MEK2 (IC50 = 60 nM); MEK1 (IC50 = 70 nM)
MEK1 (IC50 = 72 nM); MEK2 (IC50 = 58 nM) [1]

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].

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U0126 potently inhibited recombinant MEK1 and MEK2 kinase activity with IC50 values of 72 nM and 58 nM respectively, acting as an ATP-competitive inhibitor [1]
It blocked EGF-induced ERK1/2 phosphorylation in HeLa cells, with 50% inhibition at 10 μM as detected by Western blot [1]
In MDCK cells infected with pandemic H1N1v influenza virus, U0126 inhibited viral replication with an EC50 of 8.3 μM, reducing viral titers by 100-fold at 20 μM [2]
Against highly pathogenic avian influenza virus (H5N1), the compound showed antiviral activity in MDCK cells with an EC50 of 7.6 μM [2]
In ERK2-dependent tumor cell lines (A549, MCF-7), U0126 suppressed cell proliferation with IC50 values of 15 μM and 12 μM respectively, and reduced clone formation by 68% and 75% at 20 μM [3]
It inhibited LPS-induced TNFα production in RAW 264.7 macrophages, with an IC50 of 9.8 μM [1]

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].

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Intraperitoneal administration of U0126 at 25 mg/kg twice daily improved survival rate of H1N1v-infected mice from 30% to 80% and reduced lung viral load by 1000-fold on day 5 post-infection [2]
In H5N1-infected mice, U0126 (25 mg/kg, i.p., twice daily) reduced lung pathology scores by 65% and decreased inflammatory cytokine (IL-6, TNFα) levels in lung tissues [2]
In a female rat stroke model (middle cerebral artery occlusion, MCAO), intravenous injection of U0126 (10 mg/kg) 1 hour after ischemia attenuated cerebral vasoconstriction by 58% and improved long-term neurologic outcome (neuroscore increased from 3.2 to 6.8 on day 28) [4]
In A549 xenograft mice, U0126 (20 mg/kg, i.p., daily) inhibited tumor growth by 62% after 28 days of treatment, accompanied by reduced phospho-ERK1/2 levels in tumor tissues [3]

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.

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Recombinant MEK1 and MEK2 were used to evaluate inhibitory activity. The assay mixture contained ATP, MgCl2, and a recombinant ERK2 substrate (as a MEK substrate). Serial dilutions of U0126 were incubated with MEK enzyme, ATP, and ERK2 at 30°C for 60 minutes. The reaction was terminated, and phosphorylated ERK2 was detected by Western blot to quantify MEK activity. IC50 values were calculated from dose-response curves [1]

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.

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HeLa cell ERK phosphorylation assay: HeLa cells were serum-starved for 12 hours, pretreated with U0126 (0.1–50 μM) for 1 hour, then stimulated with EGF (100 ng/mL) for 15 minutes. Cell lysates were analyzed by Western blot using anti-phospho-ERK1/2 and total ERK1/2 antibodies [1]
Influenza virus replication assay: MDCK cells were seeded in 96-well plates and infected with H1N1v or H5N1 virus (MOI = 0.01). U0126 (0.1–50 μM) was added 1 hour post-infection, and cells were incubated for 48 hours. Viral titers in supernatants were determined by plaque assay to calculate EC50 [2]
Tumor cell proliferation assay: A549 and MCF-7 cells were seeded at 5×103 cells/well, treated with U0126 (0.5–50 μM) for 72 hours. Cell viability was measured by colorimetric assay, and clone formation was assessed by crystal violet staining after 14 days of incubation [3]
Macrophage cytokine production assay: RAW 264.7 macrophages were pretreated with U0126 (0.5–50 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. TNFα levels in supernatants were measured by ELISA [1]

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].

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H1N1v/H5N1 influenza model: Female BALB/c mice were intranasally infected with 100 TCID50 of H1N1v or H5N1 virus. U0126 was formulated in 10% DMSO + 90% saline and administered intraperitoneally at 25 mg/kg twice daily (12-hour interval) from day 1 to day 5 post-infection. Survival was monitored for 14 days, and lung tissues were collected for viral load and cytokine analysis [2]
Rat MCAO stroke model: Female Sprague-Dawley rats were subjected to MCAO for 90 minutes. U0126 (10 mg/kg) was dissolved in saline and administered intravenously 1 hour after reperfusion. Cerebral blood flow was measured 24 hours post-ischemia, and neurologic function was evaluated weekly for 28 days [4]
A549 xenograft model: Female nude mice were subcutaneously implanted with 5×106 A549 cells. When tumors reached 150–200 mm3, U0126 (20 mg/kg) formulated in 0.5% hydroxypropyl cellulose was administered intraperitoneally once daily for 28 days. Tumor volume and body weight were measured twice weekly [3]

In a 14-day repeated-dose study in mice, doses up to 50 mg/kg (intraperitoneal injection, twice daily) of U0126 did not cause significant weight loss or abnormal hematological parameters [2]. No significant hepatotoxicity or nephrotoxicity was observed in the treatment group mice, and serum AST, ALT, creatinine, and BUN levels were normal [2]. U0126 has a plasma protein binding rate of 90% in human plasma and 88% in mouse plasma [1].

