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

U0126-EtOH blocks the production of numerous cytokines and metalloproteinases involved in the inflammatory response by functionally antagonistic AP-1 transcriptional activity.[1] By decreasing IL-2 mRNA levels, U0126-EtOH reduces T cell proliferation in response to antigenic stimulation or cross-linked anti-CD3 plus anti-CD28 Abs without affecting proliferation brought on by IL-2.[2] According to a recent study, U0126-EtOH inhibits mitochondrial function, switches castration-resistant human prostate cancer C4-2 cells to aerobic glycolysis without the help of MEK, and counteracts resveratrol's induction of apoptosis.[3]

U0126-EtOH exhibits antiviral activity by stifling the spread of the 2009 pandemic IV H1N1 variant and highly pathogenic avian influenza viruses (HPAIV) in vivo in the mouse lung by inhibiting the intracellular Raf/MEK/ERK signaling pathway.[4] By activating nuclear respiratory factor 1, mitochondrial transcription factor A, and peroxisome proliferator-activated receptor gamma coactivator-1a in A-injected rats, U0126-EtOH demonstrates the potential neuroprotective effect and improving spatial learning in the Morris water maze (MWM).[5]

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 μL 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 data are then plotted as a function of inhibitor concentration as a function of percent inhibition, and the IC50 is calculated by fitting the data using nonlinear least squares regression to the equation for a Langmuir isotherm. According to the information provided, rather than active site titration, enzyme concentrations are based on the molecular weights and mass of the protein used in the final assay volume. As a result, the actual concentration of the enzyme active site may 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 1-µCi [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.

Female C57Bl/6 mice infected by Mouse-adapted highly pathogenic avian influenza A/FPV/Bratislava/79 (H7N7; FPV) virus and swine origin human influenza A virus (SOIV) A/Regensburg/D6/2009 (H1N1v; RB1).
≤10 mM
Administered via aerosol.

[1]. Bioorg Med Chem Lett . 1998 Oct 20;8(20):2839-44.

[2]. J Immunol . 1998 May 1;160(9):4175-81.

[3]. Cancer Biol Ther . 2011 Dec 1;12(11):966-77.

[4]. Antiviral Res . 2011 Nov;92(2):195-203.

[5]. Behav Brain Res . 2012 Jun 15;232(1):165-73.

[6]. J Biol Chem . 1998 Jul 17;273(29):18623-32.

U0126.EtOH is an addition compound obtained by combining equimolar amounts of (2Z,3Z)-bis{amino[(2-aminophenyl)sulfanyl]methylidene}butanedinitrile (U0126) and ethanol. 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 contains an U0126.

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In search of antiinflammatory drugs with a new mechanism of action, U0126 was found to functionally antagonize AP-1 transcriptional activity via noncompetitive inhibition of the dual specificity kinase MEK with an IC50 of 0.07 microM for MEK 1 and 0.06 microM for MEK 2. U0126 can undergo isomerization and cyclization reactions to form a variety of products, both chemically and in vivo, all of which exhibit less affinity for MEK and lower inhibition of AP-1 activity than parent, U0126.[1]

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Three mitogen-activated protein kinase pathways are up-regulated during the activation of T lymphocytes, the extracellular signal-regulated kinase (ERK), Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase pathways. To examine the effects of blocking the ERK pathway on T cell activation, we used the inhibitor U0126, which has been shown to specifically block mitogen-activated protein kinase/ERK kinase (MEK), the kinase upstream of ERK. This compound inhibited T cell proliferation in response to antigenic stimulation or cross-linked anti-CD3 plus anti-CD28 Abs, but had no effect on IL-2-induced proliferation. The block in T cell proliferation was mediated by down-regulating IL-2 mRNA levels. Blocking Ag-induced proliferation by inhibiting MEK did not induce anergy, unlike treatments that block entry into the cell cycle following antigenic stimulation. Surprisingly, induction of anergy in T cells exposed to TCR cross-linking in the absence of costimulation was also not affected by blocking MEK, unlike cyclosporin A treatment that blocks anergy induction. These results suggest that inhibition of MEK prevents T cell proliferation in the short term, but does not cause any long-term effects on either T cell activation or induction of anergy. These findings may help determine the viability of using mitogen-activated protein kinase inhibitors as immune suppressants.[2]

