PF-562271 is an effective ATP-competitive, reversible inhibitor of FAK(IC50=1.5 nM) and Pyk2 kinase(IC50=13 nM).

FAK:1.5 nM, PYK2:13 nM

在胫骨植入MDA-MB-231细胞的大鼠中,口服 PF-562271（5 mg/kg）,引起骨钙素和松质骨增加,从而使肿瘤细胞生长减缓.在携带H125肺部异种移植肿瘤、异种移植PC3M-luc-C6的小鼠模型中,口服 PF-562271 （25 mg/kg）抑制肿瘤细胞生长,引起细胞凋亡.在U87 mg的小鼠中,PF-562271（口服< 33 mg/kg）能够以时间和剂量依赖的方式抑制肿瘤中FAK磷酸化.在BxPc3异种移植小鼠、PC3-M异种移植小鼠中口服 PF-562271（50 mg/kg）,能够抑制肿瘤生长.

PF-562271能够结合在ATP与FAK结合的部位,并在激酶铰链区形成抑制剂与主链原子之间的氢键。在鸡胚绒毛尿囊膜中,PF-562271（1 nM ）阻断bFGF刺激的血管生成。在PC3-M细胞中,PF-562271（3.3 μM）能够使细胞停止在G1期。对于A431 细胞,PF-562271（250 nM ）能够抑制细胞侵入胶原蛋白。

The purified-activated FAK kinase domain (amino acid 410-689) is reacted with 50 μM ATP and 10 μg per well of a random peptide polymer of Glu and Tyr, p(Glu/Tyr), in kinase buffer (50 mM HEPES pH 7.5, 125 mM NaCl, and 48 mM MgCl2) for 15 min. Phosphorylation of p(Glu/Tyr) is challenged with serially diluted compound at 1/2-Log concentrations starting at a top concentration of 1 μM. Each concentration is tested in triplicate. Phosphorylation of p(Glu/Tyr) is detected with a general antiphospho-tyrosine (PY20) antibody followed by horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody. HRP substrate is added, and absorbance readings at 450 nm are obtained after addition of stop solution (2 M H2SO4). IC50 values are determined using the Hill-Slope Model[1].

PF-562271 (Haoyuan Chemexpress Co., Ltd.) is dissolved in DMSO and stored, and then diluted with appropriate media before use[2]. Ewing sarcoma cells are plated in 10-cm dishes, allowed to adhere for 24 hours, and then treated with PF-562271, PD0325901, or Dasatinib. ATP content is measured as a surrogate for cell number using the CellTiter-Glo Luminescent Cell Viability Assay. Luminescence readings are obtained using the FLUOstar Omega microplate reader. For experiments with small-molecule treatment, 1.25×103 Ewing sarcoma cells are seeded in each well and treated with a range of concentrations. IC50 values are calculated from ATP measurements obtained after 3 days of treatment using log-transformed, normalized data in GraphPad Prism 5.0. Cell lines are also treated with compound in 6-cm dishes, trypsinized, and counted by light microscopy using trypan blue exclusion. For experiments using shRNA-transduced cells, 1.25×103 cells are seeded per well into 384-well plates on day 3 posttransduction. ATP content is measured on days 3, 6, and 8 posttransduction[2].