FAK (IC50 = 1.5 nM)

PND-1186 inhibition of FAK is distinct from effects of Src PTK inhibitors. PND-1186 inhibits 4T1 breast carcinoma motility in vitro. Nanomolar PND-1186 levels promote 4T1 apoptosis in suspended but not adherent conditions. PND-1186 inhibition of FAK and p130Cas tyrosine phosphorylation under spheroid growth conditions. PND-1186 decreases soft agar colony number and size at nanomolar concentrations. [1]

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PND-1186's IC50 in breast cancer cells was found to be less than 100 nM using anti-phospho-specific immunoblotting against FAK Tyr-397[1]. FAK has a crucial role in generating an invasive and metastatic cell phenotype in murine 4T1 breast cancer cells. When PND-1186 is introduced to 4T1 cells at increasing concentrations (0.1 to 1.0 µM), FAK Tyr-397 phosphorylation (pY397) is inhibited, and within an hour, total FAK protein levels are raised[1].

PND-1186 suppresses the growth of 4T1 subcutaneous tumors by inducing apoptosis when injected subcutaneously in the neck region (30 mg/kg or 100 mg/kg)[1].

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PND-1186 inhibits 4T1 subcutaneous tumor growth by induction of apoptosis [1]
To determine the sensitivity of 4T1 tumor growth to PND-1186 administration, mCherry fluorescently-labeled 4T1 cells were grown subcutaneously in BALB/c mice (Fig. 7). After allowing eight days for primary tumor establishment, vehicle or PND-1186 at 30 mg/kg or at 100 mg/kg was administered every 12 h (twice-daily, b.i.d.) for 5 days after which time, mCherry 4T1 tumors were visualized in situ followed by extraction and weighing (Fig. 7A and B). Whereas vehicle-treated 4T1 tumors were brightly fluorescent, generally multi-lobed and had become invasive to the surrounding tissues, tumors in mice treated with 100 mg/kg PND-1186 contained dark non-fluorescent centers, were generally rounded, and were loosely adherent to sub-dermal tissues (Fig. 7A). 100 mg/kg PND-1186 treatment significantly reduced final 4T1 tumor weight 2-fold (n=8, p<0.05) whereas 30 mg/kg PND-1186 slightly reduced final tumor weight but was not significantly different compared to control (n=8, p>0.05). To determine if the loss of mCherry fluorescence in the center of 100 mg/kg PND-1186 tumors was associated with increased cell apoptosis, medial sections were analyzed by TUNEL (Fig. 7C and D) and anti-cleaved caspase 3 (Fig. 7E) staining. Both 30 and 100 mg/kg administration of PND-1186 significantly increased tumor TUNEL staining compared to vehicle-treated controls (Fig. 7D). As elevated cleaved caspase-3 staining was also found in the tumors of PND-1186-treated mice (Fig. 7E), these results parallel our in vitro analyses and show that PND-1186 promotes apoptosis of 4T1 cells in 3D conditions resulting in the inhibition of tumor growth in vivo.

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PND-1186 inhibits ovarian carcinoma tumor growth in vivo [1]
During ovarian carcinoma tumor cell progression, cells can dissociate from the primary tumor and grow as multi-cellular spheroids within the peritoneal space32. As PND-1186 selectively promotes 4T1 breast carcinoma apoptosis in 3D environments, PND-1186 effects on murine ID8 ovarian carcinoma cell growth were evaluated in vitro and in vivo (Fig. 8). There was a dose-dependent reduction of ID8 cell number when grown in suspended cell culture with 0.1 µM PND-1186 showing significant inhibition at 72 h (Fig. 8A) and promoting a dramatic reduction in viable cells after 15 days (Fig. 8B). To determine if low levels of PND-1186 could affect ID8 growth in vivo, cells were intraperitoneally-injected into C57BL6 mice and after 11 days, mice were provided 0.5 mg/ml PND-1186 in 5% sucrose in lieu of drinking water on an ad libitum basis. No adverse effects and no body weight loss were noted with PND-1186 and sucrose addition to the drinking water. After 30 treatment days, PND-1186 had significantly reduced the number of ascites-associated cells compared to sucrose controls (Fig. 8C) and this was associated with the almost complete inhibition of FAK pY397 by 0.5 mg/ml PND-1186 administration (Fig. 8D and E). Taken together, our results show that PND-1186 inhibits the growth of breast and ovarian carcinoma cells in vitro and in vivo. The selective effects on PND-1186 in promoting apoptosis of cells in three dimensional environments points to a novel role for FAK activity in generating anchorage-independent survival signals.

