IC50: 30 nM (FLAP)[3] IC50: 3 nM (Leukotriene biosynthesis in intact leukocytes) and 1.1 μM (Leukotriene biosynthesis in human whole blood)[2] PPARα[1]

In a culture of mouse primary keratinocytes, MK-886 sodium salt (0.5-2 μM; 15 hours) treatment lowers keratin-1 expression[1]. 10 μM MK-886 sodium salt can reduce Wy-14643 activation of PPARα by approximately 80% in monkey kidney fibroblast CV-1 cells, mouse keratinocyte 308 cells, and human lung adenocarcinoma A549 cells using a transient transfection technique. In the stable transfection system, MK-886 sodium salt also reduces PPARα activation by fatty acids [1]. Despite the expression of all PPAR isoforms in Jurkat cells, apoptosis induced by MK-886 sodium salt cannot be prevented by different PPARα and PPARγ agonists[1].

Male Sprague-Dawley rats treated with MK-886 sodium salt (L 663536; 5 mg/kg; oral administration) show a strong inhibition of antigen-induced dyspnea in inbred rats that have previously received methysergide treatment[2]. Using rat pleurisy (ED50, 0.2 mg/kg po), inflammatory paw (ED50, 0.8 mg/kg), and a model of leukotriene excretion in rat bile after antigen provocation, MK-886 sodium salt (L 663536) suppresses leukotriene production in vivo[2].

PPAR ligand binding assay: Binding of MK886 to PPARs was determined using the coactivator-dependent receptor ligand assay (CARLA). A construct containing the PPARα ligand binding domain cloned in frame with glutathione S-transferase (GST) was procured from Walter Wahli. The GST–PPAR ligand binding domain fusion protein was expressed in Escherichia coli BL2 DE3 (pLysS). Bacterial pellets containing the fusion protein were resuspended in 10 ml of lysis buffer [PBS containing 1% (v}v) Triton X-100 and 0.5 mM PMSF] and lysed by repeated freeze–thawing. DNA and insoluble matter were removed by centrifugation at 4500 g. The fusion protein was purified using GSH–Sepharose beads at 4 °C, washed three times in lysis buffer, and equilibrated in 20 mM Tris}HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40 and 1 mM dithiothreitol (DTT) supplemented with 1% (w}v) dry milk. The amount of protein used per reaction was 1–3 µg. The beads were incubated with different concentrations of MK886 and $&S-radiolabelled steroid receptor coactivator-1 (SRC-1) prepared with a pSG5 plasmid construct that can express SRC-1 (procured from Walter Wahli) in Šitro using a coupled transcription}translation rabbit reticulocyte lysate system. Labelling was achieved by incubating for 1 h in the presence of [$&S]methionine, and the beads were recovered by centrifugation. The beads were then washed and analysed for interaction with SRC-1 using SDS}PAGE. Coomassie Brilliant Blue staining of GST–ligand binding domain fusions allowed standardization between different reactions. The SRC-1 protein complex was visualized by autoradiography. Relative band densities were determined using a scanned image and UN-SCAN-IT software[1].

Western Blot Analysis[1]
Cell Types: Primary Keratinocytes
Tested Concentrations: 0.5 µM, 1 µM or 2 µM
Incubation Duration: 15 hrs (hours)
Experimental Results: diminished keratin-1 expression.

Animal/Disease Models: Male SD (SD (Sprague-Dawley)) rat (300-400 g), antigen-induced dyspnea [1]
Doses: 5 mg/kg
Route of Administration: Oral
Experimental Results:Inhibition of antigen-induced dyspnea.

[1]. Inhibition of peroxisome-proliferator-activated receptor (PPAR)alpha by MK886. Biochem J. 2001 Jun 15;356(Pt 3):899-906.
[2]. L-663,536 (MK-886) (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2 - dimethylpropanoic acid), a novel, orally active leukotriene biosynthesis inhibitor. Can J Physiol Pharmacol. 1989 May;67(5):456-64.
[3]. Mancini JA, et al. 5-Lipoxygenase-activating protein is the target of a novel hybrid of two classes of leukotriene biosynthesis inhibitors. Mol Pharmacol. 1992 Feb;41(2):267-72.

