生物活性

MK-2206 is a highly selective inhibitor of Akt1, Akt2, and Akt3 with IC50 values of 8 nM, 12 nM and 65 nM, respectively. 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 shows synergistic responses in combination with cytotoxic agents such as erlotinib or lapatinib in lung NCI-H460 or ovarian A2780 tumor cells.

体外研究

体内研究

15% Captisol

激酶实验

Total PKB and PKBβ assay[1]

Total PKB and PKBβ activities were measuredas follows. Muscle lysates (containing500 μg of protein) wereimmunoprecipitated either with 2 μg of anti-(PKB PH domain) antibody (for totalactivity) or with 2 μg of anti-PKBβ antibody (for PKBβ activity) pre-bound to20 μl of Protein G–Sepharose. The immunoprecipitates were washed twice with 0.5ml of lysis buffer and once with 0.5 ml of buffer A (containing 10 mM Mops, pH7.0, 0.5 mM EDTA, 0.1% 2- mercaptoethanol and 10 mM magnesium acetate). Proteinkinase activity was then measured in a final volume of 50 μl of buffer A supplementedwith 2 mM dithiothreitol, 0.02%Brij 35, 200 μM substrate peptide and 0.1 mM [γ-³²P]ATP (specific radioactivity of 1000 c.p.m./pmol). After 30 minof incubation at 30◦C with continual gentle mixing, 20 μl aliquots ofsupernatant were taken and spotted on to Whatman P81 papers for thedetermination of ³²P-incorporation by scintillation counting.

细胞实验

Cell lines[2]

MV-4-11, MOLM-13, OCI/AML3, and U937 humanleukemia cell lines were cultured in RPMI 1640 medium containing 10 % fetalbovine serum in a 37 ^oC incubator supplied with 5 % CO₂.

Chemicals

MK-2206 and MG132 were dissolved in DMSO,and the final concentration of DMSO was kept below 0.01 % (v/v) during cellculture treatment. Lithium chloride was dissolved in double-distilled water andstored at 4^oC.

Cellviability assay

Cell viability was assessed using the MTSassay. Briefly, leukemia cells and peripheral blood mononuclear cells (PBMCs)were seeded in 96-well plates at 3000–5000 cells per well and were exposed tovarious concentrations of drugs for the indicated times. At the end of each setof experiments, 20μL of CellTiter 96 AQueous One Solution Reagent was added to eachwell, and the cells were incubated at 37^oC for 2 h. The absorbanceat 490 nm was determined for each well using an ELISA plate reader.Determination of cell viability was calculated by measuring relative changes inabsorbance. IC50s were calculated by CompuSyn software.

Westernblotting

After drug treatment, leukemia cells werelysed in lysis buffer [50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1 % NP-40, 0.5 %sodium deoxycholate, 0.1 % SDS, protease inhibitor cocktail, and phosphataseinhibitor], incubated on ice for 30 min, and followed by centrifugation for 10min at 18,000xg. Protein concentrations were measured using the Quick StartBradford Protein Assay. Equal amounts of proteins from each sample weresubjected to SDS-PAGE and then transferred to polyvinylidene fluoride membranes,followed by 5 % nonfat milk blocking in PBS-T (phosphate-buffered saline with0.1 % Tween-20) for 1 h. After overnight incubation at 4^oC withappropriate primary antibodies, the membranes were washed with PBS-T, incubatedwith a horseradish peroxidase-conjugated secondary antibody, exposed to theWestern Lightning Plus-ECL enhanced chemiluminescence substrate, and thenimaged by an LAS-4000 image reader for signal detection. Target proteins weredetected using individual specific primary antibodies. The band density inwestern blots was quantified by ImageJ software.

Apoptosisassay

The FITC Annexin V Apoptosis Detection KitI, propidium iodide (PI, 50 lg/mL), and Epics XL flow cytometer were used todetermine early or late phases of cell apoptosis. The fraction of apoptoticcells after drug treatment was assessed by sub-G1 fraction analysis and AnnexinV analysis in a flow cytometer. Briefly, leukemia cells were treated with drugsfor 72 h at the indicated concentrations and were then collected, washed twicein phosphate-buffered saline, and resuspended in 100μL 19 AnnexinV Binding Buffer. Then, 5 μL Annexin V-FITCand 5μL PI were added to the resuspended cells, followed by incubation for15 min at room temperature in the dark. Finally, 400μL 19 Annexin VBinding Buffer was added. For each sample, 10,000 cells were analyzed by flowcytometry.

动物实验

In Vivo Xenograft Experiments[3]

NOD.Cg-Prkdc^scidI12rg^tm1Wjl/SzJ(NSG) or NOD.CB17-Prkdc^scid/J (non-obese diabetic/severe combined immunodeficiency[NOD/SCID]) mice were used at 6–8 weeks of age. Fluorescence- activated cellsorting (FACS)-sorted SW480 cells (CD133^high, CD133⁺,CD133^-) were resuspended 1:1 in Matrigel and serum-free medium, andwere injected subcutaneously into the flank at 5 x 10⁴ cells per mouse.For the in vivo treatment, MK-2206 (100 mg/kg) was administered orally threetimes a week when tumor size reached 0.2–0.3 cm in diameter. Tumor growth was measuredtwice weekly using a caliper, and mice were sacrificed when tumor size reacheda diameter of 1 cm.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Lai YC, Liu Y, Jacobs R, Rider MH. A novel PKB/Akt inhibitor, MK-2206, effectively inhibits insulin-stimulated glucose metabolism and protein synthesis in isolated rat skeletal muscle. Biochem J. 2012;447(1):137-147.
[2] Lu JW, Lin YM, Lai YL, et al. MK-2206 induces apoptosis of AML cells and enhances the cytotoxicity of cytarabine. Med Oncol. 2015;32(7):206.
[3] Malkomes P LI, Luetticke A, Oppermann E, Haetscher N, Serve H, Holzer K, Bechstein WO, Rieger MA. Selective AKT Inhibition by MK-2206 Represses Colorectal Cancer-Initiating Stem Cells. Ann Surg Oncol. 2016;23(9):2849-2857.

体内溶解度

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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