Baculovirus FAK catalytic domain and in vitro kinase assays [1]
The FAK catalytic domain region (411–686) was generated by polymerase chain reaction using the primers 5’-cgatcgaattctcgaccagggattatgagattca-3’ 5’-tagctgtcgacttactgcaccttctcctcctccagg-3’, cloned into pGEX4T as a fusion with GST, and moved into the pAcG2T baculovirus expression vector. Virus clones were identified by plaque assays and amplified. For protein expression, SF9 cells were transduced at a multiplicity of infection of 2–5 pfu/cell and cultured at 27°C for 48 h. Glutathione agarose affinity chromatography were used to purify GST-FAK (411–686) followed by size fractionation using hiload 16/60 Superdex chromatography. Protein was concentrated and stored frozen in 50 mM Tris pH 8.0, 150 mM NaCl, 1 mM Na orthovanadate, 0.5 mM EDTA, 0.5 mM EGTA, 0.1% β-mercaptoethanol, and 20% glycerol. Purity was estimated at >90% by SDS-PAGE. GST-FAK in vitro kinase activity was 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 were 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 at ½-Log concentrations (starting at 1 µM) were tested in triplicate. Substrate phosphorylation was measured using horseradish peroxidase-conjugated anti-pTyr antibodies (PY20, Santa Cruz Biotechnology) with spetrophometic color quantitation. IC50 values were determined using the Hill-Slope Model. Kinase selectivity profiling was performed by using the KinaseProfiler service.
PND-1186 was identified through high-throughput kinase activity screens and conventional medicinal chemistry approaches. PND-1186 was synthesized and prepared as a HCl salt as described in a patent application41.

Western Blot Analysis[1]
Cell Types: 4T1 breast carcinoma cells
Tested Concentrations: 0.1, 0.2, 0.4, 0.6 and 1.0 µM
Incubation Duration: 1 hour
Experimental Results: Inhibited FAK Tyr-397 phosphorylation (pY397) and resulted in elevated levels of total FAK protein.

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Anchorage-dependent, spheroid, and soft agar cell growth assays [1]
Cells (2×105) were plated per 35 mm well under adherent (tissue culture-treated) and non-adherent conditions (poly-HEMA-coated) in 6-well plates in growth media. Between 24 and 168 h, all cells were collected, a single cell suspension was prepared by limited trypsin-EDTA treatment, and viable cells were enumerated by trypan blue staining and counting. For spheroid area determination, cells were imaged after 72 h in phase contrast using an Olympus IX51 microscope. Area was calculated using Image J software (version 1.43). For soft agar assays, 48-well plates were coated with a 1:4 mix of 2% agar in 0.2 ml growth media (bottom layer). 5×104 cells were 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 was added containing DMSO or PND-1186 (final concentration for 0.6 ml). In separate experiments, PND-1186 was added after 4 days. After 10 days, colonies were 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 were performed in triplicate and were experiments were repeated at least two times.

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Cell growth and apoptosis assays [1]
For cell growth analyses, adherent or suspended cells were treated with PND-1186 for the indicated times, collected as a single cell suspension by limited trypsin treatment, fixed with 70% ethanol, collected by centrifugation and washed with PBS. Cell pellets were resuspended in 300 µl of PBS containing propidium iodide (PI) (10 µg/ml), DNAse-free RNAse (100 µg/ml, Qiagen), and then incubated at 37 °C with agitation for 1 h. Samples were analyzed by flow cytometry and cell cycle analyses were performed by ModFit LT3.2 software. Hypodiploid DNA content as a measure of cell apoptosis was detected by PI staining as described 28. For cell apoptosis analyses, adherent or suspended cells were treated with PND-1186 and collected as above, stained for phycoerythrin (PE)-conjugated annexin V binding and 7-amino-actinomycin (7-AAD) reactivity, and analyzed within 1 h by flow cytometry. Quadrant gates were positioned based on cell autofluorescence (negative) staurosporine-treated (positive) controls. Apoptosis was calculated to be the percent of annexin V-positive cells. In the soft agar assays, apoptosis was quantified by visual inspection of at least 200 cells and was defined as the appearance of membrane blebbing or cell shrinkage. Apoptosis was also detected by appearance of cleaved caspase-3 antibody reactivity in protein lysates by immunoblotting.

Animal/Disease Models: balb/c (Bagg ALBino) mouse[1]
Doses: 30 mg /kg or 100 mg/kg
Route of Administration: Injected (100 µL) subcutaneously (sc) in the neck region; every 12 h (twice-daily, bid) for 5 days.
Experimental Results: 100 mg/kg treatment Dramatically decreased final 4T1 tumor weight 2-fold whereas 30 mg/kg treatment slightly decreased final tumor weight but was not Dramatically different compared to control.

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Mouse tumor studies [1]
Six to eight week old female C57BL6 and BALB/c mice were used. All in vivo studies were carried out under an approved institutional experimental animal care and use protocol. Growing tumor cells were harvested by limited trypsinization, washed in PBS, and counted using a ViCell XR (Beckman) prior to injection. Cell viability as measured by trypan blue exclusion was >95%. For subcutaneous tumor growth, 1×106 mCherry-labeled 4T1 cells in 100 µl PBS were injected into the hindflank of Balb/C mice. After 8 days, mice with equal volume tumors (as measured using vernier calipers and determined by length × width2/2) were grouped (n=8 per group) and PND-1186 solubilized in polyethylene glycol 400 (PEG400) in PBS (1:1) was injected (100 µl) subcutaneously in the neck region at 30 mg/kg or 100 mg/kg every 12 hours. Control animals received PEG400:PBS injections and at 13 days, tumors were imaged in situ using an Olympus OV100 Intravital Fluorescence Molecular Imaging System, tumors were excised and weighed, half was frozen in OCT, and half was solubilized in protein lysis buffer for FAK phosphorylation analyses. For ID8 ovarian carcinoma tumor growth, 0.8 mL of 1×107 ID8 cells in PBS was intraperitoneal injected into C57BL6 mice. After 11 days, 0.5 mg/mL PND-1186 dissolved in 5% sucrose in water was provided for drinking and control mice received 5% sucrose (n=8 per group). Administration continued ad libitum for 30 days after which mice were euthanized, ascites fluid collected, cells obtained by centrifugation (2000 rpm for 5 min), cell volume measured by pipet, and then solubilized in protein lysis buffer for immunoblotting analyses.

