Recombinant kinase assay

Purified-activated FAK kinase domain (amino acid 410 ~ 689) was reacted with 50 μmol/L ATP and 10 μg p(Glu/Tyr), in kinase buffer [50 mmol/L HEPES (pH 7.5), 125 mmol/L NaCl and 48 mmol/L MgCl2] for 15 mins. Phosphorylation of p(Glu/Tyr) was challenged with serially diluted PF-562271 at 1/2-Log concentrations starting at a top concentration of 1 μmol/L. Each concentration was tested in triplicate. Phosphorylation of p(Glu/Tyr) was detected with a general antiphospho-tyrosine (PY20) antibody followed by horseradish peroxidase (HRP)-conjugated goat antimouse IgG antibody. HRP substrate was added, and absorbance readings at 450 nm were obtained after addition of stop solution (2 mol/L H2SO4). IC50 values were determined using the Hill-Slope Model.

Cell lines

PC3-M cells

Preparation method

The solubility of this compound in DMSO is >25.4 mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below - 20 ℃ for several months.

Reacting condition

1.1 or 3.3 μmol/L; 48 hrs

Applications

After 48-hour exposure, PF-562271 at the concentration of 3.3 μM altered the cell cycle progression of PC3-M cells. However, the inhibitory activity of PF-562271 against cdk5/p35 enzyme was undetected.

Animal models

Nude mice bearing U87MG human glioblastoma cells

Dosage form

3.3, 10 or 33 mg/kg; p.o.

Applications

In nude mice bearing U87MG human glioblastoma cells, PF-562271 inhibited FAK phosphorylation in a dose- and time-dependent manner. After 1-hr exposure to 33 mg/kg PF-562271, maximal pFAK inhibition (78%) was achieved. However, inhibition effect of PF-562271 on FAK phosphorylation was sustained (>50%) for >4 hrs after this single p.o. dose. The calculated EC50 value was 93 ng/mL.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

PF-562271 is a potent, ATP-competitive and reversible inhibitor of both focal adhesion kinase (FAK), a non-receptor tyrosine kinase involved in a variety of cellular events, and proline-rich tyrosine kinase 2 (Pyk2), an FAK homolog containing 48% amino acid identity, with half maximal inhibitory concentration (IC50) of 1.5 nmol/L and 14 nmol/L respectively. As a potential therapeutic agent either alone or in combination with other agents for the treatment of cancer, PF-562271 has been reported to effectively inhibit the proliferation of tumors in both xenograft and transgenic mouse models, in which it dose-dependently inhibits FAK phosphorylation in tumor-bearing mice with half maximal effective concentration (EC50) of 93 ng/mL.

References:
[1]Stokes JB, Adair SJ, Slack-Davis JK, Walters DM, Tilghman RW, Hershey ED, Lowrey B, Thomas KS, Bouton AH, Hwang RF, Stelow EB, Parsons JT, Bauer TW. Inhibition of focal adhesion kinase by PF-562,271 inhibits the growth and metastasis of pancreatic cancer concomitant with altering the tumor microenvironment. Mol Cancer Ther. 2011 Nov;10(11):2135-45. doi: 10.1158/1535-7163.MCT-11-0261. Epub 2011 Sep 8.
[2]Roberts WG, Ung E, Whalen P, Cooper B, Hulford C, Autry C, Richter D, Emerson E, Lin J, Kath J, Coleman K, Yao L, Martinez-Alsina L, Lorenzen M, Berliner M, Luzzio M, Patel N, Schmitt E, LaGreca S, Jani J, Wessel M, Marr E, Griffor M, Vajdos F. Antitumor activity and pharmacology of a selective focal adhesion kinase inhibitor, PF-562,271. Cancer Res. 2008 Mar 15;68(6):1935-44. doi: 10.1158/0008-5472.CAN-07-5155.