Dacomitinib (PF299)(PF299804; PF-00299804) is a highly selective, orally bioavailable small-molecule inhibitor of the HER family of tyrosine kinases with IC50 of 6, 45.7 and 73.7 nM for EGFR, ERBB2, and ERBB4, respectively.

ErbB2:45.7 nM, ERB4:73.7 nM, EGFR:6.0 nM

PF-299804（15 mg/kg,p.o.）具有较强的抗肿瘤活性.其可引起各种人肿瘤异种移植模型中明显的肿瘤退化,如表达和/或过表达ERBB家族成员,或与耐埃罗替尼和吉非替尼相关的含有双重突变(L858R/T790M)的ERBB1 (EGFR)异种移植模型.

PF299804对EGFR信号传导有抑制作用,并可促使含EGFR T790M的H3255 GR细胞系凋亡。PF299804对表达T790M 突变体的H3255和 HCC827细胞的生长具有抑制作用。在HER2-扩增的胃癌细胞中,PF299804诱导细胞凋亡和G1期阻滞,并抑制HER家族及其下游信号通路,包括STAT3,AKT和细胞外信号调节激酶(ERK)中受体的磷酸化。PF299804还可阻断SNU216细胞中EGFR/HER2,HER2/HER3和HER3/HER4异质二聚体形成,以及HER3与p85α的结合。在大多数敏感细胞系中,PF299804对HER2,EGFR,HER4,AKT和ERK的磷酸化水平具有降低作用。PF299804通过G0/G1期阻滞和对细胞凋亡的诱导发挥其抗增殖作用。在47种人乳腺癌和永生的乳腺上皮细胞系中,相对于非扩增细胞系(RR = 3.39, p < 0.0001),PF299804优先抑制HER-2-扩增的乳腺癌细胞系的生长。

ELISA-Based ERBB Kinase Assay: The ERBB1, ERBB 2, and ERBB4 cytoplasmic fusion proteins are made by cloning the ERBB1 sequence (Met-668 to Ala-1211), ERBB2 (Ile-675 to Val-1256), and ERBB4 sequence (Gly-259 to Gly-690) into the baculoviral vector pFastBac using PCR. Proteins are expressed in baculovirusinfected Sf9 insect cells as GST fusion proteins. The proteins are purified by affinity chromatography using glutathione sepharose beads. Inhibition of ERBB tyrosine kinase activity is assessed using an ELISA-based receptor tyrosine kinase assay. Kinase reactions (50 mM HEPES, pH 7.4, 125 mM NaCl, 10 mM MgCl2, 100 μM sodium orthovanadate, 2 mM dithiothreitol, 20 μM ATP, PF299804 or vehicle control, and 1-5 nM GST-erbB per 50 μL of reaction mixture) are run in 96-well plates coated with 0.25 mg/mL poly-Glu-Tyr. The reactions are incubated for 6 minutes at room temperature while being shaken. Kinase reactions are stopped by removal of the reaction mixture, and then the wells are washed with wash buffer (0.1% Tween 20 in PBS). Phosphorylated tyrosine residues are detected by adding 0.2 μg/mL antiphosphotyrosine antibody (Oncogene Ab-4; 50 μL/well) coupled to horseradish peroxidase (HRP) diluted in PBS containing 3% BSA and 0.05% Tween 20 for 25 minutes while being shaken at room temperature. The antibody is removed, and plates are washed in wash buffer. HRP substrate (SureBlue3,3?,5,5?-tetramethyl benzidine or TMB) is added (50 μL per well) and incubated for 10-20 minutes while it is shaken at room temperature. The TMB reaction is stopped with the addition of 50 μL of stop solution (0.09 N H2SO4). The signal is quantified by measuring absorbance at 450 nm. IC50 values are determined for PF299804 using the median effect method.

Growth and inhibition of growth is assessed by 5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. This assay, a colorimetric method fordetermining the number of viable cells, is based on the bioreduction of MTS by cells to a formazan product that is soluble in cell culture medium, can be detected spectrophotometrically. The cells are exposed totreatment for 72 hours, and the number of cells used per experiment is determined empirically. All experimental points are set up in 6 to 12 wells, and all experiments are repeated at least thrice. The data are graphically displayed using GraphPad Prism version 3.00 for Windows (GraphPad Software). The curves are fitted using a nonlinear regression model with a sigmoidal dose response.(Only for Reference)