Inactive analog of the MEK inhibitor U0126

The compound U0126 (1,4-diamino-2,3-dicyano-1, 4-bis[2-aminophenylthio]butadiene) was identified as an inhibitor of AP-1 transactivation in a cell-based reporter assay. U0126 was also shown to inhibit endogenous promoters containing AP-1 response elements but did not affect genes lacking an AP-1 response element in their promoters. These effects of U0126 result from direct inhibition of the mitogen-activated protein kinase kinase family members, MEK-1 and MEK-2. Inhibition is selective for MEK-1 and -2, as U0126 shows little, if any, effect on the kinase activities of protein kinase C, Abl, Raf, MEKK, ERK, JNK, MKK-3, MKK-4/SEK, MKK-6, Cdk2, or Cdk4. Comparative kinetic analysis of U0126 and the MEK inhibitor PD098059 (Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci U. S. A. 92, 7686-7689) demonstrates that U0126 and PD098059 are noncompetitive inhibitors with respect to both MEK substrates, ATP and ERK. We further demonstrate that the two compounds bind to deltaN3-S218E/S222D MEK in a mutually exclusive fashion, suggesting that they may share a common or overlapping binding site(s). Quantitative evaluation of the steady state kinetics of MEK inhibition by these compounds reveals that U0126 has approximately 100-fold higher affinity for deltaN3-S218E/S222D MEK than does PD098059. We further tested the effects of these compounds on the activity of wild type MEK isolated after activation from stimulated cells. Surprisingly, we observe a significant diminution in affinity of both compounds for wild type MEK as compared with the deltaN3-S218E/S222D mutant enzyme. These results suggest that the affinity of both compounds is mediated by subtle conformational differences between the two activated MEK forms. The MEK affinity of U0126, its selectivity for MEK over other kinases, and its cellular efficacy suggest that this compound will serve as a powerful tool for in vitro and cellular investigations of mitogen-activated protein kinase-mediated signal transduction.[1]

In Vitro Kinase Assays[1]
The amount of immunoprecipitated wild type MEK used in these assays was adjusted to give a similar amount of activity units as obtained with 10 nm recombinant MEK (see below). All other assays were performed with a recombinant, constitutively activated mutant MEK-1 (ΔN3-S218E/S222D) or constitutively active MEK-2(S222E/S226D). Reaction velocities were measured using a 96-well nitrocellulose filter apparatus as described below. Unless otherwise noted, reactions were carried out at an enzyme concentration of 10 nm, in 20 mm Hepes, 10 mm MgCl2, 5 mm β-mercaptoethanol, 0.1 mg/ml BSA, pH 7.4, at room temperature. Reactions were initiated by the addition of [γ-33P]ATP into the premixed MEK/ERK/inhibitor reaction mixture, and an aliquot of 100 μl was taken every 6 min and transferred to the 96-well nitrocellulose membrane plate which had 50 mm EDTA to stop the reaction. The membrane plate was drawn and washed 4 times with buffer (see above) under vacuum. Wells were then filled with 30 μl of Microscint-20 scintillation fluid, and the radioactivity of33P-phosphorylated ERK was counted with a Top Count scintillation counter. Velocities were obtained from the slopes of radioactivity versus time plots. Concentrations of ERK and ATP were 400 nm and 40 μm, respectively, unless otherwise indicated.
For MKK-3 and MKK-6, a coupled assay was used in which 200 nm MKK-3(S189E/T193D) or MKK-6(S207E/T211E) was preincubated with 100 nm wild type p38 in the presence of 20 μm cold ATP with or without U0126 for 15 min. The coupled reaction was then initiated with the addition of 3 μm myelin basic protein (for MKK-3) or ATF (for MKK-6) and 2 μCi of [γ-33P]ATP. For SEK, 700 nmMEKK, 143 nm SEK, 400 nm JNK, and 1 μm c-Jun were mixed with or without compound and initiated with 10 μm ATP and 1 μCi of [γ-33P]ATP. All reactions were carried out and analyzed as described for the immune complex kinase assays. The effect of U0126 on Abl kinase activity was determined by using baculovirus expressed c-Abl. Inhibition of c-Abl autophosphorylation was measured. Effects of U0126 on Cdk2 and Cdk4 were determined using recombinant proteins as described.
For all of the in vitro enzyme assays, the percent inhibition was calculated 100 (1 −Vi/Vo) whereVi and Vo are the initial reaction velocities in the presence and absence of inhibitor, respectively. The data were then plotted as percent inhibition as a function of inhibitor concentration and fit, by nonlinear least squares regression, to the standard equation for a Langmuir isotherm to determine the IC50. As reported, enzyme concentrations were based upon molecular weights and mass of protein used in the final assay volume and not on active site titration. Thus, the actual enzyme active site concentration may differ from that reported.[1]

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

C8H10N4S2

226.3218

226.035

C, 42.46; H, 4.45; N, 24.76; S, 28.33

108923-79-1

5842955

White to off-white solid powder

1.338g/cm3

344.9ºC at 760mmHg

162.4ºC

6.37E-05mmHg at 25°C

1.647

2.5

2

6

3

14

339

0

CS/C(=C(/C(=C(/SC)\N)/C#N)\C#N)/N

LBQNBMSPTURKGS-KQQUZDAGSA-N

InChI=1S/C8H10N4S2/c1-13-7(11)5(3-9)6(4-10)8(12)14-2/h11-12H2,1-2H3/b7-5+,8-6+

(2Z,3Z)-2,3-bis[amino(methylsulfanyl)methylidene]butanedinitrile

u0124; 108923-79-1; (2Z,3Z)-bis[amino(methylsulfanyl)methylidene]butanedinitrile; 1,4-Diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; Bis[amino(methylthio)methylene]Butanedinitrile; Bis[amino(methylthio)methylene]Succinonitrile (6CI); Bis[amino(methylthio)methylene]Butanedinitrile (9CI); 2,3-Bis[amino(methylthio)methylene]butanedinitrile; U-0124; Bis[amino(methylthio)methylene]butanedinitrile; 2,3-bis[amino(methylsulfanyl)methylidene]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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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