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.

JAK3:1 nM, JAK1:112 nM, JAK2:20 nM

方法: 人类 MM 细胞系 MM.1S 与永生化 BMSC 系 HS5 和 HS27A 共培养，用 Tofacitinib (0-10 µM) 处理 24 h，通过 CellTiter-Glo reagent 检测细胞活力。
结果: 与单培养生长相比，MM.1S 细胞数量显著增加，证实了该细胞系中基质诱导的增殖信号。Tofacitinib 处理以剂量依赖的方式减少了 MM.1S 细胞数量。Tofacitinib 单独对 MM.1S 细胞存活率或单独对基质细胞没有影响。[1]
方法: NK-92 细胞用 Tofacitinib (50 nM) 处理过夜，再用 IL-15 (2.33 nM) 处理 2 h，检测 STAT1 磷酸化。
结果: Tofacitinib 阻断 NK-92 细胞中的 IL-15 信号传导。Tofacitinib 通过阻断 STAT 蛋白磷酸化来破坏 JAK/STAT 信号传导。[2]

方法: 为检测体内抗肿瘤活性，用 Tofacitinib (21.5 mg/kg，50% DMSO+10% PEG 400+40% water) 皮下注射给携带原位扩散 MM.1S 异种移植物的 NSG 小鼠，每天一次，持续四周。
结果: 小鼠存活率显著提高，并且基于生物发光成像定量，肿瘤负荷显著降低。[1]

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.

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)