GSK2606414   中文名称: GSK-2606414

EIF2AK3 (PERK) (IC50 = 0.4 nM); EIF2AK1 (HRI) (IC50 = 420 nM); EIF2AK2 (PKR) (IC50 = 696 nM)

GSK2606414 抑制细胞中 PERK 激活[1]。

GSK2606414（50 和 150 mg/kg，口服）可防止小鼠肿瘤异种移植物生长[1]。

GST-PERK 胞质结构域是购买的。 6-His 全长人 eIF2 从 Sf9 昆虫细胞中的杆状病毒表达中纯化。 eIF2 蛋白通过透析到 PBS 中进行缓冲液交换，通过 NHS-LC-生物素进行化学修饰，然后通过透析到 50 mM Tris、pH 7.2、250 mM NaCl 和 5 mM DTT 中进行缓冲液交换。将蛋白质等分并保存在 80°C。将新鲜配制的淬灭溶液添加到反应中后，会产生 4 nM eIF2 磷酸 Ser51 抗体、已用 Eu-1024 标记的 4 nM 抗兔 IgG、40 nM 链霉亲和素 Surelight APC 和 15 mM 的终浓度乙二胺四乙酸。反应在黑色 384 孔聚苯乙烯低体积板中进行，最终体积为 10 μL。将所研究的化合物溶解在 DMSO 中至浓度为 1.0 mM，然后在 DMSO 中连续稀释 1-3 至 11 倍。将每个浓度以 0.1 μL 的量转移至测定板的相应孔中。结果产生 0.00017 至 10 μM 的最终化合物浓度范围。为了测定含有化合物的板，在室温下预孵育 30 分钟后添加 GST-PERK 溶液。添加 ATP 和 eIF2 底物溶液以启动反应。孵育1小时后添加淬灭溶液。在信号检测之前，将板在室温下覆盖 2 小时。在 Viewlux 阅读器上，产生的信号被量化。通过使用 APC/Eu 计算转换数据，APC 信号被归一化为铕信号。浓度响应曲线的信息绘制为使用数据缩减公式 100 × [1 – (U1 – C2)/(C1 – C2)] 与化合物浓度计算的抑制百分比，其中 U 是未知值，C1 是平均值C2 是 1% DMSO 获得的对照值，C2 是 0.1 M EDTA 获得的平均对照值。使用最小响应、最大响应、斜率因子、化合物浓度和抑制浓度 (IC50) 将数据拟合到曲线。每种化合物的结果均以 IC50 值列出。

PERK 抑制剂（GSK2606414，化合物 38）孵育后，用毒胡萝卜素刺激细胞。两小时后裂解细胞并检查 PERK 自磷酸化抑制。

Female nude mice have their right flank subcutaneously implanted with exponentially growing BxPC3 tumor cells (10×10⁶ cells/mouse) cultivated in cell culture. Mice with tumors measuring less than 200 mm³ are randomly assigned to one of eight treatment groups sixteen days post-implantation. The animals are given the compound at 50 or 150 mg/kg, b.i.d., for 21 days along with a vehicle (0.5% hydoxypropylmethylcellulose, 0.1% Tween 80 in water, pH 4.8). Using calipers, the tumor volume is measured twice a week and computed. When the dosage is finished, the results are expressed as a percent inhibition, or 100[1-(average growth of the drug-treated population)/(average growth of the vehicle-treated control population)]. In statistical analysis, the two-tailed t test is employed.

[1]. J Med Chem . 2012 Aug 23;55(16):7193-207.

[2]. Cell Host Microbe . 2017 Dec 13;22(6):766-776.e4.

C24H20F3N5O

451.44

451.16199

C, 63.85; H, 4.47; F, 12.63; N, 15.51; O, 3.54

1337531-36-8

white solid powder

CN1C=C(C2=C(N=CN=C21)N)C3=CC4=C(C=C3)N(CC4)C(=O)CC5=CC(=CC=C5)C(F)(F)F

SIXVRXARNAVBTC-UHFFFAOYSA-N

InChI=1S/C24H20F3N5O/c1-31-12-18(21-22(28)29-13-30-23(21)31)15-5-6-19-16(11-15)7-8-32(19)20(33)10-14-3-2-4-17(9-14)24(25,26)27/h2-6,9,11-13H,7-8,10H2,1H3,(H2,28,29,30)

1-[5-(4-amino-7-methylpyrrolo[2,3-d]pyrimidin-5-yl)-2,3-dihydroindol-1-yl]-2-[3-(trifluoromethyl)phenyl]ethanone

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.54 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one),clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above system and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (5.54 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one),suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.54 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one),clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 4: in 5% DMSO+45% PEG 300+ddH2O: 20mg/mL

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Solubility in Formulation 1: 3.33 mg/mL (7.38 mM) in 0.5% HPMC 0.2%Tween80 (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。