MEK2 (IC50 = 60 nM); MEK1 (IC50 = 70 nM)

U0126 处理可有效降低 A549 细胞中所有检查毒株的子代病毒滴度。虽然 nM 浓度的 U0126 可有效减少 H1N1v 和 H5N1 (MB1)，但需要 μM 浓度的 U0126 才能降低 H5N1 (GSB) 和 H7N7 病毒滴度。 U0126-EtOH 针对 H1N1v 的 EC50 值在 A549 细胞中为 1.2±0.4 μM，在 MDCKII 细胞中为 74.7±1.0 μM[2]。胎牛血清（FCS）刺激的大鼠肝癌细胞（FAO）表现出显着的S期细胞比例（32.62％），而U0126显着降低了S期细胞比例（9.92％）并增加了G0-细胞比例G1 期以及较小程度的 G2/M[3]。

每天给小鼠施用U0126-EtOH（U0126；腹膜内注射，10.5 mg/kg）。整个对照实验中的肿瘤大小要么恒定，要么略有增加。另一方面，在所有 U0126-EtOH 实验中，植入和早期肿瘤生长均显着减少。此外，注射后 9 天及此后，用 U0126-EtOH 治疗的肿瘤体积减少了 60-70%[3]。对大鼠进行短暂大脑中动脉闭塞（tMCAO）120分钟，并在再灌注后0和24小时给予U0126-EtOH（U0126；腹膜内注射，30mg/kg）。接受U0126-EtOH治疗后，S6c的血管收缩显着降低[4]。

在这些测定中，调整免疫沉淀的野生型 MEK 的量以产生与 10 nM 重组 MEK 相同数量的活性单位。 96 孔硝化纤维过滤器装置用于测量反应速度，如下详述。除非另有说明，所有反应均使用 10 nM 酶浓度、20 mM Hepes、10 mM MgCl2、5 mM β-巯基乙醇、0.1 mg/mL BSA、pH 7.4 和室温。向预混合的 MEK/ERK/抑制剂反应混合物中添加 [γ-33P]ATP 以启动反应。然后每 6 分钟取出 100 μL 等分试样，转移至含有 50 mM EDTA 的 96 孔硝酸纤维素膜板中以终止反应。将膜板拉出并用缓冲液真空清洗四次。然后将30μL Microscint-20闪烁液倒入孔中，使用Top Count闪烁计数器测量33P-磷酸化ERK的放射性。根据放射性与时间图的斜率，可以计算出速度。除非另有说明，ERK 和 ATP 浓度分别为 400 nM 和 40 μM。存在和不存在抑制剂时的初始反应速度分别称为 Vi 和 Vo，它们用于计算所有体外酶测定的抑制百分比。然后通过使用非线性最小二乘回归将数据拟合到 Langmuir 等温线的标准方程来计算 IC50。然后将数据绘制为抑制剂浓度与抑制百分比的函数关系。如上所述，酶浓度不是通过活性位点滴定，而是通过最终测定体积中使用的分子量和蛋白质质量来确定。因此，酶活性位点的实际浓度可能与报告值不同。

A.E7 或 Th17 细胞与已用丝裂霉素 C 以及不同浓度的鸽子细胞色素 c、PR8 Ag 或 5 U/mL 人 rIL-2 处理的 B10.BR 或 BALB/c 脾细胞一起孵育。为了确定 MEK 抑制对 T 细胞增殖的直接影响，一些测定还包含 U0126 或无活性类似物 U0124。每个孔在培养开始两天后接受 1μCi [3H]TdR 脉冲，并在第二天收获培养物。在不使用液体闪烁混合物的情况下，在 Packard Matrix 96 直接 β 计数器上测量 [3H]TdR 掺入 DNA 的情况。

Athymic female nude mice (SWISS, nu/nu)[3].
10.5 mg/kg.
Intraperitoneal injection daily.

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Sex differences are well known in cerebral ischemia and may impact the effect of stroke treatments. In male rats, the MEK1/2 inhibitor U0126 reduces ischemia-induced endothelin type B (ETB) receptor upregulation, infarct size and improves acute neurologic function after experimental stroke. However, responses to this treatment in females and long-term effects on outcome are not known. Initial experiments used in vitro organ culture of cerebral arteries, confirming ERK1/2 activation and increased ETB receptor-mediated vasoconstriction in female cerebral arteries. Transient middle cerebral artery occlusion (tMCAO, 120 minutes) was induced in female Wistar rats, with U0126 (30 mg/kg intraperitoneally) or vehicle administered at 0 and 24 hours of reperfusion, or with no treatment. Infarct volumes were determined and neurologic function was assessed by 6-point and 28-point neuroscores. ETB receptor-mediated contraction was studied with myograph and protein expression with immunohistochemistry. In vitro organ culture and tMCAO resulted in vascular ETB receptor upregulation and activation of ERK1/2 that was prevented by U0126. Although no effect on infarct size, U0126 improved the long-term neurologic function after experimental stroke in female rats. In conclusion, early prevention of the ERK1/2 activation and ETB receptor-mediated vasoconstriction in the cerebral vasculature after ischemic stroke in female rats improves the long-term neurologic outcome [4].

