生物活性

GF 109203X is a potent and selective inhibitor of protein kinase C, selective for the α and β1 isoforms (IC50 values are 0.0084, 0.0180, 0.210, 0.132, and 5.8 μM for α, β1, δ, ε and ζ isoforms respectively). Selective over MLCK, PKG and PKA (IC50 values are 0.6, 4.6, and 33 μM respectively). Potent antagonist at the 5-HT3 receptor (Ki = 29.5 nM). Anti-inflammatory in vivo. It inhibits PKC within intact platelets and T cells, Fas-mediated apoptosis, and T cell-mediated autoimmune diseases, as well as GSK-3. See sc-24004 for water soluble form of GF 109203X. GF 109203X is an inhibitor of PHK/CaM, PKC α, PKC β, PKC δ, PKC ε, PKC γ and SR/HTR3.

Inhibitionof PKC subtypes and various receptor tyrosine kinases by GF 109203X[1]

Kinase activities of GFI09203X

The current inhibition of bisindolylmaleimideI

体外研究

体内研究

10% DMSO in saline

激酶实验

GSK-3 activity assay[2]

GSK-3 activity was measured in cell lysatesand in GSK-3βimmunoprecipitates. GSK-3βwas immunoprecipitated fromcell lysates by tumbling with 4μl of anti-GSK-3βmonoclonal antibody and 3.75 mg protein A-Sepharose for 2 h at 4oC.The resulting immunoprecipitates were washed three times in kinase assay bu¡er(20 mM HEPES, pH 7.5, 20 mMβ-glycerophosphate and 1 mM EDTA) and finally resuspended in 300μlof kinase assay bu¡er containing 0.1% mercaptoethanol and 2.5μMcAMP-dependent protein kinase inhibitor peptide (IP20). The activity of GSK-3was measured in duplicate in 20μl of cell lysate or 20μlof GSK-3βimmunoprecipitate using the synthetic peptide substrateRRAAEELDSRAGS (P) PQL (0.71mg/ml) in the absence or in the presence of theGSK-3 inhibitor, lithium chloride (50mM). The assay was terminated after 15 minincubation with [γ-32P] ATP by spotting onto P81 ion-exchange paper. Thepaper was washed four times in 0.6% phosphoric acid and bound radioactivity quanti¢edby scintillation counting. Phosphorylation of peptide by adipocyte lysates andby GSK-3βimmunoprecipitates was essentially completely inhibited by lithiumchloride. The average activity of GSK-3 in the extracts was 1220±144 pmol peptide phosphorylated/ min/g dry weight of adipocytes(n=11). The average activity of GSK-3βin immunoprecipitates was 276±54 pmol peptide phosphorylated/ min/g dry weight of adipocytes(n=11).

细胞实验

In vitro osteoclastogenesis[8]

Freshly isolated bone marrow macrophages(BMM) were cultured in completeα-MEM with M-CSF. BMM cells wereseeded on to 96-well plate (6x103) and cultured with GF109203X inthe presence of 100ng/ml RANKL and 25ng/ml M-CSF. Medium was changed every 2days. After 6–7 days of culture, the cells were fixed with 2.5% glutaraldehydeand histochemically stained to detect TRAP. TRAP positive multinucleated cellswith more than three nuclei were counted as osteoclasts.

MTSassay for cell proliferation and viability

The effects of compounds on BMM cellproliferation were examined using MTS assay. The MTS assay was performed accordingto manufacturer’s introduction. Briefly, 6x103BMM cells per wellwere seeded onto a 96-well plate, followed by overnight incubation. Cells were incubatedwith GF109203X for 48 h. MTS/PMS mixture was then added and cells incubated fora further 2 h. MTS stain was measured by spectrophotometric absorbance at 490nm using an ELISA plate reader.

Analysisof osteoclast activity

BMM-derived osteoclasts were generated on a30mm diameter collagen-coated culture plate at the density of 1x105cells/well.Cells were differentiated with 100ng/ml RANKL and M-CSF until osteoclasts wereevident at 5 days incubation. Osteoclasts were then dissociated from thecollagen plate using 1 ml/well cell dissociation solution and the same numberof cells was cultured in each well of a hydroxyapatite-coated plate. Osteoclastswere cultured in the presence or absence of GF109203X for 48 h with 100ng/mlRANKL (n=6). Then, half of the wells were fixed with 2.5% glutaraldehyde andhistochemically stained for TRAP and osteoclasts counted as above. The rest ofthe wells were bleached with 10% bleach solution. The areas of hydroxyapatite resorptionwere observed by light microscopy and analysed using Image J software.

动物实验

Animals[9]

Female Swiss Webster mice were used in allstudies. The mice were housed in Parke-Davis animal care facilities and werefed ad Iibitum.

PMA-inducedear edema

Ear edema was induced in female SwissWebster mice. Ear swelling was measured at various time points after PMAadministration with a micrometer. Test compounds were applied 1 h before PMAtreatment in 20μl of acetone.

Eicosanoidmeasurement

PMA-treated mice were euthanized and earbiopsies were harvested with a punch. The ears were homogenized with 10 mg/mlBW755c to stop further arachidonic metabolism. After acetonitrile precipitationthe samples were resuspended in acidified water and loaded onto a prewashed C18Sep-Pak column. The samples were loaded onto the column and the eicosanoidswere eluted with 90% methanol. Eicosanoids were quantified by radioimmunoassay.Extraction efficiencies for PGE2, LTC4 and LTB 4 were 100±4.5, 67±3.3 and 91±6.7, respectively.

Myeloperoxidaseassay

Tissue myeloperoxidase was used as anindicator of neutrophil accumulation in the PMA-treated mice. Ear samples wereobtained and processed. Briefly, ear punch samples from PMA-treated mice were homogenizedand freeze thawed three times. The homogenate was centrifuged 50000 x g) andthe homogenate was assayed for myeloperoxidase using o-tolidine as substrate.Units of myeloperoxidase were calculated from a standard curve constructed usinghorseradish peroxidase.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Toullec D PP, Coste H, Bellevergue P, Grand-Perret T, Ajakane M, Baudet V, Boissin P, Boursier E, Loriolle F. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem. . 1991;266(24):15771-15781.
[2] Hers I TJ, Denton RM. The protein kinase C inhibitors bisindolylmaleimide I (GF 109203x) and IX (Ro 31-8220) are potent inhibitors of glycogen synthase kinase-3 activity. FEBS Lett. . 1999 460(3):433-436.
[3] Park WS, Son YK, Ko EA, et al. The protein kinase C inhibitor, bisindolylmaleimide (I), inhibits voltage-dependent K+ channels in coronary arterial smooth muscle cells. Life Sci. 2005;77(5):512-527.
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体内溶解度

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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