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

激酶测定：Akt 激酶通过 GSK 衍生的生物素化肽底物进行测定。肽磷酸化的程度通过均相时间分辨荧光 (HTRF) 测定，使用对磷酸肽具有特异性的镧系元素螯合物 (Lance) 偶联单克隆抗体，并结合链霉亲和素连接的别藻蓝蛋白 (SA-APC) 荧光团，该荧光团将与生物素结合肽上的部分。当喷枪和 APC 靠近时，会发生从喷枪到 APC 的非辐射能量转移，然后从 APC 发出 655 nm 的光。工作溶液：100X 蛋白酶抑制剂混合物 (PIC)：1mg/mL 苯甲脒、0.5 mg/mL 胃酶抑素、0.5 mg/mL 亮肽素、0.5 mg/mL 抑肽酶； 10X 检测缓冲液：500 mM HEPES，pH7.5，1% PEG，16.6 mM EDTA，1 mM EGTA，1% BSA，20 mM 9-甘油磷酸盐；淬灭缓冲液 50 mM HEPES pH 7.3、16.6 mM EDTA、0.1% BSA、0.1% Triton X-100、0.17 nM 标记单克隆抗体、0.0067 mg/mL SA-APC； ATP/MgCl2 工作溶液：1X 检测缓冲液、1 mM DTT、1X PIC、5% 甘油、活性 Akt；肽工作溶液：1X 检测缓冲液、1 mM DTT、1X PIC、5% 甘油、2 TM GSK 生物素化肽。通过向适当的孔中添加 16 µL ATP/MgCl2 工作溶液来组装反应。添加 MK-2206 或载体 (1.0 µL)，然后添加 10 µL 肽工作溶液。加入 13 μL 酶工作液并混合开始反应。允许反应进行 50 分钟，然后通过添加 60 µL HTRF 淬灭缓冲液来停止。终止的反应在室温下孵育至少30分钟，然后在仪器中读数。 细胞测定：MK-2206溶解在DMSO中作为储备溶液，并在使用前用培养基稀释。将细胞（A431、HCC827、NCI-H292、NCI-H358、NCI-H23、NCI-H1299、Calu-6 和 NCI-H460 细胞）以 2-3 × 103 的密度接种在 96 孔板中，并孵育 24小时。然后将 MK-2206（0、0.3、1 和 3 μM）添加到细胞中。 72 或 96 小时后测定细胞增殖。MK-2206 是一种变构抑制剂，由 pleckstrin 同源结构域激活。 MK-2206 抑制 Akt T308 和 S473 的自身磷酸化。 MK-2206 还可以防止 Akt 介导的下游信号分子磷酸化，包括 TSC2、PRAS40 和核糖体 S6 蛋白。与 Ras 突变细胞系（NCI-H358、NCI-H23、NCI-H1299 和 Calu-6）相比，MK-2206 更有效地抑制 Ras 野生型 (WT) 细胞系（A431、HCC827 和 NCI-H292） ）。 MK-2206 还在肺 NCI-H460 或卵巢 A2780 肿瘤细胞中与细胞毒性药物（例如厄洛替尼或拉帕替尼）联合显示出协同反应。MK-2206 或 siRNA 介导的 Akt 抑制可强烈激活人神经胶质瘤细胞中的自噬。然而，真核延伸因子 2 (eEF-2) 沉默会抑制 MK-2206 诱导的自噬，从而促进细胞凋亡。

MK-2206 在 240 mg/kg 剂量下显示 60% TGI，并抑制 A2780 卵巢癌异种移植物中超过 70% 的磷酸-Akt1/2（T308 和 S473）。 MK-2206 与厄洛替尼或拉帕替尼联合使用，在 NCI-H292 异种移植物中表现出显着的抗肿瘤活性。