[1]. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem. 1998 Jul 17;273(29):18623-32.

[2]. Antiviral activity of the MEK-inhibitor U0126 against pandemic H1N1v and highly pathogenic avian influenza virus in vitro and in vivo. Antiviral Res. 2011, 92(2), 195-203.

[3]. RNAi-mediated ERK2 knockdown inhibits growth of tumor cells in vitro and in vivo. Oncogene. 2008 Sep 11;27(40):5315-25.

[4]. U0126 attenuates cerebral vasoconstriction and improves long-term neurologic outcome after stroke in female rats. J Cereb Blood Flow Metab. 2015 Mar;35(3):454-60.

U0126 is an aryl sulfide that is (2Z,3Z)-bis[amino(sulfanyl)methylidene]butanedinitrile in which the sulfanyl hydrogens are replaced by 2-aminophenyl groups. An inhibitor of mitogen-activated protein kinase that also exhibits anti-cancer properties. It has a role as an EC 2.7.11.24 (mitogen-activated protein kinase) inhibitor, an apoptosis inducer, an antineoplastic agent, an antioxidant, an osteogenesis regulator and a vasoconstrictor agent. It is an enamine, an aryl sulfide, a substituted aniline and a dinitrile.
U-0126 is a direct inhibitor of the mitogen-activated protein-kinase kinase family members, MEK-1 and MEK-2.
U-0126 is a synthetic organic compound that selectively inhibits the kinase activity of Mitogen-Activated Protein kinase, preventing cytokine and prostaglandin E2 production.

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The emergence of the 2009 H1N1 pandemic swine influenza A virus is a good example of how this viral infection can impact health systems around the world in a very short time. The continuous zoonotic circulation and reassortment potential of influenza A viruses (IAV) in nature represents an enormous public health threat to humans. Beside vaccination antivirals are needed to efficiently control spreading of the disease. In the present work we investigated whether the MEK inhibitor U0126, targeting the intracellular Raf/MEK/ERK signaling pathway, is able to suppress propagation of the 2009 pandemic IV H1N1v (v=variant) as well as highly pathogenic avian influenza viruses (HPAIV) in cell culture and also in vivo in the mouse lung. U0126 showed antiviral activity in cell culture against all tested IAV strains including oseltamivir resistant variants. Furthermore, we were able to demonstrate that treatment of mice with U0126 via the aerosol route led to (i) inhibition of MEK activation in the lung (ii) reduction of progeny IAV titers compared to untreated controls (iii) protection of IAV infected mice against a 100× lethal viral challenge. Moreover, no adverse effects of U0126 were found in cell culture or in the mouse. Thus, we conclude that U0126, by inhibiting the cellular target MEK, has an antiviral potential not only in vitro in cell culture, but also in vivo in the mouse model.[2]

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Sex differences are well known in cerebral ischemia and may impact the effect of stroke treatments. In male rats, the MEK1/2 inhibitor U0126 reduces ischemia-induced endothelin type B (ETB) receptor upregulation, infarct size and improves acute neurologic function after experimental stroke. However, responses to this treatment in females and long-term effects on outcome are not known. Initial experiments used in vitro organ culture of cerebral arteries, confirming ERK1/2 activation and increased ETB receptor-mediated vasoconstriction in female cerebral arteries. Transient middle cerebral artery occlusion (tMCAO, 120 minutes) was induced in female Wistar rats, with U0126 (30 mg/kg intraperitoneally) or vehicle administered at 0 and 24 hours of reperfusion, or with no treatment. Infarct volumes were determined and neurologic function was assessed by 6-point and 28-point neuroscores. ETB receptor-mediated contraction was studied with myograph and protein expression with immunohistochemistry. In vitro organ culture and tMCAO resulted in vascular ETB receptor upregulation and activation of ERK1/2 that was prevented by U0126. Although no effect on infarct size, U0126 improved the long-term neurologic function after experimental stroke in female rats. In conclusion, early prevention of the ERK1/2 activation and ETB receptor-mediated vasoconstriction in the cerebral vasculature after ischemic stroke in female rats improves the long-term neurologic outcome.[4]

C18H16N6S2

380.49

380.087

C, 56.82; H, 4.24; N, 22.09; S, 16.85

109511-58-2

U0126-EtOH;1173097-76-1

3006531

White to off-white solid powder

1.4±0.1 g/cm3

565.1±50.0 °C at 760 mmHg

295.6±30.1 °C

0.0±1.5 mmHg at 25°C

1.762

-1.07

202.26

4

8

5

26

610

0

N#CC(/C(C#N)=C(N)/SC1=CC=CC=C1N)=C(N)\SC2=CC=CC=C2N

DVEXZJFMOKTQEZ-JYFOCSDGSA-N

InChI=1S/C18H16N6S2/c19-9-11(17(23)25-15-7-3-1-5-13(15)21)12(10-20)18(24)26-16-8-4-2-6-14(16)22/h1-8H,21-24H2/b17-11+,18-12+

(2Z,3Z)-2,3-bis[amino-(2-aminophenyl)sulfanylmethylidene]butanedinitrile

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.57 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (6.57 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.57 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 10% DMSO+50% PEG 300+ddH2O: 28mg/mL

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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。