FAK (IC50 = 4 nM)

PF-573228 suppresses the purified recombinant catalytic portion of FAK with an IC50 of 4 nM [1]. PF-573228 suppresses FAK phosphorylation on Tyr397 at an IC50 of 30-100 nM[1]. PF-573228 significantly lowers FAK Tyr397 phosphorylation [1]. PF-573228 decreases chemotaxis and chemotactic migration while also reducing focal adhesion turnover [1].

In several human s.c. xenograft models, PF-562271 exhibits dose-dependent tumor growth inhibition, and produces maximum tumor inhibition for PC-3M, BT474, BxPc3, and LoVo ranging from 78% to 94% inhibition at doses of 25 to 50 mg/kg twice daily, without weight loss, morbidity, or death. PF-562271 (25 mg/kg by p.o.) leads to a significant decrease in tumor progression in both subcutaneous and bone metastasis PC3M-luc-C6 xenograft models. In a Huh7.5 hepatocellular carcinoma xenograft model, combination therapy of sunitinib and PF-562271 targets angiogenesis and tumor aggressiveness, and produces more significant anti-tumor effect than single agent by blocking tumor growth and impacting the ability of the tumor to recover upon withdrawal of the therapy.

Recombinant Kinase Assay[1]
Purified activated FAK kinase domain (amino acids 410–689) was reacted with 50 μm ATP, and 10 μg/well of a random peptide polymer of Glu and Tyr (molar ratio of 4:1), poly(Glu/Tyr) in kinase buffer (50 mm HEPES, pH 7.5, 125 mm NaCl, 48 mm MgCl2) for 15 min. Phosphorylation of poly(Glu/Tyr) was challenged with serially diluted compounds at ½-Log concentrations starting at a top concentration of 1 μm. Each concentration was run in triplicate. Phosphorylation of poly(Glu/Tyr) was detected with a general anti-phospho-tyrosine (PY20) antibody, followed by horseradish peroxidase-conjugated goat anti-mouse IgG antibody. The standard horseradish peroxidase substrate 3, 3′, 5, 5′-tetramethylbenzidine was added, and Optical Density readings at 450 nm were obtained following the addition of stop solution (2 m H2SO4). The IC50 values were determined using the Hill slope model. Broad kinase selectivity profiling was performed using the KinaseProfiler™ selectivity screening service available through Upstate Biotechnology, Inc. For more information please see: www.upstate.com/discovery/services/kp_overview.q.

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Cellular Kinase Assays[1]
Using the GeneSwitch inducible system from Invitrogen, stable A431 epithelial carcinoma clones were generated to express either wild type V5-tagged FAK protein or mutant FAK Y397F V5-tagged protein under the inducible regulation of mifepristone. Stable clones were grown in Dulbecco’s modified Eagle’s medium, 10% fetal bovine serum, 750 μg/ml Zeocin, and 50 μg/ml Hygromycin. One day prior to running the FAK cell ELISA, A431·FAKwt cells were seeded at 1.2 × 106 cells/ml in growth medium in 96-well U-bottom plates. After 4–6 h at 37 °C, 5% CO2, FAK expression was induced with 0.1 nm miferpristone. Uninduced controls were included. Goat anti-mouse or anti-rabbit plates were subsequently coated with either anti-V5 or anti-FAK (1.0 μg/ml) or an irrelevant antibody control in Superblock Tris-buffered saline buffer. Anti-V5- or anti-FAK-coated plates were blocked in 3% bovine serum albumin, 0.5% Tween for 1 h at room temperature. The cells were treated with ½-Log serial dilutions starting at a top concentration of 1 μm for 30 min at 37 °C, 5% CO2. Lysates from cells treated with indicated concentrations of compound were prepared in P-lysis buffer (50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm EDTA, 1 mm Na3VO4, 1 mm NaF, and protease inhibitors) and transferred to the anti-V5- or anti-FAK-coated plates to capture total induced or total FAK protein. Anti-phosphospecific FAK[Y397] was used to detect autophosphorylated FAK Tyr397, followed by secondary reporter antibody. Horseradish peroxidase substrate was added, and plates were read at 450 nm. The IC50 values were determined using the Hill slope model. For Western blot analysis, REF52 cells were treated with the indicated concentrations of inhibitor for the indicated periods of time prior to lysis in CH buffer (50 mm HEPES, 0.15 m NaCl, 2 mm EDTA, 1% Nonidet P-40, and 0.5% sodium deoxycholate, pH 7.2) containing 1 mm phenylmethylsulfonyl fluoride, 100 mm leupeptin, and 0.05 TIU/ml aprotinin, 1 mm Na3VO4, 40 mm NaF, and 10 mm Na4P2O7. For suspension/replating experiments, REF52 cells were suspended in serum-free medium in the presence or absence of the indicated concentrations of inhibitor and were allowed to reattach to plates coated with 5 μg/ml FN for 30 min in the continued presence or absence of inhibitor. The cells were lysed in CH-buffer, and Western blot analysis of whole cell lysates was performed using 25–50 μg of protein.