Akt 激酶通过 GSK 衍生的生物素化肽底物进行检测。通过将磷酸肽特异的镧系螯合物 (Lance) 偶联单克隆抗体与链霉亲和素连接的别藻蓝蛋白 (SA-APC) 荧光团（可与肽的生物素部分结合）相结合，可以使用均相时间分辨荧光 (HTRF)以确定磷酸化程度。当喷枪和APC靠近时，喷枪将非辐射能量转移到APC，然后APC发射波长为655 nm的光。蛋白酶抑制剂混合物 (PIC) 100X：苯甲脒 1 mg/mL、胃酶抑素 0.5 mg/mL、亮肽素 0.5 mg/mL、抑肽酶 0.5 mg/mL； 10X 检测试剂：20 mM 9-甘油磷酸、50 mM HEPES、pH 7.3、16.6 mM EDTA、0.1% BSA、0.1% Triton X-100、0.17 nM 标记单克隆抗体和 0.0067 mg/mL SA-APC 组成淬灭缓冲液。 ATP/MgCl2 测定的工作溶液：1X 测定缓冲液、1 mM DTT、1X PIC、5% 甘油、活性 Akt；肽工作溶液：2 TM GSK 生物素化肽、1X 检测缓冲液、1 mM DTT、1X PIC 和 5% 甘油。通过向适当的孔中添加 16 µL ATP/MgCl2 工作溶液来组装反应。添加 MK-2206 或载体 (1.0 µL)，然后添加 10 µL 肽工作溶液。加入 13 μL 酶工作液并混合开始反应。允许反应进行 50 分钟，然后通过添加 60 µL HTRF 淬灭缓冲液来停止。停止的反应在室温下孵育至少30分钟，然后在仪器中读数。

在 96 孔板中，接种 2-3 × 103 个细胞，然后将板孵育 24 小时。之后，向细胞添加 MK-2206（0、0.3、1 和 3 μM）。 72或96小时后，评估细胞增殖。

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细胞增殖试验和组合指数测定[2]
在96孔板中以每孔2至3×103的密度接种细胞。镀敷后24小时，将不同浓度的药物（作为单一试剂或组合）加入孔中。在给药后72或96小时使用CellTiter-Glo测定法测定细胞增殖。根据Chou和Talalay的方法，使用组合指数（CI）评估药物相互作用的性质。从Calcusyn获得了一个商业软件包。在与多西他赛的组合中，我们测试了三种治疗序列：（a）MK-2206，然后是多西他赛——细胞暴露于MK-2206 24小时，然后在MK-2206洗脱后，细胞用多西他赛再治疗72小时；（b） 多西他赛，然后是MK-2206——细胞暴露于多西他赛24小时，然后在多西他赛洗脱后，细胞用MK-2206再处理72小时；以及（c）同时治疗——细胞暴露于MK-2206和多西他赛72小时。[2]
在添加10%胎牛血清的培养基中培养的LN229和T98G细胞用一系列浓度的MK-2206处理，并通过Western blot检测磷酸化-eEF-2和eEF-2的水平。使用微管蛋白作为负荷控制。（B） 用非靶向RNA或靶向eEF-2激酶的siRNA转染LN229和T98 G细胞，然后用MK-2206处理24小时。Western检测eEF-2激酶、LC3和p62。使用微管蛋白作为负荷控制。[3]

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MK-2206对人脑胶质瘤细胞自噬的影响[3]
（A） 在添加10%胎牛血清的培养基中培养的LN229和T98G细胞用MK2206处理24小时，通过Western blot检测LC3水平。（B） 在10nM巴非霉素A1存在或不存在的情况下，用MK2206处理LN229和T98G细胞24小时，并通过Western blot检测LC3水平。使用微管蛋白作为负荷控制。（C） LN229和T98G细胞用GFP-LC3质粒转染，然后用2.5或5μM MK2206处理24小时。治疗结束时，在60倍放大镜下检查细胞中GFP-LC3斑点的数量。条形图是具有10个或更多GFP-LC3斑点的细胞百分比的量化。每次治疗中至少对100个细胞进行评分。*p<0.05；**p<0.01，t检验，MK-2206与赋形剂相比。（D） 用10μM MK-2206处理LN229和T98G细胞24小时，用流式细胞术分析处理细胞中的AO荧光强度。（E） 用MK-2206（2.5μM）或载体处理的LN229细胞通过胰蛋白酶消化收获，固定并包埋在刺树脂中。切割90nm薄切片，用JEOL 1200EX透射电子显微镜在80Kv下检查。箭头表示自噬空泡。

