| 名稱 | Tofacitinib |
| 描述 | Tofacitinib (Tasocitinib) is a Janus kinase inhibitor that inhibits JAK3/2/1 (IC50=1/20/112 nM) and is orally active. Tofacitinib is used for the treatment of moderate to severe rheumatoid arthritis. |
| 細胞實驗 | To measure IL-2-dependent proliferation, isolated lymphocytes are resuspended to a cell density of 1-2 × 106/mL in complete RPMI medium (RPMI 1640 containing 10% (w/v) fetal calf serum (FCS), 1%(w/v) penicillin and treptomycin).Phytohemagluttinin (PHA) is added to a final concentration of 10 mg/mL, and the culture incubated for 3 days at 37 °C in a humidified 5% (v/v) CO2 incubator to upregulate IL-2R and JAK3 expression. IL-2 (200U/mL), with or without CP-690,550 is then added and the cells are incubated for 72 hours at 37 °C in a humidified 5% (v/v) CO2 incubator, after which 50 mL of 3H-thymidine (5mCi/mL) is added. The plates are incubated for an additional 18 hours, harvested with a 96-well harvester, and counted on a scintillation counter. HUO3 cells are maintained in culture with granulocyte-macrophage colony stimulating factor and human foreskin fibroblasts are maintained in culture with 10% fetal calf serum. CP-690550 is added to freshly plated cells and cultured for 4 days. 3Hthymidine is added during the last 18 hours of the culture period. (Only for Reference) |
| 激酶實驗 | JAK3 Kinase Assay: A fragment encoding the catalytic domain of human JAK3 (785aa to 1125aa, JH1 catalytic domain) is amplified by PCR from the full length cDNA and cloned into the EcoRI site of the baculovirus transfer vector pVL1393. Recombinant baculovirus is used to infect Sf9 (Spodoptera frugipedra) cells and recombinant GSTJAK3 fusion protein is isolated on glutathione sepharose. The fusion protein is eluted with reduced glutathione and stored in buffer containing 50 mM Tris, pH 7.5, 10 mM DTT and 10% glycerol. JAK3 kinase activity is measured by ELISA as follows: Plates are coated overnight with a random L-glutamic acid and tyrosine co-polymer (4:1) (100 ug/mL). The plates are washed and recombinant JAK3 JH1:GST (100 ng/well) with or without inhibitors is incubated at room temperature for 30 minutes, after which HRP-conjugated PY20 anti-phosphotyrosine antibody (ICN) is added and developed by TMB (3,3',5,5'-tetramethylbenzidine). Other kinases (Table 1) are produced in E. coli or in insect cells, depending upon what is found to be optimal for the given kinase. The catalytic activity of tyrosine kinases is easured using the aftorementioned ELISA, whereas serine/threonine kinases are assayed using radioactive enzyme assays. |
| 體外活性 | 方法: 人類 MM 細胞系 MM.1S 與永生化 BMSC 系 HS5 和 HS27A 共培養(yǎng)，用 Tofacitinib (0-10 μM) 處理 24 h，通過 CellTiter-Glo reagent 檢測細胞活力。
結(jié)果: 與單培養(yǎng)生長相比，MM.1S 細胞數(shù)量顯著增加，證實了該細胞系中基質(zhì)誘導的增殖信號。Tofacitinib 處理以劑量依賴的方式減少了 MM.1S 細胞數(shù)量。Tofacitinib 單獨對 MM.1S 細胞存活率或單獨對基質(zhì)細胞沒有影響。[1]
方法: NK-92 細胞用 Tofacitinib (50 nM) 處理過夜，再用 IL-15 (2.33 nM) 處理 2 h，檢測 STAT1 磷酸化。
結(jié)果: Tofacitinib 阻斷 NK-92 細胞中的 IL-15 信號傳導。Tofacitinib 通過阻斷 STAT 蛋白磷酸化來破壞 JAK/STAT 信號傳導。[2] |
| 體內(nèi)活性 | 方法: 為檢測體內(nèi)抗腫瘤活性，用 Tofacitinib (21.5 mg/kg，50% DMSO+10% PEG 400+40% water) 皮下注射給攜帶原位擴散 MM.1S 異種移植物的 NSG 小鼠，每天一次，持續(xù)四周。
結(jié)果: 小鼠存活率顯著提高，并且基于生物發(fā)光成像定量，腫瘤負荷顯著降低。[1] |
| 存儲條件 | Keep away from moisture,Keep away from direct sunlight,
Pure form: -20°C for 3 years | In solvent: -80°C for 1 year
Shipping with blue ice/Shipping at ambient temperature. |
| 溶解度 | H2O : < 1 mg/mL (insoluble or slightly soluble)
10% DMSO+40% PEG300+5% Tween 80+45% Saline : 4 mg/mL (12.81 mM), Sonication is recommended.
DMSO : 125 mg/mL (400.17 mM), Sonication is recommended.
Ethanol : 0.76 mg/mL (2.43 mM), Sonication is recommended.
|
| 關鍵字 | Tofacitinib | ROCK2 | Lck | Janus kinase | JAK3 | JAK2 | JAK1 | JAK | Inhibitor | inhibit | CP690550 | CP 690550 | Apoptosis |
| 相關產(chǎn)品 | Urea | Formamide | Dimethyl phthalate | Aceglutamide | Alginic acid | Dextran sulfate sodium salt (MW 5000) | Metronidazole | Sildenafil citrate | Citric Acid Triammonium | Stavudine | Tamoxifen | Adenosine |
| 相關庫 | 經(jīng)典已知活性庫 | 抗癌上市藥物庫 | 已知活性化合物庫 | 激酶抑制劑庫 | 抗衰老化合物庫 | FDA 上市藥物庫 | TGF-β/Smad靶點化合物庫 | 膜蛋白靶向化合物庫 | 免疫/炎癥分子化合物庫 | 酪氨酸激酶分子庫 | 藥物功能重定位化合物庫 | 抗癌藥物庫 |