| 名稱(chēng) | Dacomitinib |
| 描述 | 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. |
| 細(xì)胞實(shí)驗(yàn) | 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) |
| 激酶實(shí)驗(yàn) | 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. |
| 體外活性 | PF-299804（15 mg/kg,p.o.）具有較強(qiáng)的抗腫瘤活性.其可引起各種人腫瘤異種移植模型中明顯的腫瘤退化,如表達(dá)和/或過(guò)表達(dá)ERBB家族成員,或與耐埃羅替尼和吉非替尼相關(guān)的含有雙重突變(L858R/T790M)的ERBB1 (EGFR)異種移植模型. |
| 體內(nèi)活性 | PF299804對(duì)EGFR信號(hào)傳導(dǎo)有抑制作用,并可促使含EGFR T790M的H3255 GR細(xì)胞系凋亡。PF299804對(duì)表達(dá)T790M 突變體的H3255和 HCC827細(xì)胞的生長(zhǎng)具有抑制作用。在HER2-擴(kuò)增的胃癌細(xì)胞中,PF299804誘導(dǎo)細(xì)胞凋亡和G1期阻滯,并抑制HER家族及其下游信號(hào)通路,包括STAT3,AKT和細(xì)胞外信號(hào)調(diào)節(jié)激酶(ERK)中受體的磷酸化。PF299804還可阻斷SNU216細(xì)胞中EGFR/HER2,HER2/HER3和HER3/HER4異質(zhì)二聚體形成,以及HER3與p85α的結(jié)合。在大多數(shù)敏感細(xì)胞系中,PF299804對(duì)HER2,EGFR,HER4,AKT和ERK的磷酸化水平具有降低作用。PF299804通過(guò)G0/G1期阻滯和對(duì)細(xì)胞凋亡的誘導(dǎo)發(fā)揮其抗增殖作用。在47種人乳腺癌和永生的乳腺上皮細(xì)胞系中,相對(duì)于非擴(kuò)增細(xì)胞系(RR = 3.39, p < 0.0001),PF299804優(yōu)先抑制HER-2-擴(kuò)增的乳腺癌細(xì)胞系的生長(zhǎng)。 |
| 存儲(chǔ)條件 | Powder: -20°C for 3 years | In solvent: -80°C for 1 year | Shipping with blue ice. |
| 溶解度 | 1eq. HCl : 47 mg/mL (100 mM)
DMSO : 45 mg/mL (95.76 mM)
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| 關(guān)鍵字 | Apoptosis | PF 299 | HER1 | EGFR | PF 00299804 | PF299804 | Epidermal growth factor receptor | inhibit | Inhibitor | Dacomitinib | PF 299804 | PF-299 | PF00299804 | ErbB-1 |
| 相關(guān)產(chǎn)品 | L-Glutamic acid | Gefitinib | Metronidazole | 5-Fluorouracil | Dextran sulfate sodium salt (MW 4500-5500) | Stavudine | Tributyrin | Myricetin | L-Ascorbic acid | Acetylcysteine | Salicylic acid | Sodium 4-phenylbutyrate |
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