Pelitinib (EKB-569) (EKB-569) is an effective irreversible EGFR inhibitor (IC50: 38.5 nM).

EGFR:38.5 nM

在达EGFR的A431异种移植模型中,Pelitinib（10 mg/kg,p.o.）可有效抑制EGFR磷酸化,1 h内抑制90%,24 h后抑制率>50%.小鼠气道上皮细胞重构模型,可被病毒感染诱导,具有延迟但永久性转变为杯状细胞化生的特点,Pelitinib（20 mg/kg/day）可完全阻断纤毛细胞的增加和Clara细胞的减少,从而从3个方面校正上皮细胞重构,并明显抑制杯状细胞的发生.Pelitinib（20 mg/kg/day）抑制APCMin/+小鼠体内87%的肿瘤发生,与2倍剂量EKI-785（40 mg/kg/day）的作用接近,与体内活性较高一致. Pelitinib对体内气道上皮细胞中EGFR信号有选择性抑制效果.

Pelitinib（75-500 nM）可特异性抑制AKT和ERK1/2的活化,而对NF-κB通路无影响。在正常人角质细胞中,Pelitinib也可有效抑制TGF-α介导的EGFR活化（IC50：56 nM）,抑制STAT3和ERK1/2的活化（IC50：60 nM和62 nM）。在A431和正常人角质细胞中,Pelitinib对EGF诱导的EGFR磷酸化有抑制作用（IC50：20-80 nM）,还抑制STAT3的磷酸化（IC50：30-70 nM）。 Pelitinib对正常人角质细胞（IC50：61 nM）,以及A431（IC50：125 nM）和MDA-468（IC50：260 nM）肿瘤细胞的增殖有明显抑制作用,但对MCF-7细胞活性较低（IC50：3.6 μM）。

Autophosphorylation of EGFR in cells: For experiments using cells in culture, A431 cells are treated with various concentrations of Pelitinib for 2.75 hours before co-incubation with 100 ng/mL EGF for 0.25 hour. Cells are washed twice with cold phosphate-buffered saline (PBS) before adding to lysis buffer (10 mM Tris, pH 7.5, 5 mM ethylenediamine tetra-acetic acid (EDTA), 150 mM NaCl, 1% Triton X-100, 1% Sodium deoxycholate, 0.1 % SDS, 1 mM PMSF, 10 mg/mL pepstatin A, 10 mg/mL leupeptin, 20 KIU/mL aprotinin, 2 mM sodium orthovanadate, and 100 mM sodium fluoride) for 20 minutes on ice, before immunoprecipitation and SDS-PAGE-immunoblotting. For immunoprecipitation, cultured cells are placed in cold lysis buffer and immediately homogenized on ice with a polytron with several pulses. The homogenate is first centrifuged at 2500 rpm (20 minutes, 4 °C) and then again at 14,000 rpm in a microcentrifuge (10 minutes, 4 °C). Supernatants (1000 μg protein) are incubated for 2 hours at 4 °C with 15 mL of EGFR polyclonal antibody. After 2 hours, 50 μL of protein G plus/protein A agarose beads is added and incubated with constant rotation for 2 hours at 4 °C. After washing with lysis buffer, beads are boiled for 2 minutes in Laemmli sample buffer. Proteins are then resolved by SDS-PAGE, transferred to immobilon membrane and probed overnight with an anti-phosphotyrosine antibody conjugated with horseradish peroxidase (HRP). Membranes are developed using the ECL reagent. Total EGFR protein is determined by stripping membranes and re-probing with receptor-specific antibodies. Quantitation of bands is done by densitometry, using ImageQuant software with a Molecular Dynamics laser transmittance scanner.

Cells are seeded in 96-well dishes, and after 2 hours, Pelitinib is added and incubated for 5 days. After incubation, the medium is removed from each well and fresh medium (150 μL) + 1 mg/mL MTT solution (50 μL) is added. After incubation for 2 hours at 37 °C, the medium is replaced with 150 μL DMSO, and absorbance at 540 nm in each well is determined. The IC50 is calculated by linear regression of the data. (Only for Reference)