PND-1186 (VS-4718) is a reversible and specific FAK inhibitor (IC50: 1.5 nM).

FAK:1.5 nM

在负荷4T1肿瘤的小鼠体内,PND-1186（100 mg/kg,s.c.）可诱导细胞凋亡,对4T1皮下肿瘤生长产生抑制作用.在负荷ID8肿瘤的小鼠体内,PND-1186（0.5 mg/mL,p.o.）还会抑制卵巢癌肿瘤的生长.

在体外,PND-1186对4T1乳腺癌能动性有抑制作用,且可促进悬浮状态的4T1凋亡,并使4T1软琼脂集落数量和大小减少。在HEY 和 OVCAR8细胞中,PND-1186促使G0-G1细胞周期阻滞,从而使细胞死亡。

In vitro kinase activity: GST-FAK in vitro kinase activity is measured and compared to His-tagged FAK 411–686 using the K-LISA screening kit and poly(Glu:Tyr) (4:1) copolymer as a substrate immobilized on microtiter plates. IC50 values are determined with various concentrations of test compounds in a buffer containing 50 μM ATP and 10 mM MnCl2, 50 mM HEPES (pH 7.5), 25 mM NaCl, 0.01% BSA, and 0.1 mM Na orthovanadate for 5 min at room temperature. Serial diluted compounds are tested in triplicate. Substrate phosphorylation is measured using horseradish peroxidase-conjugated anti-pTyr antibodies with spetrophotometric color quantitation. IC50 values are determined using the Hill-Slope Model. Kinase selectivity profiling is performed by using the KinaseProfiler service.

For soft agar assays, 48-well plates are coated with a 1:4 mix of 2% agar (EM Science) in 0.2 mL growth media (bottom layer). 5×104 cells are plated per well (in triplicate) in a mixture of 0.3% agar in 0.2 mL growth media (top layer). After agar solidification, 0.2 mL growth media is added containing DMSO or PND-1186 (final concentration for 0.6 mL). In separate experiments, PND-1186 is added after 4 days. After 10 days, colonies are imaged in phase contrast, enumerated by counting 9 fields (3 fields per well), and total area determined using Image J. For all analyses, experimental points are performed in triplicate and repeated at least two times. (Only for Reference)