Wound Healing Assay[1]
Confluent REF52 monolayers on 35-mm Bioptechs delta-T dishes were wounded with a 10-μl pipette tip, and cells migrating into the wound were filmed for 9 h at 37 °C by time lapse microscopy. PF-573,228 was added at the time of wounding, and filming was initiated 1 h post-wounding. Time lapse microscopy was performed using a Nikon TE200 inverted microscope with a 20× differential interference contrast objective and a Bioptechs heated stage. The images were captured with a Hamamatsu Orca camera and compiled using Improvision Openlab software. Following image capture, the nuclei of single cells were tracked over time as they migrated into the wound, and the cell speed was calculated by dividing the length of the cell track by the total time of the movie.[1]

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Immunofluorescence and Cell Spreading[1]
Adherent REF52 cells were washed and treated with increasing concentrations of PF-573,228 for 1 h in the absence of serum. For replating experiments REF52 cells were held in suspension for 30 min without serum and in the presence of increasing amounts of the FAK inhibitors. The cells were plated onto FN-coated (5 μg/ml) coverslips (for immunofluorescence) or delta-T dishes (for differential interference contrast time lapse movies) for 60 min in the continued presence of inhibitors. The cells were fixed for 20 min with fresh 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 2 min and blocked in 20% goat serum, 2% bovine serum albumin for 30 min. The cells were then stained with antibodies to paxillin or FAK Tyr(P)397. Secondary antibodies were labeled with Alexa Fluor 488 or 594 dyes. Images were acquired using a Nikon Eclipse E600 microscope with a 60× oil objective, a Hamamatsu digital camera, and the Openlab imaging software package.

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TIRF Microscopy and Adhesion Turnover Analysis[1]
NIH-3T3 cells were stably transfected with green fluorescent protein-paxillin using the FLP-In system. Confluent monolayers on delta-T dishes were treated with PF-573,228 and wounded with a pipette tip as described above. Fluorescent adhesions were visualized at 37 °C using a Nikon TE2000-E Eclipse inverted TIRF microscope equipped with a 60 × 1.45 N.A. TIRF objective, a Retiga digital camera and QCapture Pro acquisition software. The images were acquired every 5 min for 60–90 min. Image analysis was performed using Openlab software. The intensity of each adhesion was measured over time, and the peak (maximal) intensity for each adhesion was determined. Adhesion lifetime was calculated as the sum of the time required for the adhesion to reach the peak intensity from its half-peak intensity and the time required for the adhesion to return to its half-peak intensity from its peak intensity. At least seven adhesions were analyzed per cell.

To better understand the role of FAK in leukocyte recruitment, we used a FAK-specific inhibitor (PF-573228) and determined the effect on IL-4 induced eosinophil recruitment in vivo. PF-573228 prevented the expression of VCAM-1 and CCL26 expression in IL-4-stimulated human endothelial cells in vitro. As a result, eosinophil adhesion and transmigration were blocked. PF-572338 also prevented IL-4-induced VCAM-1 expression in vivo. Using brightfield intravital microscopy, we found that PF-573228 decreased leukocyte rolling flux, adhesion, and emigration. We specifically examined eosinophil recruitment in vivo by using an eosinophil-GFP reporter mouse and found PF-573228 attenuated eosinophil emigration. This study reveals that a FAK inhibitor influences inflammation through its action on eosinophil recruitment. [2]

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25 mg/kg; Oral gavage

PC3M-luc-C6 xenograft models

[1]. Cellular characterization of a novel focal adhesion kinase inhibitor. J Biol Chem. 2007 May 18;282(20):14845-52.

[2]. Inhibiting focal adhesion kinase (FAK) blocks IL-4 induced VCAM-1 expression and eosinophil recruitment in vitro and in vivo. J Leukoc Biol . 2018 Jul;104(1):147-158.

