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

Treatment with U0126 effectively lowers progeny virus titers of all examined strains in A549 cells. While nM concentrations of U0126 are effective at reducing H1N1v and H5N1 (MB1),μM concentrations of U0126 are needed to lower H5N1 (GSB) and H7N7 virus titers. The EC50 values for U0126-EtOH against H1N1v are 1.2±0.4 μM in A549 cells and 74.7±1.0 μM in MDCKII cells[2]. Rat hepatocarcinoma cells (FAO) stimulated by fetal calf serum (FCS) exhibit a significant proportion in S phase (32.62%), whereas U0126 significantly reduces the proportion of cells in S phase (9.92%) and increases the proportion of cells in G0-G1 phase and to a lesser extent in G2/M[3].

Mice are administered U0126-EtOH (U0126; i.p., 10.5 mg/kg) every day. The tumor sizes throughout the control experiment are either constant or slightly increasing. On the other hand, engraftment and early tumor growth are markedly reduced in all U0126-EtOH experiments. Additionally, 9 days after injection and thereafter, the volume of tumors treated with U0126-EtOH is reduced by 60–70%[3]. Rats are given transient middle cerebral artery occlusion (tMCAO) for 120 minutes, and at 0 and 24 hours after reperfusion, they are given U0126-EtOH (U0126; i.p., 30 mg/kg). After receiving U0126-EtOH therapy, the vasoconstriction to S6c is significantly decreased[4].

In these assays, the amount of immunoprecipitated wild type MEK is adjusted to produce an equivalent number of activity units to that of 10 nM recombinant MEK. A 96-well nitrocellulose filter apparatus is used to measure reaction velocities, as detailed below. A 10 nM enzyme concentration, 20 mM Hepes, 10 mM MgCl2, 5 mM β-mercaptoethanol, 0.1 mg/mL BSA, pH 7.4, and room temperature are used for all reactions unless otherwise specified. The pre-mixed MEK/ERK/inhibitor reaction mixture is added [γ-33P]ATP to start the reactions. An aliquot of 100 μL is then taken every 6 minutes and transferred to the 96-well nitrocellulose membrane plate containing 50 mM EDTA to stop the reaction. The membrane plate is drawn and vacuum-washed four times with buffer. After that, 30 L of Microscint-20 scintillation fluid is poured into the wells, and a Top Count scintillation counter is used to measure the radioactivity of 33P-phosphorylated ERK. From the slopes of radioactivity versus time plots, speeds are calculated. Unless otherwise stated, ERK and ATP concentrations are 400 nM and 40 μM, respectively. The initial reaction velocities in the presence and absence of the inhibitor, respectively, are called Vi and Vo, and they are used to calculate the percent inhibition for all in vitro enzyme assays. The IC50 is then calculated by fitting the data using nonlinear least squares regression to the standard equation for a Langmuir isotherm. The data are then plotted as a function of inhibitor concentration as a function of percent inhibition. As stated, rather than active site titration, enzyme concentrations are determined by the molecular weights and protein mass used in the final assay volume. The actual concentration of the enzyme active site may therefore vary from the reported value.

A.E7 or Th17 cells are incubated with B10.BR or BALB/c splenocytes that have been treated with mitomycin C, along with varying concentrations of pigeon cytochrome c, PR8 Ag, or 5 U/mL human rIL-2. In order to ascertain the direct effects of MEK inhibition on T cell proliferation, some assays also contain U0126 or an inactive analog, U0124. Each well receives a 1µCi [3H]TdR pulse two days after culture initiation, and the following day, the cultures are harvested. Without the use of liquid scintillation mixtures, the incorporation of [3H]TdR into DNA is measured on a Packard Matrix 96 direct beta counter.

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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.57 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 solution 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 (6.57 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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 (6.57 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 10% DMSO+50% PEG 300+ddH2O: 28mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)