PF-562271 besylate (PF-00562271 Besylate) 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.

FAK:1.5 nM

PF-562271对FAK和Pyk2酪氨酸激酶活性表现出选择性抑制效果，其IC50分别为1.5 nM和14 nM。在基于细胞的实验中，PF-562271对FAK的IC50表现为5 nM，与其他激酶靶标相比表现出更高的选择性。[1] 在二维(2D)培养条件下，PF-562271能够依剂量依赖性抑制FAK WT、FAK/和FAK激酶缺陷(KD)细胞的增殖，其IC50分别为3.3 μM、2.08 μM和2.01 μM。[2]

在多个人类皮下移植模型中，PF-562271显示出剂量依赖性的肿瘤生长抑制作用，对PC-3M、BT474、BxPc3和LoVo的最大肿瘤抑制率在每日两次，每次25至50 mg/kg剂量下，范围从78%至94%，且无体重下降、病态或死亡现象。[1] PF-562271（通过p.o.给药，25 mg/kg）在皮下和骨转移的PC3M-luc-C6移植模型中显著减缓了肿瘤进展。[3] 在Huh7.5肝细胞癌移植模型中，sunitinib和PF-562271的联合治疗针对血管生成和肿瘤侵袭性，比单一化合物治疗产生更显著的抗肿瘤效果，通过阻断肿瘤生长及影响肿瘤在撤销治疗后的恢复能力。[4]

Recombinant kinase assay and enzyme kinetics : Briefly, 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 minutes. Phosphorylation of p(Glu/Tyr) is challenged with serially diluted PF-562271 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. Broad kinase selectivity profiling is performed in house and by using the KinaseProfiler Selectivity Screening Service available through UpState Biotechnology.

Cells are plated for 48 hours before addition of PF-562271. After 3 days cells are fixed by addition of ice cold 25% trichloroacetic acid (TCA) solution prior to staining with Sulforhodamine B (SRB) dye solution. Plates are washed with 1% glacial acetic acid, air-dried and resuspended in 10 mM Tris buffer, pH 10.5 before reading absorbance at 540 nm. Curve fitting and generation of IC50 values is carried out using GraphPad Prism 4 software from six replicates.(Only for Reference)