resulted in stronger Akt inhibition in whole blood than placebo. The 80 mg and 100 mg dose groups reached peak Akt inhibition at 6 hours post-administration, with mean plasma concentrations >65 nM. Akt inhibition was observed within 2 to 24 hours. In summary, MK-2206 was well tolerated in healthy subjects after a single dose. The pharmacokinetic profile of MK-2206 is dose-proportional and there is clear evidence of Akt inhibition. Clinical development of MK-2206 in cancer patients is underway, with a focus on tumors with PI3K pathway activation events. [1] Serine/threonine kinase Akt is a key signaling node downstream of phosphatidylinositol-3-kinase and plays an important role in promoting cell survival and inhibiting apoptosis. Akt inhibitors may be particularly effective against cancers associated with enhanced Akt signaling and decreased sensitivity to cytotoxic drugs or receptor tyrosine kinase inhibitors. We evaluated the efficacy of a novel allosteric Akt inhibitor, MK-2206, in combination with several anticancer drugs. In vitro experiments showed that MK-2206 synergistically inhibited the proliferation of human cancer cell lines when used in combination with molecularly targeted drugs 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 Akt phosphorylation in erlotinib-insensitive cells by MK-2206 is one of its synergistic mechanisms, and this synergistic effect was observed even in erlotinib-insensitive cell lines. In lung cancer NCI-H460 or ovarian cancer A2780 cells, MK-2206 also showed synergistic effects when used in combination with cytotoxic drugs such as topoisomerase inhibitors (doxorubicin, camptothecin), antimetabolites (gemcitabine, 5-fluorouracil), antimicrotubule agents (docetaxel), and DNA cross-linking agents (carboplatin). Synergistic effects with docetaxel depend on the order of administration; administration of MK-2206 before docetaxel administration is ineffective. MK-2206 inhibits carboplatin- and gemcitabine-induced Akt phosphorylation. In vivo, MK-2206 exhibits significantly stronger tumor-suppressive activity when used in combination with these drugs than when used alone. These findings suggest that Akt inhibition may enhance the efficacy of existing anticancer therapies; therefore, MK-2206 holds promise as an effective treatment for cancer patients receiving these cytotoxic drugs and/or molecularly targeted therapies. [2]

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Inhibition of the survival kinase Akt can induce apoptosis and has been found to activate autophagy, which may interfere with tumor attack. In this study, we explored the mechanism by which MK-2206 (the first small molecule Akt allosteric inhibitor to enter clinical development) modulates apoptosis and autophagy in tumor cells by regulating Akt. In human glioma cells, Akt inhibition mediated by MK-2206 or siRNA significantly activated autophagy, while silencing eukaryotic elongation factor-2 (eEF-2) kinase (a protein synthesis regulator) attenuated this autophagy response. eEF-2 silencing inhibited MK-2206-induced autophagy while also promoting apoptosis. Similarly, siRNA-mediated eEF-2 kinase inhibition enhanced the efficacy of MK-2206 against glioma cells. In summary, these results suggest that attenuating autophagy and enhancing apoptosis by inhibiting eEF-2 kinase can modulate the sensitivity of glioma cells to Akt inhibitors. Our findings suggest that targeting eEF-2 kinase may enhance 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.)