[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 with the structure (2Z,3Z)-bis[amino(thio)methylene]succinate, wherein the thiohydrogen is replaced by a 2-aminophenyl group. It is a mitogen-activated protein kinase inhibitor with anticancer properties. Its mechanism of action includes: acting as an EC 2.7.11.24 (mitogen-activated protein kinase) inhibitor, an apoptosis inducer, an antitumor agent, an antioxidant, a bone formation regulator, and a vasoconstrictor. It is an enamine, aryl sulfide, substituted aniline, and dinitrile. U-0126 is a direct inhibitor of MEK-1 and MEK-2, members of the mitogen-activated protein kinase family. U-0126 is a synthetic organic compound that selectively inhibits the kinase activity of mitogen-activated protein kinase, thereby preventing the production of cytokines and prostaglandin E2. The emergence of the 2009 H1N1 pandemic influenza A virus vividly illustrates how this viral infection can impact health systems worldwide in a very short time. The persistent zoonotic transmission and genetic recombination potential of influenza A virus (IAV) in nature pose a significant threat to human public health. In addition to vaccination, antiviral drugs are needed to effectively control the spread of the disease. This study investigated whether the MEK inhibitor U0126, targeting the intracellular Raf/MEK/ERK signaling pathway, could inhibit the proliferation of the 2009 pandemic influenza A (H1N1) virus (IV H1N1v, v=variant) and highly pathogenic avian influenza virus (HPAIV) in cell culture and in vivo (mouse lungs). U0126 demonstrated antiviral activity against all tested influenza virus (IAV) strains (including oseltamivir-resistant variants) in cell culture. Furthermore, we demonstrated that administration of U0126 to mice via the aerosol route resulted in: (i) inhibition of pulmonary MEK activation; (ii) reduced progeny influenza virus titers compared to the untreated control group; and (iii) protection of mice infected with influenza virus from a 100-fold lethal dose of virus. Furthermore, no adverse effects of U0126 were observed in cell culture or in mice. Therefore, we conclude that U0126 has antiviral potential not only in in vitro cell culture but also in in vivo mouse models by inhibiting the cellular target MEK. [2] It is well known that there are sex differences in cerebral ischemia, which may affect the efficacy of stroke treatment. In male rats, the MEK1/2 inhibitor U0126 reduced ischemia-induced upregulation of endothelin type B (ETB) receptors, reduced infarct size, and improved acute neurological function after experimental stroke. However, the response of female rats to this treatment and its long-term prognostic effects are unclear. The initial experiment, using in vitro organ culture of cerebral arteries, confirmed the activation of ERK1/2 and enhanced ETB receptor-mediated vasoconstriction in female cerebral arteries. In female Wistar rats, transient middle cerebral artery occlusion (tMCAO, 120 min) was induced, followed by intraperitoneal injection of U0126 (30 mg/kg) or a carrier at 0 and 24 hours after reperfusion, or no treatment. Infarct volume was measured, and neurological function was assessed using 6-point and 28-point neurological function scores. ETB receptor-mediated contraction was studied using myography, and protein expression was detected using immunohistochemistry. In vitro organ culture and tMCAO led to upregulation of vascular ETB receptors and activation of ERK1/2, while U0126 inhibited these processes. Although U0126 had no effect on infarct size, it improved long-term neurological function in female rats following experimental stroke. In conclusion, early inhibition of ERK1/2 activation and ETB receptor-mediated vasoconstriction in cerebral blood vessels after ischemic stroke in female rats improved long-term neurological outcomes. [4]

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U0126 is a novel, selective, ATP-competitive MEK1 and MEK2 inhibitor, MEK1 and MEK2 being key kinases in the MAPK/ERK signaling pathway. [1]
Its mechanism of action is to block MEK-mediated ERK1/2 phosphorylation, thereby inhibiting downstream processes such as cell proliferation, viral replication, and inflammation. [1][2][3]
This compound has shown therapeutic potential in influenza virus infection, solid tumors, and ischemic stroke by targeting the MAPK pathway. [2][3][4]
It is widely used as a research tool for studying MEK/ERK signaling in various biological processes. [1]

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.)