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

FAK:1.5 nM

In mice implanted with 4T1 tumors, PND-1186 (100 mg/kg, s.c.) induces apoptosis and inhibits the growth of subcutaneous 4T1 tumors. Additionally, in mice carrying ID8 tumors, PND-1186 (0.5 mg/mL, p.o.) suppresses the growth of ovarian cancer tumors.

In vitro, PND-1186 demonstrates an inhibitory effect on the motility of 4T1 breast cancer cells, promotes apoptosis in suspended 4T1 cells, and reduces both the number and size of 4T1 soft agar colonies. Additionally, in HEY and OVCAR8 cells, PND-1186 induces G0-G1 cell cycle arrest, leading to cell death.

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)