生物活性

SB202190 is a highly selective, potent, cell-permeable inhibitor of p38 MAP kinases, inhibiting p38α (SAPK2A, MAPK14) and p38β (SAPK2B, MAPK11) with IC50 values of 50 and 100 nM, respectively. Research demonstrates that when one residue of ERK2, a related p38 not inhibited by pyridinyl imidazoles, is changed it allows SB202190 to bind. Studies have revealed that SB202190 can improve leukemia cell growth by activating MEK/MAPK, as well as phosphorylate and activate MLK3.

Thekinase activity of SB203580

Theinhibition of p38 MAP kinase[3]

SB203580inhibited IL-2-driven T cell proliferation with an IC50 of 3–5 μM.[1]

IC50value of SB203580 on human breast cancer cell line MDA-MB-231 is 85.1μM.[4]

体外研究

体内研究

Dissolved in drinking water

激酶实验

PKB Kinase Assay[1]

Cells were lysed in Buffer A for Westernblotting and PKB kinase assays. Kinase assays were performed according to themanufacturer’s instructions. Briefly, 4μg of sheepanti-PKBαwas immobilized on 25μl of protein G-Sepharoseovernight (or 1.5 h) and washed in Buffer A (50mM Tris, pH 7.5, 1mM EDTA, 1mMEGTA, 0.5mM Na3VO4, 0.1%β-mercaptoethanol,1%Triton X-100, 50mM sodium fluoride, 5mM sodium pyrophosphate, 0.1mMphenylmethylsulfonyl fluoride, 1μg/ml aprotinin, pepstatin,leupeptin, and 1μM microcystin). The immobilized anti-PKB was then incubated with0.5ml of lysate (from 5 x 106cells) for 1.5 h and washed threetimes in 0.5ml of Buffer A supplemented with 0.5M NaCl, two times in 0.5ml ofBuffer B (50mM Tris-HCl, pH 7.5, 0.03% (w/v) Brij-35, 0.1mM EGTA, and 0.1%β-mercaptoethanol),and twice with 100 ml of assay dilution buffer; 5x assay dilution buffer is100mM MOPS, pH 7.2, 125mMβ-glycerophosphate, 25mM EGTA, 5mM sodium orthovanadate, 5mM DTT. Tothe PKB enzyme immune complex was added 10 ml of assay dilution buffer, 40μMprotein kinase A inhibitor peptide, 100μMPKB-specific substrate peptide, and 10μCi of [γ-32P]ATP,all made up in assay dilution buffer. The reaction was incubated for 20 min atroom temperature with shaking, then samples were pulse spun, and 40μlof reaction volume were removed into another tube to which was added 20μl of 40% trichloroacetic acid to stop the reaction. This was mixedand incubated for 5 min at room temperature, and 40 ml was transferred onto P81phosphocellulose paper and allowed to bind for 30 s. The P81 pieces were washedthree times in 0.75% phosphoric acid then in acetone at room temperature.γ-32Pincorporation was then measured by scintillation counting.

细胞实验

Cellculture maintenance[4]

MDA-MB-231 human breast cancer cell linewas cultured in RPMI-1640 containing 4.5 g/L glucose, 10 mM HEPES, 1 mM sodiumpyruvate, 0.15% sodium bicarbonate, 100μg/mlstreptomycin and100 U/ml penicillin. The medium was supplemented with 10% fetalbovine serum (FBS). The cell line was grown in 25 cm2corning flasksfor passages. For the experiments, the cells were grown in 35 x 10 mm Corningdishes, 60 x 15 mm Corning dishes or E-Plate L8. The cells were subculturedupon reaching about 70% confluence.

MTT-basedcytotoxicity assay

MDA-MB-231 cells (100,000) were seeded ineach 35 x 10 mm dish. Cells were treated at the logarithmic phase of the growthwith various doses of SB203580 (0.1, 0.5, 1, 5, 10, 25, 50, 100μM)or SB202190 (0.1, 0.5, 1, 5, 10, 25, 50, 100μM) for 24 h.Following the inhibitor exposure period, the medium was changed with RPMI-1640containing 0.5% FBS + 0.5 mg/ml MTT, the cells were incubated for another 4 hat 37oC with 5% CO2. After MTT treatment, cells werelysed with 200μl 3%SDS plus 1 ml 40 mM HCl/isopropanol for 15 min.  After dissolving the MTT-formazan crystals,the cell lysate was diluted with 3% SDS + 40 mM HCl/isopropanol. Finally, eachsample absorbance was recorded at 570 nm with a SmartSpec Plusspectrophotometer. The data were analyzed and graphed with GraphPad Prism 5program. The IC50 values were then obtained by

GraphPad Prism 5 program using nonlinearregression to fit the data to the log(inhibitor) versus normalized response.

动物实验

Mice[5]

Female MRL/lpr mice aged 12 weeks wererandomized into two groups (n=10 per group) and were fed control diet (named asgroup 2 in the following) or diet with SB203580 (named as group 3 in thefollowing) starting at the age of 14 weeks and continuing for up to22 weeks.SB203580 dissolved in drinking water (250μmol/L), was orally administered (0.4ml/day). Ten C57BL/6 female mice were used as negative controls (named as group1 in the following). Two mice inMRL/lpr group 2 were dead at 16 weeks and 18weeks of age respectively. Two mice in MRL/lpr group 3 were dead at 19 weeks ofage. Significant increase of urine protein (300–2000 mg/dl) was found in eachmouse before death, indicating a probable renal failure be the cause of death.Ultimately, 10 mice in group 1, 8 mice in group 2 and group 3 were included instatistical analysis.

Immunofluorescenceexamination

One of the two kidneys of each mouse wassnap-frozen in OCT (optimum cutting temperature) compound for cryostatsectioning, and 5 μm frozen sections were stained with FITC (fluoresceinisothiacyanate)-conjugated antibodies (Southern Biotech, Birmingham, AL, USA)to detect murine IgG(diluted to 1:50), IgM(diluted to 1:75), IgA(dilutedto1:25) and C3 (diluted to 1:50) depositions. The fluorescence intensity wasassessed in at least 30 glomeruli and graded semiquantitatively (0, none; 1,slight; 2, mild; 3, moderate; 4, strong).

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] Lali FV HA, Turner SJ, Foxwell BM. The pyridinyl imidazole inhibitor SB203580 blocks phosphoinositide-dependent protein kinase activity, protein kinase B phosphorylation, and retinoblastoma hyperphosphorylation in interleukin-2-stimulated T cells independently of p38 mitogen-activated protein kinase. J Biol Chem. 2000;275(10):7395-7402.
[2] Clerk A SP. The p38-MAPK inhibitor, SB203580, inhibits cardiac stress-activated protein kinases/c-Jun N-terminal kinases (SAPKs/JNKs). FEBS Lett. . 1998;426(1):93-96.
[3] Kumar S MP, Gum RJ, Hand AT, Lee JC, Young PR. Novel homologues of CSBP/p38 MAP kinase: activation, substrate specificity and sensitivity to inhibition by pyridinyl imidazoles. Biochem Biophys Res Commun. 1997;235(3):533-538.
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体内溶解度
250 μM

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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