FAK (IC50 = 1.5 nM)
PND-1186 hydrochloride targets focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase that plays a critical role in cell adhesion, migration, invasion, proliferation, and survival. FAK is overexpressed and hyperactivated in many cancer types and is associated with poor prognosis. By inhibiting FAK activity, PND-1186 hydrochloride disrupts downstream signaling pathways including PI3K/AKT and MAPK/ERK, leading to reduced cell survival, proliferation, and migration. The compound's selectivity for FAK over other kinases makes it a valuable tool for studying FAK biology.

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].

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In vitro studies demonstrate that PND-1186 hydrochloride is a potent inhibitor of FAK with an IC₅0 of 1.5 nM. The compound selectively induces apoptosis in tumor cells while sparing normal cells. Its in vitro activity is assessed using purified FAK enzyme assays with a peptide substrate and ATP, measuring phosphorylation by scintillation counting or fluorescence. Cellular assays are conducted using cancer cell lines, where the compound inhibits FAK phosphorylation (pY397), reduces cell viability, and induces apoptosis. The compound's effects on cell migration and invasion are assessed using wound healing or transwell migration assays.

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.

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In vivo activity of PND-1186 hydrochloride has been evaluated in animal models of cancer. As a potent and selective FAK inhibitor, the compound has shown efficacy in reducing tumor growth, metastasis, and angiogenesis in various xenograft models. Oral administration of PND-1186 has been investigated for the treatment of solid tumors and acute myeloid leukemia. The compound's anti-tumor activity is attributed to its ability to inhibit FAK-mediated signaling pathways essential for tumor cell survival and metastasis. In vivo efficacy is typically assessed by measuring tumor volume, metastasis burden, and survival outcomes.

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 application.

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In vitro enzyme/receptor binding assays for PND-1186 hydrochloride involve measuring its inhibition of FAK kinase activity. The assay typically uses purified recombinant FAK enzyme, a peptide substrate (e.g., poly(Glu,Tyr)), and ATP. The compound is incubated with the enzyme and substrates at 30-37degC, and phosphorylation is quantified by ELISA or scintillation counting using radiolabeled ATP. IC₅0 values are calculated from dose-response curves. Selectivity profiling is performed using panels of other kinases to assess the compound's specificity for FAK over other kinases. Binding affinity may be assessed using surface plasmon resonance.

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.

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Cellular assays for PND-1186 hydrochloride are conducted using cancer cell lines to evaluate its effects on FAK signaling, cell viability, and apoptosis. Cells are treated with varying concentrations of the compound (typically 0.1 nM to 10 microM) for 24-72 hours. FAK phosphorylation (pY397) is measured by Western blotting or ELISA to confirm target engagement. Cell viability is assessed using MTT, CCK-8, or CellTiter-Glo assays. Apoptosis is evaluated by Annexin V/PI staining, caspase-3/7 activity, or PARP cleavage. Cell migration and invasion are assessed using scratch wound healing or transwell assays.

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.

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In vivo animal studies with PND-1186 hydrochloride are conducted in xenograft mouse models using human tumor cell lines. Tumor-bearing mice are treated with the compound via oral gavage at various doses (typically 10-100 mg/kg), reflecting its oral bioavailability. Tumor volume is measured regularly using calipers, and body weight is monitored for tolerability. At study termination, tumors are excised and analyzed for FAK phosphorylation, apoptosis markers, proliferation markers (Ki-67), and angiogenesis (CD31). Metastasis models may be used to assess the compound's ability to inhibit tumor dissemination.

Pharmacokinetic properties of PND-1186 hydrochloride have been characterized in preclinical studies. The compound is orally bioavailable, making it suitable for oral administration in animal studies. Its molecular weight is approximately 538.0 g/mol. The hydrochloride salt form enhances aqueous solubility. Detailed PK parameters such as half-life, Cmax, AUC, and bioavailability have been determined in pharmacokinetic studies. The compound is typically formulated using appropriate vehicles such as DMSO, PEG, or saline for in vivo administration. Standard bioanalytical methods such as LC-MS/MS are used to quantify compound concentrations in plasma and tissues.

PND-1186 has low inherent toxicity to mice.

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Toxicological data for PND-1186 hydrochloride have been evaluated in preclinical studies. The compound is generally well-tolerated at therapeutic doses in animal models, with no significant toxicity reported at standard dosing ranges. As a FAK inhibitor, the compound's toxicity profile is expected to be related to FAK inhibition in normal tissues, though FAK is predominantly expressed in tumor cells. Standard toxicity assessments include body weight monitoring, organ histopathology, serum biochemistry, and hematology in animal studies. The compound is not an FDA-approved drug but has been investigated in clinical trials as an anti-cancer agent.

