生物活性

PF-562271 is a potent, ATP-competitive, reversible inhibitor of FAK with IC50 of 1.5 nM, ~10-fold less potent for Pyk2 than FAK and >100-fold selectivity against other protein kinases, except for some CDKs.

Invitro profile of PF-562,271[1]

cellviability[2]

体外研究

体内研究

30% PEG400+0.5% Tween80+5% Propylene glycol

激酶实验

Invitro kinase assay[3]

For kinase assays, 200ng of recombinantactive kinase (FAK or Pyk2) were incubated with purified ASC proteins(wild-type and mutant) and 30 μ l of kinase buffer containing 60 mM HEPES, pH7.5, 10 mM MgCl2, 5 mM MnCl2, 0.3 mM Na3VO4,1.25 mM DTT, 0.2 mM ATP and 10 μ Ci of [γ P32] ATP. The kinase reactionswere performed for 30 min at room temperature (RT) with shaking and stopped bythe addition of SDS sample buffer, and the samples were heated at 95 °C for 5min. The proteins were resolved by 12.5% SDS-PAGE, and phosphorylated proteinswere detected by autoradiography.

细胞实验

Cellculture[2]

A673, EW8, SKNEP, EWS834, and RDES cellswere grown in Dulbeccos' Modified Eagle's Media (DMEM) with10% FBS. A673 mediumwas supplemented with 1 mmol/L sodium pyruvate. TC71 and TC32 were grown inRPMI with 10% FBS and TTC466, EWS502, and CADO-ES-1 with 15% FBS.

All cell lines have unique genotypes andEWS/FLI and EWS/ERG rearrangements were confirmed by RNA sequencing.

Small-moleculetreatment in vitro

Ewing sarcoma cells were plated in 10-cmdishes, allowed to adhere for 24 hours, and then treated with PF-562271 .

Cellviability

ATP content was measured as a surrogate forcell number using the CellTiter-Glo Luminescent Cell Viability Assay. Luminescencereadings were obtained using the FLUOstar Omega microplate reader. For experimentswith small-molecule treatment, 1.25x103Ewing sarcoma cells wereseeded in each well and treated with a range of concentrations. IC50 valueswere calculated from ATP measurements obtained after 3 days of treatment usinglog-transformed, normalized data in GraphPad Prism5.0. Cell lines were alsotreated with compound in 6-cm dishes, trypsinized, and counted by lightmicroscopy using trypan blue exclusion. For experiments using shRNA-transducedcells, 1.25 x103cells were seeded per well into 384-well plates onday 3 posttransduction. ATP content was measured on days 3, 6, and 8 posttransduction.

Colonyformation in methylcellulose matrix

Approximately 3.75x103cellswere dissolved into 1.5 mL of methylcellulose matrix, plated into gridded 6-cmplates, and incubated for at least 10 days. Colonies from 100 squares werecounted using a Nikon inverted microscope.

Flowcytometry

Cells undergoing apoptosis were identifiedby flow cytometry using the ApoAlert Annexin V-FITC Apoptosis Kit. Forintracellular phosphoprotein staining, cells were fixed and permeabilized usingthe BD Cytofix/Cytoperm Kit and stained with phycoerythrin (PE) anti-phospho-S6and analyzed by flow cytometry.

动物实验

Animalexperiments[3]

Female wild-type C57BL/6J, Sykflox/flox,and LysM-Cre mice were maintained under specific pathogen–free conditions andused at 6–9 weeks of age. For MSU-induced peritonitis, mice wereintraperitoneally treated with dimethyl sulfoxide or PF-562271 (25mg per kg body weight) at 2 h before and 30 minutes after challenge with MSU (1mg). At 6 h after MSU treatment, peritoneal cells were collected. The number oftotal cells was determined by trypan blue staining. Neutrophils (CD11b+/Ly6G+)and macrophages (CD11b+/F4/80+) were subjected to antibody staining andanalyzed with a FACSCalibur.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Roberts WG, Ung E, Whalen P, et al. Antitumor activity and pharmacology of a selective focal adhesion kinase inhibitor, PF-562,271. Cancer Res. 2008;68(6):1935-1944.
[more]

体内溶解度
约28 mg/mL

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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