Mubritinib (TAK-165) (TAK-165) is a potent inhibitor of HER2/ErbB2 with IC50 of 6 nM.

HER2/ErbB2:6.0 nM

Mubritinib在对其他酪氨酸激酶（例如EGFR、FGFR、PDGFR、Jak1、Src及Blk）表现出高达4000倍的选择性。即使在低浓度0.1 μM下，Mubritinib显著阻断了HER2的磷酸化作用，导致在高HER2水平的细胞系BT474中PI3K-Akt和MAPK途径的下调。Mubritinib不仅在ErbB2过表达的癌细胞系BT474中展现出高效的抗增殖作用，IC50为5 nM，而且还在HER2表达较弱的细胞系中表现出显著的抗增殖效果，分别对LNCaP、LN-REC4和T24的IC50为53 nM、90 nM和91 nM。对于HER2表达极微弱的PC-3细胞，Mubritinib没有抑制活性，IC50为4.62 μM，同样对于EGFR过表达的HT1376和ACHN细胞系也是如此，IC50均>25 μM。[1]

Mubritinib 显著抑制了LN-REC4异种移植瘤，治疗/对照组瘤体体积比为26.5%。尽管在体外无法有效抑制UMUC-3和ACHN细胞的生长（IC50分别为1.812 和 >25 μM），但Mubritinib通过口服（每天10或20 mg/kg）显著抑制了UMUC-3和ACHN异种移植瘤的生长，治疗/对照组瘤体体积比分别为22.9%和26%，与此同时，Herceptin（20 mg/kg）对UMUC-3肿瘤生长无效。[1]

Inhibition of HER2/erbB2 tyrosine kinase activity: BT-474 cells are seeded on 24-well plates and cultured overnight. Mubritinib is then added at various concentrations. After incubation for 2 hours, the cells are harvested directly into sodium dodecyl sulfate (SDS)-sample buffer (200 μL). Aliquots containing equal amounts of total cell extract are run on 7.5% to 15% gradient SDS–polyacrylamide gel electrophoresis (PAGE). Following electrophoresis, proteins are transferred onto a polyvinylidene fluoride (PVDF) membrane, for western blot analysis using a relevant primary antibody. Detection of protein is accomplished by an enhanced chemiluminescent (ECL) detection method. The extent of tyrosine phosphorylation of HER2/erbB2 is measured by the LAS-1000 plus lumino-image analyser. The concentration of Mubritinib that inhibits HER2/erbB2 phosphorylation by 50% (IC50) is calculated from a dose–response curve generated by least-squares linear regression of the response using SAS software.

Cells are seeded into 6-well plates and cultured overnight. Mubritinib is then added at various concentrations, and the cells are treated continuously for 72 hours. After the incubation period, cells are counted for the measurement of antiproliferative activity.(Only for Reference)