3-[3-(tert-butylthio)-1-(4-chlorobenzyl)-5-(propyl-2-yl)-1H-indole-2-yl]-2,2-dimethylpropionic acid belongs to the indole class of compounds. Its structure is 1H-indole, with substitutions at the 5-position (isopropyl), 3-position (tert-butylthio), 1-position (4-chlorobenzyl), and 2-position (2-carboxy-2-methylpropyl). It is an inhibitor of arachidonic acid 5-lipoxygenase. It possesses various pharmacological activities, including as an EC 1.13.11.34 (arachidonic acid 5-lipoxygenase) inhibitor, an antitumor drug, and a leukotriene antagonist. It is an aryl sulfide, belonging to the indole, monocarboxylic acid, and monochlorobenzene classes. MK-886 is an experimental inhibitor of leukotriene synthesis. Although MK886 was initially identified as an inhibitor of 5-lipoxygenase-activated protein (FLAP), recent data suggest that this activity is not the underlying cause of its apoptosis-inducing activity [Datta, Biswal, and Kehrer (1999) Biochem. J. 340, 371-375]. Since FLAP is a fatty acid-binding protein, MK886 may affect other such proteins. Peroxisome proliferator-activated receptors (PPARs), a class of nuclear receptors activated by fatty acids and their metabolites, are involved in apoptosis and may represent a target for MK886. The ability of MK886 to inhibit PPAR-α, -β, and -γ activity was evaluated using a reporter gene assay system (peroxisome proliferator-luciferase). In monkey kidney fibroblast CV-1 cells, mouse keratinocyte 308 cells, and human lung adenocarcinoma A549 cells, using transient transfection systems, 10–20 μM MK886 inhibited Wy14,643-activated PPAR-α by approximately 80%. Similar inhibitory effects of MK886 on PPAR-α were also observed in a stable reporter gene transfection system in CV-1 cells. The inhibitory effects of MK886 on PPAR-β and PPAR-γ were very mild. MK886 inhibits PPAR-α through a non-competitive mechanism, as demonstrated by its effect on the binding of arachidonic acid to the PPAR-α protein and in a dose-response study of transient reporter gene transfection in COS-1 cells. An experiment evaluating PPAR ligand-receptor interactions showed that MK886 prevents the conformational changes required for the formation of the active complex. In mouse primary keratinocyte cultures, MK886 reduced the expression of keratin-1 (a protein encoded by a PPARα-responsive gene), suggesting that PPAR inhibition has functional consequences in normal cells. Although Jurkat cells express all PPAR isoforms, multiple PPARα and PPARγ agonists failed to prevent MK886-induced apoptosis. This is consistent with the mechanism of action of MK886 as a non-competitive inhibitor of PPARα, but may also indicate that PPARα is not directly involved in MK886-induced apoptosis. Although multiple PPAR activators have been identified, the results show that MK886 can inhibit PPARα, making it the first compound found to have this effect. [1] L-663,536 (3-[1-(4-chlorobenzyl)-3-tert-butylthio-5-isopropylindol-2-yl]-2,2-dimethylpropionic acid) is a potent inhibitor of leukotriene (LT) biosynthesis in intact human polymorphonuclear leukocytes (PMNs) (IC50 of 2.5 nM). Similarly, L-663,536 inhibited A23187-induced LTB4 production in rat peripheral blood and PMN. At a concentration that inhibits leukotriene biosynthesis in human whole blood (1.1 μM), no effect on cyclooxygenase or 12-lipoxygenase was observed, a result also observed in washed human platelets. The compound had no effect on 5-lipoxygenase in rats or pigs, indicating that L-663,536 is not a direct inhibitor of 5-lipoxygenase. In vivo, L-663,536 effectively inhibited antigen-induced dyspnea in meseido-pretreated inbred rats (ED50: 0.036 mg/kg, orally) and ascariasis-induced bronchoconstriction in squirrel monkeys (1 mg/kg, orally). The compound inhibited leukotriene biosynthesis in vivo in the following models: rat pleurisy model (ED50, 0.2 mg/kg orally), rat paw inflammation model (ED50, 0.8 mg/kg), rat bile leukotriene excretion model after antigen challenge, and guinea pig ear model (in which leukotriene synthesis was induced by local stimulation with ionocarrier A23187) (ED50, 2.5 mg/kg orally and 0.6 μg locally). The results showed that L-663,536 is a potent leukotriene biosynthesis inhibitor, effective both in vitro and in vivo, suggesting that the compound is suitable for studying the role of leukotrienes in a variety of pathological conditions. [2]

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An 18 kDa leukocyte membrane protein called 5-lipoxygenase activator protein (FLAP) has recently been identified as a target for two structurally different classes of leukotriene biosynthesis inhibitors. These two classes of inhibitors are based on indole and quinoline structures, respectively, represented by MK-886 and L-674,573. Recently, a novel hybrid compound based on indole and quinoline inhibitors, known as quinoline compounds, has been developed. These compounds, such as L-689,037, are potent leukotriene biosynthesis inhibitors, effective both in vitro and in vivo. In this study, we developed and characterized a highly efficient radiolabeled iodine-based photoaffinity analog, L-689,037, named [125I]L-691,678. Immunoprecipitation experiments with FLAP antiserum using this compound demonstrated that quinolone leukotriene biosynthesis inhibitors can directly interact with FLAP. Furthermore, we found that MK-886, L-674,573, and L-689,037 can specifically and competitively bind to FLAP in a concentration-dependent manner with [125I]L-691,678 and the MK-886 photoaffinity analog [125I]L-669,083. These results indicate that these three classes of leukotriene biosynthesis inhibitors share a common binding site on FLAP, further demonstrating that FLAP is a suitable target for structurally diverse leukotriene biosynthesis inhibitors. [3]

C27H33CLNNAO2S

494.06

493.181

C, 65.64; H, 6.73; Cl, 7.18; N, 2.84; Na, 4.65; O, 6.48; S, 6.49

118427-55-7

MK-886;118414-82-7

4519262

Typically exists as solid at room temperature

6.675

0

3

8

33

645

0

CC(C)C1=CC2=C(C=C1)N(CC3=CC=C(C=C3)Cl)C(=C2SC(C)(C)C)CC(C)(C)C(=O)[O-].[Na+]

CBNCIYNCWVGEKJ-UHFFFAOYSA-M

InChI=1S/C27H34ClNO2S.Na/c1-17(2)19-10-13-22-21(14-19)24(32-26(3,4)5)23(15-27(6,7)25(30)31)29(22)16-18-8-11-20(28)12-9-18;/h8-14,17H,15-16H2,1-7H3,(H,30,31);/q;+1/p-1

sodium;3-[3-tert-butylsulfanyl-1-[(4-chlorophenyl)methyl]-5-propan-2-ylindol-2-yl]-2,2-dimethylpropanoate

MK-886 sodium salt; 118427-55-7; MK886 sodium; MK-886 (sodium salt); PNR27O326B; sodium;3-[3-tert-butylsulfanyl-1-[(4-chlorophenyl)methyl]-5-propan-2-ylindol-2-yl]-2,2-dimethylpropanoate; CHEMBL416657; MK 886 sodium;

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.)