6-[[4-[(3-methylsulfonylphenyl)methylamino]-5-(trifluoromethyl)-2-pyrimidinyl]amino]-3,4-dihydro-1H-quinolin-2-one is a member of quinolines.

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Focal adhesion kinase (FAK) is a member of a family of non-receptor protein-tyrosine kinases that regulates integrin and growth factor signaling pathways involved in cell migration, proliferation, and survival. FAK expression is increased in many cancers, including breast and prostate cancer. Here we describe perturbation of adhesion-mediated signaling with a FAK inhibitor, PF-573,228. In vitro, this compound inhibited purified recombinant catalytic fragment of FAK with an IC(50) of 4 nM. In cultured cells, PF-573,228 inhibited FAK phosphorylation on Tyr(397) with an IC(50) of 30-100 nM. Treatment of cells with concentrations of PF-573,228 that significantly decreased FAK Tyr(397) phosphorylation failed to inhibit cell growth or induce apoptosis. In contrast, treatment with PF-573,228 inhibited both chemotactic and haptotactic migration concomitant with the inhibition of focal adhesion turnover. These studies show that PF-573,228 serves as a useful tool to dissect the functions of FAK in integrin-dependent signaling pathways in normal and cancer cells and forms the basis for the generation of compounds amenable for preclinical and patient trials.[1]

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Leukocyte recruitment plays a critical role during both normal inflammation and chronic inflammatory diseases, and ongoing studies endeavor to better understand the complexities of this process. Focal adhesion kinase (FAK) is well known for its role in cancer, yet it also has been shown to regulate aspects of neutrophil and B16 melanoma cell recruitment by rapidly influencing endothelial cell focal adhesion dynamics and junctional opening. Recently, we found that FAK related non-kinase (FRNK), a protein that is often used as a FAK dominant negative, blocked eosinophil transmigration by preventing the transcription of vascular cell adhesion molecule-1 (VCAM-1) and eotaxin-3 (CCL26). Surprisingly, the blocking occurred even in the absence of endogenous FAK. To better understand the role of FAK in leukocyte recruitment, we used a FAK-specific inhibitor (PF-573228) and determined the effect on IL-4 induced eosinophil recruitment in vitro and in vivo. PF-573228 prevented the expression of VCAM-1 and CCL26 expression in IL-4-stimulated human endothelial cells in vitro. As a result, eosinophil adhesion and transmigration were blocked. PF-572338 also prevented IL-4-induced VCAM-1 expression in vivo. Using brightfield intravital microscopy, we found that PF-573228 decreased leukocyte rolling flux, adhesion, and emigration. We specifically examined eosinophil recruitment in vivo by using an eosinophil-GFP reporter mouse and found PF-573228 attenuated eosinophil emigration. This study reveals that a FAK inhibitor influences inflammation through its action on eosinophil recruitment.[2]

C22H20F3N5O3S

491.49

491.123

C, 53.76; H, 4.10; F, 11.60; N, 14.25; O, 9.77; S, 6.52

869288-64-2

11612883

White to off-white solid powder

1.5±0.1 g/cm3

1.616

1.03

3

10

6

34

822

0

HESLKTSGTIBHJU-UHFFFAOYSA-N

InChI=1S/C22H20F3N5O3S/c1-34(32,33)16-4-2-3-13(9-16)11-26-20-17(22(23,24)25)12-27-21(30-20)28-15-6-7-18-14(10-15)5-8-19(31)29-18/h2-4,6-7,9-10,12H,5,8,11H2,1H3,(H,29,31)(H2,26,27,28,30)

3,4-Dihydro-6-[[4-[[[3-(methylsulfonyl)phenyl]methyl]amino]-5-(trifluoromethyl)-2-pyrimidinyl]amino]-2(1H)-quinolinone

PF573,228; PF 573,228; PF-573,228; PF573228; 869288-64-2; PF-573228; PF 573228; PF573228; PF-228; 3,4-Dihydro-6-[[4-[[[3-(methylsulfonyl)phenyl]methyl]amino]-5-(trifluoromethyl)-2-pyrimidinyl]amino]-2(1H)-quinolinone; 6-((4-((3-(Methylsulfonyl)benzyl)amino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-3,4-dihydroquinolin-2(1H)-one; 6-(4-(3-(methylsulfonyl)benzylamino)-5-(trifluoromethyl)pyrimidin-2-ylamino)-3,4-dihydroquinolin-2(1H)-one; PF 573228; PF-573228;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 2.5 mg/mL (5.09 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.23 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.23 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% propylene glycol:30 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)