| Protocol | In Vitro | In vitro activity: In vitro, PND-1186 inhibits 4T1 breast carcinoma motility, promotes 4T1 apoptosis in suspended conditions, and decreases 4T1 soft agar colony number and size. In HEY and OVCAR8 cells, VS-4718 promotes G0-G1 cell-cycle arrest followed by cell death. Kinase Assay: 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. Cell Assay: 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. |
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| In Vivo | In mice bearing 4T1 tumors, PND-1186 (100 mg/kg s.c.) inhibits 4T1 subcutaneous tumor growth by induction of apoptosis. In mice bearing ID8 tumors, PND-1186 (0.5 mg/mL for p.o.) also causes ovarian carcinoma tumor growth inhibition. |
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| Animal model | Mice bearing ID8 tumors or 4T1 tumors |
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| Formulation | Dissolved in polyethylene glycol 400 (PEG400) in PBS (1:1) for s.c. |
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| Dosages | 5% sucrose for p.o.; 100 mg/kg every 12 hours for s.c.; 0.5 mg/mL for p.o.; Administered via s.c. or p.o. |
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These protocols are for reference only. InvivoChem does not
independently validate these methods.
Preparing Stock Solutions
| Solvent volume to be added |
Mass (the weight of a compound) |
| Mother liquor concentration |
1mg | 5mg | 10mg | 20mg |
| 1mM | 1.9940 mL | 9.9701 mL | 19.9402 mL | 39.8804 mL |
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| 5mM | 0.3988 mL | 1.9940 mL | 3.9880 mL | 7.9761 mL |
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| 10mM | 0.1994 mL | 0.9970 mL | 1.9940 mL | 3.9880 mL |
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| 20mM | 0.0997 mL | 0.4985 mL | 0.9970 mL | 1.9940 mL |
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Quality Control Documentation
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