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

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Efficacy studies in mouse xenograft models[2] Human tumor cells were suspended in 50% Matrigel (BD) and 50% PBS and were injected s.c. into the left flank of the mice.[2]
When the mean tumor size reached 0.13 cm3 for the SK-OV-3 or 0.2 cm3 for the NCI-H292, HCC70, PC-3, and NCI-H460 models, the mice were randomized into control and treatment groups with approximately equivalent ranges of tumor volume between groups (n = 5 animals per group). The following vehicles were used to dose the compounds: 30% Captisol for MK-2206; 0.5% methylcellulose + 0.1% Tween 80 for erlotinib; distilled water for lapatinib; 0.73% ethanol in saline for docetaxel; and saline for carboplatin and gemcitabine. The control group received vehicle only. Tumor volume was measured with calipers twice a week. Animal body weight and physical signs were monitored during the experiments.

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Briefly, 4–6 week-old female nude mice were inoculated subcutaneously with LN229 cells (5 × 106 cells/per site) with or without silencing of eEF-2 kinase. At day 7 after inoculation, MK-2206 (120 mg/kg, p.o.) was administered to the tumor-bearing mice. Tumors were harvested 24 h post drug administration for analysis of autophagy and apoptosis. Apoptosis was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling tetramethylrhodamine red apoptosis kit from Roche, and using Western blot analysis of cleaved caspase 3. Autophagy was detected by Western blot analysis of LC3 II.[3]

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

[2]. Mol Cancer Ther. 2010 Jul;9(7):1956-67.

[3]. Cancer Res. 2011 Apr 1;71(7):2654-63.

MK-2206 is an organic heterotricyclic compound that is [1,2,4]triazolo[3,4-f][1,6]naphthyridin-3(2H)-one substituted at positions 8 and 9 respectively by 4-(1-aminocyclobutyl)phenyl and phenyl groups. It has a role as an EC 2.7.1.137 (phosphatidylinositol 3-kinase) inhibitor. It is functionally related to a 1,6-naphthyridine.
Akt Inhibitor MK2206 is an orally bioavailable allosteric inhibitor of the serine/threonine protein kinase Akt (protein kinase B) with potential antineoplastic activity. Akt inhibitor MK2206 binds to and inhibits the activity of Akt in a non-ATP competitive manner, which may result in the inhibition of the PI3K/Akt signaling pathway and tumor cell proliferation and the induction of tumor cell apoptosis. Activation of the PI3K/Akt signaling pathway is frequently associated with tumorigenesis and dysregulated PI3K/Akt signaling may contribute to tumor resistance to a variety of antineoplastic agents.

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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]

C25H21N5O

407.46714

407.175

C, 73.69; H, 5.19; N, 17.19; O, 3.93

1032349-93-1

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

24964624

Solid powder

5.355

89.33

2

4

3

31

760

0

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

ULDXWLCXEDXJGE-UHFFFAOYSA-N

InChI=1S/C25H21N5O/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)

1,2,4-Triazolo(3,4-f)(1,6)naphthyridin-3(2H)-one, 8-(4-(1-aminocyclobutyl)phenyl)-9-phenyl-

MK 2206; MK-2206; 1032349-93-1; 8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-2H-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3-one; MK 2206; MK-2206 free base; MK-2206 (free base); 51HZG6MP1K; MK2206

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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