[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 currently undergoing clinical trial NCT02215629 (Dose-escalation study for acute myeloid or B-cell acute lymphoblastic leukemia). The FAK inhibitor VS-4718 is an orally bioavailable focal adhesion kinase (FAK) inhibitor with potential antitumor activity. After administration, VS-4718 inhibits FAK, blocking fibronectin-stimulated autophosphorylation of FAK at the Tyr397 site and potentially preventing activation of multiple integrin-mediated downstream signaling pathways, including ERK, JNK/MAPK, and PI3K/Akt. This leads to a reduction in the number of cancer stem cells (CSCs) and inhibits tumor cell migration, proliferation, and survival. Cytoplasmic tyrosine kinase FAK is an integrin signaling molecule that is persistently activated in various tumor cell types; it is involved in tumor cell invasion, migration, and proliferation, and plays a crucial role in the development, function, and survival of cancer stem cells (CSCs). Tumor cells can grow in an anchor-independent manner. This is partly mediated by survival signals that bypass normal growth-limiting mechanisms controlled by integrin cell surface receptors. Follicular adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinase that binds to integrins and regulates a variety of cellular processes, including growth, survival, and migration. Since increased FAK expression and tyrosine phosphorylation are associated with tumor progression, FAK inhibitors are being tested for their antitumor effects. This article analyzes PND-1186, a substituted pyridine reversible FAK activity inhibitor with an in vitro half-maximal inhibitory concentration (IC50) of 1.5 nM. Using antiphosphorylation-specific immunoblotting to detect FAK Tyr-397, the IC50 of PND-1186 in breast cancer cells was approximately 100 nM. PND-1186 did not alter the tyrosine phosphorylation levels of c-Src or p130Cas in adherent cells but inhibited cell migration. Notably, 1.0 µM of PND-1186 (a concentration more than 5 times higher than the IC50) had a limited effect on cell proliferation. However, under non-adherent conditions, such as in globular and soft agar clones, 0.1 µM of PND-1186 blocked tyrosine phosphorylation of FAK and p130Cas, promoted caspase-3 activation, and induced apoptosis. PND-1186 inhibited the growth of subcutaneous tumors of 4T1 breast cancer, which was associated with increased tumor cell apoptosis and caspase-3 activation. PND-1186 was well tolerated in drinking water in mice and was able to inhibit the growth of ascites and peritoneal-associated ovarian cancer tumors, which was associated with the inhibition of FAK Tyr-397 phosphorylation. Our results from treatment with low doses of PND-1186 support the conclusion that FAK activity selectively promotes the survival of tumor cells in a three-dimensional environment. [1]

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PND-1186 hydrochloride (VS-4718 hydrochloride) is a potent, highly specific, and reversible FAK inhibitor with an IC₅0 of 1.5 nM. It selectively promotes tumor cell apoptosis and has been investigated as an anti-cancer agent. The compound has been studied in clinical trials for the treatment of solid tumors and acute myeloid leukemia. Its mechanism of action involves inhibition of FAK-mediated signaling pathways essential for tumor cell survival and metastasis. The compound is orally bioavailable and has shown efficacy in preclinical models. It is available in high purity (≥99.92%) and is typically stored at -20degC.

C25H27CLF3N5O3

537.96

537.175

C, 55.82; H, 5.06; Cl, 6.59; F, 10.59; N, 13.02; O, 8.92

1356154-94-3

PND-1186;1061353-68-1

56654067

White to off-white solid powder

6.196

4

10

7

37

709

0

OWQFAUOQRRIFLB-UHFFFAOYSA-N

InChI=1S/C25H26F3N5O3.ClH/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);1H

2-[[2-(2-methoxy-4-morpholin-4-ylanilino)-5-(trifluoromethyl)pyridin-4-yl]amino]-N-methylbenzamide;hydrochloride

PND-1186 hydrochloride; 1356154-94-3; PND-1186 (Hydrochloride); VS-4718 hydrochloride; SR-2516 hydrochloride; S803K9N9TL; 2-(2-(2-Methoxy-4-morpholinophenylamino)-5-(trifluoromethyl)pyridine-4-ylamino)-N-methylbenzamide hydrochloride; UNII-S803K9N9TL;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : 200 mg/mL (371.77 mM)
H2O : 20 mg/mL (37.18 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (9.29 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 50.0 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: ≥ 5 mg/mL (9.29 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 50.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)