Bafetinib (INNO-406) (INNO-406) is an effective and specific dual Bcr-Abl/Lyn inhibitor (IC50: 5.8/19 nM), and no inhibition of the phosphorylation of the T315I mutant and less effective to c-Kit and PDGFR.

Abl:5.8 nM, Lyn:19 nM

Bafetinib通过阻断WT Bcr-Abl自磷酸化及其下游激酶活性，对K562和293T细胞的IC50分别为11 nM和22 nM，有效抑制包括K562、KU812和BaF3/wt在内的Bcr-Abl阳性细胞系的生长，对Bcr-Abl阴性的U937细胞系增殖无影响。此外，Bafetinib对Bcr-Abl点突变细胞系（例如BaF3/E255K细胞）展示了剂量依赖的抗增殖效果。[1] 在Bcr-Abl阳性白血病细胞系中，Bafetinib通过阻断Bcr-Abl的磷酸化，诱导包括半胱天冬酶介导和非半胱天冬酶依赖的细胞死亡。[2]

在Bcr-Abl阳性的KU812小鼠模型中，Bafetinib（0.2 mg/kg/天）显著抑制肿瘤生长，在20 mg/kg/天的剂量下能完全抑制肿瘤生长，并且没有不良反应。对于Balb/c小鼠，Bafetinib的最大耐受剂量为200 mg/kg/d，生物可利用度（BA）值为32%。[1] 在携带Ba/F3/wt bcr-ablGFP、Ba/F3/Q252H或Ba/F3/M351T细胞的中枢神经系统（CNS）白血病模型中，Bafetinib（60 mg/kg）和环孢霉素A（CsA）（50 mg/kg）的组合治疗比单独使用Bafetinib或CsA更显著地抑制了大脑中白血病的生长。[3]

Kinase assay : Bcr-Abl kinase assays are performed in 25 μL of reaction mixture containing 250 μM peptide substrate, 740 Bq/μL [γ-33P]ATP, and 20 μM cold adenosine triphosphate (ATP) by using the SignaTECT protein tyrosine kinase assay system. Each Bcr-Abl kinase is used at a concentration of 10 nM. Kinase assays for Abl, Src, and Lyn are carried out with an enzyme-linked immunosorbent assay (ELISA) kit. The inhibitory effects of NS-187 against 79 tyrosine kinases are tested with KinaseProfiler.

K562, BaF3/wt, BaF3/E255K, and BaF3/T315I cells are plated at 1 × 103 in 96-well plates, whereas KU812 and U937 cells are plated at 5 × 103 in 96-well plates. Cells are incubated with serial dilutions of Bafetinib for 3 days. Cell proliferation is measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Nacalai Tesque) assay, and the 50% inhibitory concentration (IC50) values are calculated by fitting the data to a logistic curve. (Only for Reference)