PND-1186 has low intrinsic toxicity to mice

[1]. PND-1186 FAK inhibitor selectively promotes tumor cell apoptosis in three-dimensional environments. Cancer Biol Ther. 2010 May 15;9(10):764-77.

VS-4718 is under investigation in clinical trial NCT02215629 (Dose Escalation Study in Acute Myeloid or B-Cell Acute Lymphoblastic Leukemia).
FAK Inhibitor VS-4718 is an orally bioavailable focal adhesion kinase (FAK) inhibitor with potential antineoplastic activity. Upon administration, VS-4718 inhibits FAK, blocks fibronectin-stimulated FAK autophosphorylation of Tyr397, and may prevent the integrin-mediated activation of several downstream signal transduction pathways, including ERK, JNK/MAPK and PI3K/Akt. This results in the reduction of the number of cancer stem cells (CSCs) and inhibits tumor cell migration, proliferation and survival. The cytoplasmic tyrosine kinase FAK is a signal transducer for integrins and is constitutively activated in various tumor cell types; it is involved in tumor cell invasion, migration and proliferation and plays a key role in the development, function and survival of CSCs.

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Tumor cells can grow in an anchorage-independent manner. This is mediated in part through survival signals that bypass normal growth restraints controlled by integrin cell surface receptors. Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase that associates with integrins and modulates various cellular processes including growth, survival, and migration. As increased FAK expression and tyrosine phosphorylation are associated with tumor progression, inhibitors of FAK are being tested for anti-tumor effects. Here, we analyze PND-1186, a substituted pyridine reversible inhibitor of FAK activity with a 50% inhibitory concentration (IC50) of 1.5 nM in vitro. PND-1186 has an IC50 of ~100 nM in breast carcinoma cells as determined by anti-phospho-specific immunoblotting to FAK Tyr-397. PND-1186 did not alter c‑Src or p130Cas tyrosine phosphorylation in adherent cells, yet functioned to restrain cell movement. Notably, 1.0 µM PND-1186 (>5-fold above IC50) had limited effects on cell proliferation. However, under non-adherent conditions as spheroids and as colonies in soft agar, 0.1 µM PND-1186 blocked FAK and p130Cas tyrosine phosphorylation, promoted caspase-3 activation, and triggered cell apoptosis. PND-1186 inhibited 4T1 breast carcinoma subcutaneous tumor growth correlated with elevated tumor cell apoptosis and caspase 3 activation. Addition of PND-1186 to the drinking water of mice was well tolerated and inhibited ascites- and peritoneal membrane-associated ovarian carcinoma tumor growth associated with the inhibition of FAK Tyr-397 phosphorylation. Our results with low-level PND-1186 treatment support the conclusion that FAK activity selectively promotes tumor cell survival in three-dimensional environments.[1]

C25H26F3N5O3

501.5

501.198

C, 59.87; H, 5.23; F, 11.36; N, 13.96; O, 9.57

1061353-68-1

PND-1186 hydrochloride;1356154-94-3

25073775

White to gray solid powder

1.3±0.1 g/cm3

654.0±55.0 °C at 760 mmHg

349.3±31.5 °C

0.0±2.0 mmHg at 25°C

1.609

5.64

3

10

7

36

709

0

IGUBBWJDMLCRIK-UHFFFAOYSA-N

InChI=1S/C25H26F3N5O3/c1-29-24(34)17-5-3-4-6-19(17)31-21-14-23(30-15-18(21)25(26,27)28)32-20-8-7-16(13-22(20)35-2)33-9-11-36-12-10-33/h3-8,13-15H,9-12H2,1-2H3,(H,29,34)(H2,30,31,32)

2-((2-((2-methoxy-4-morpholinophenyl)amino)-5-(trifluoromethyl)pyridin-4-yl)amino)-N-methylbenzamide.

SR-2156; VS-4718; PND-1186; PND 1186; 1061353-68-1; 2-((2-((2-Methoxy-4-morpholinophenyl)amino)-5-(trifluoromethyl)pyridin-4-yl)amino)-N-methylbenzamide;PND1186; SR 2516; SR2516; VS4718; VS 4718.

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 (4.99 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 25.0 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 2: ≥ 2.5 mg/mL (4.99 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.99 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 4: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O: 5mg/mL

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