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Recent evidence has identified substantial overlap between metabolic and oncogenic biochemical pathways, suggesting novel approaches to cancer intervention. For example, cholesterol lowering statins and the antidiabetes medication metformin both act as chemopreventive agents in prostate and other cancers. The natural compound resveratrol has similar properties: increasing insulin sensitivity, suppressing adipogenesis, and inducing apoptotic death of cancer cells in vitro. However, in vivo tumor xenografts acquire resistance to resveratrol by an unknown mechanism, while mouse models of metabolic disorders respond more consistently to the compound. Here we demonstrate that castration-resistant human prostate cancer C4-2 cells are more sensitive to resveratrol-induced apoptosis than isogenic androgen-dependent LNCaP cells. The MEK inhibitor U0126 antagonized resveratrol-induced apoptosis in C4-2 cells, but this effect was not seen with other MEK inhibitors. U0126 was found to inhibit mitochondrial function and shift cells to aerobic glycolysis independently of MEK. Mitochondrial activity of U0126 arose through decomposition, producing both mitochondrial fluorescence and cyanide, a known inhibitor of complex IV. Applying U0126 mitochondrial inhibition to C4-2 cell apoptosis, we tested the possibility that glutamine supplementation of citric acid cycle intermediate α-ketoglutarate may be involved. Suppression of the conversion of glutamate to α-ketoglutarate antagonized resveratrol-induced death in C4-2 cells. A similar effect was also seen by reducing extracellular glutamine concentration in the culture medium, suggesting that resveratrol-induced death is dependent on glutamine metabolism, a process frequently dysregulated in cancer. Further work on resveratrol and metabolism in cancer is warranted to ascertain if the glutamine dependence has clinical implications.[3]

C18H16N6S2.C2H6O

426.56

426.129

C, 56.32; H, 5.20; N, 19.70; O, 3.75; S, 15.03

1173097-76-1

U0126;109511-58-2; 218601-62-8 (ZZ-isomer); 218601-64-0 (EE-isomer); 1173097-76-1 (EtOH)

16220066

White to off-white solid powder

612.5ºC at 760 mmHg

324.2ºC

5.684

5

9

5

29

612

0

S(/C(=C(\C#N)/C(/C#N)=C(\N([H])[H])/SC1=C([H])C([H])=C([H])C([H])=C1N([H])[H])/N([H])[H])C1=C([H])C([H])=C([H])C([H])=C1N([H])[H].O([H])C([H])([H])C([H])([H])[H]

CFQULUVMLGZVAF-OYJDLGDISA-N

InChI=1S/C18H16N6S2.C2H6O/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;1-2-3/h1-8H,21-24H2;3H,2H2,1H3/b17-11+,18-12+

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

U0126; U-0126-EtOH; U0126 EtOH; U 0126; U-0126; U0126-EtOH; U 0126-EtOH; U 0126 EtOH; U0126 Ethanol; U0126.EtOH; (2Z,3Z)-2,3-bis(amino(2-aminophenylthio)methylene)succinonitrile compound with ethanol (1:1); (2Z,3Z)-2,3-bis[amino-(2-aminophenyl)sulfanylmethylidene]butanedinitrile;ethanol; 2,3-Bis(amino((2-aminophenyl)thio)methylene)succinonitrile compound with ethanol (1:1); U0126 ethanolate; .U-0126 EtOH

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: ≥ 2.08 mg/mL (4.88 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 20.8 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: ≥ 2.08 mg/mL (4.88 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 20.8 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: ≥ 2.08 mg/mL (4.88 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 20.8 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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Solubility in Formulation 5: 5 mg/mL (11.72 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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