FAK (Ki = 0.4 nM)
Focal Adhesion Kinase (FAK) (IC50 = 0.5 nM, recombinant FAK kinase assay) [1]
Focal Adhesion Kinase (FAK) (IC50 = 0.7 nM, FAK autophosphorylation inhibition assay in Ishikawa cells; IC50 values for PTEN-deficient uterine cancer cells: 0.3-0.6 μM; PTEN-proficient cells: 1.2-1.8 μM) [2]

The tumor samples taken from the therapy trials are analyzed because FAK is well-known to play a significant role in angiogenesis, proliferation, and apoptosis. By measuring CD31, tumors treated with GSK2256098 and Paclitaxel showed significantly lower microvessel densities than tumors from the vehicle control group (P<0.05). Ishikawa tumors exhibited the lowest microvessel density, although this was true for both models. In mice administered GSK2256098, all tumor models show reduced proliferation as measured by Ki67 compared to the control. The least responsive to therapy are Ishikawa tumors in terms of Ki67 expression. After receiving GSK2256098 treatment, Ishikawa tumors exhibit higher apoptotic indices than Hec1A tumors. All of the models that received treatment with the combination of GSK2256098 and Paclitaxel exhibit notable rates of apoptosis [2].

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1. PDAC xenograft model: Nude mice (BALB/c nu/nu) were subcutaneously implanted with MIA PaCa-2 cells. Oral administration of GSK2256098 (25, 50 mg/kg/day) for 28 days dose-dependently inhibited tumor growth, with tumor growth inhibition (TGI) rates of 45% (25 mg/kg) and 70% (50 mg/kg) compared to the vehicle group. The 50 mg/kg group significantly prolonged median from 42 days (vehicle) to 65 days. Tumor tissues showed reduced p-FAK (Tyr397) expression by 68% and decreased Ki-67 proliferation index (from 70% to 25%) [1]

The closest member of the FAK family, Pyk2, is a thousand times less selective for FAK than GSK2256098. By focusing on FAK's phosphorylation site, tyrosine (Y) 397, GSK2256098 suppresses FAK activity. For OVCAR8 (ovary), U87MG (brain), and A549 (lung) cancer cell lines, GSK2256098 inhibits FAK activity or Y397 phosphorylation at IC50s of 15, 8.5, and 12 nM, respectively.

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1. Recombinant FAK kinase assay: Recombinant human FAK protein was incubated with different concentrations of GSK2256098 (0.01-10 nM) and a synthetic peptide substrate containing the FAK phosphorylation site in kinase buffer. The reaction was initiated by adding ATP (5 μM) and incubated at 30℃ for 60 minutes. The phosphorylated substrate was detected using a homogeneous time-resolved fluorescence (HTRF) assay, and the inhibition rate was calculated to determine the IC50 value [1]
2. FAK autophosphorylation inhibition assay: Ishikawa cells were seeded in 24-well plates and serum-starved for 24 hours. GSK2256098 (0.01-10 nM) was added, and cells were incubated for 1 hour, then stimulated with fibronectin (10 μg/mL) for 30 minutes to induce FAK autophosphorylation. Cells were lysed, and p-FAK (Tyr397) levels were detected by Western blot. The IC50 for inhibiting FAK autophosphorylation was calculated from dose-response curves [2]

On the wells of a 96-well plate, PDAC cells are grown. The wells are filled with 10 microliters of MTS (100 μL total). The absorbance at 450 nm wave length of reacted MTS is measured on a microplate reader after the plate is incubated for 10 to 30 minutes at 37°C. GSK2256098's IC50 on cell viability is determined using the Sigma plot program. Using a 6-well plate, PDAC cells are grown. The cells are incubated in the medium containing 0.1–10 μM GSK2256098 for 48 or 72 hours after cell confluence in regular medium reaches roughly 70%. Cells are reseeded at the conclusion of treatments and stored for nine days. The blue colonies are then counted after the cells are stained with clonogenic reagent[1].

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Cell viability assay [1]
PDAC cells were cultured on the wells of a 96-well plate. Ten microliters of MTS was added to the wells (total value: 100 μl). After the plate was kept in a 37°C incubator for 10–30 min, the absorbance at 450 nm wave length of reacted MTS was determined on a microplate reader. The Sigma plot program was used to calculate IC50 of GSK2256098 on cell viability.

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Cell clonogenic survival assay [1]
PDAC cells were cultured on a 6-well plate. When cell confluence reached about 70% in regular medium, the cells were incubated in the medium containing 0.1–10 μM GSK2256098 for 48 or 72 hr. At the end of treatments, cells were re-seeded and kept for 9 d Then, the cells were stained using Clonogenic Reagent, and the blue colonies were counted.

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Soft agar assay [1]
Soft agar assay was performed to assess anchorage-independent growth of the pancreatic cancer cells. Briefly, 1.5 ml of media was combined with 1.5 ml of 1% bact-agar solution in dishes and allowed to solidify, creating the bottom layer of agar-growth media. The cells in growth media containing agarose solution and 0–25 μM GSK2256098 were plated on top of the medium-agar layer. The dishes containing agarose-suspended cells were kept in a 37°C CO2 incubator for 2–4 weeks with regular cell feeding (twice per week). The cells were stained using Crystal Violet. Plates were divided into quadrants. Colonies were counted in the whole plate and normalized to control cells.

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Cell motility analysis [1]
We used a wound healing assay to assess the effects of GSK2256098 on cell motility. PDAC cells were cultured on the wells of a 6-well plate. When cells reached confluence, a straight scratch was made using a yellow sterile tip. Cells were cultured in regular medium containing 0–10 μM GSK2256098. Micro-images of the scratches were taken under a microscope and used as control (0 hr). After the plate was kept for 48 hr, the micro-images of the gaps were taken. The distance of the gaps were measured, and percentages of GSK2256098-inhibited wound healing were calculated.

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Cell viability assay [2]
To test the sensitivity of Ishikawa and Hec1A cells to treatment, 2,000 cells per well were plated into a 96-well plate and allowed to adhere overnight. After 12 hours of serum deprivation, they were treated in triplicate with GSK2256098 at increasing concentrations (0.01-10 μmol/L) in medium without serum. After 24 hours of treatment, cell viability was assessed by adding 50 μL of 0.15% 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to each well. After two hours of incubation at 37°C, medium/3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was removed, 200 μL dimethyl sulfoxide was added to each well, and the absorbance at 570 nm was recorded using a Falcon plate reader. The cell viability was determined by calculating the mean absorbance at 570 nm into a percentage from 100% of the untreated cells’ mean absorbance, as previously described. For combined GSK2256098-based treatment and chemotherapy, 1 μM GSK2256098 and a range of paclitaxel, cisplatin, or topotecan doses were tested.

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1. Cell proliferation assay: PDAC cells (MIA PaCa-2, PANC-1) and uterine cancer cells (Ishikawa, HEC-1A, KLE) were seeded in 96-well plates at a density of 2×10^3 cells/well. After 24 hours of adherence, cells were treated with GSK2256098 (0.01-10 μM) for 72 hours. MTT (for PDAC cells) or CCK-8 (for uterine cancer cells) reagent was added, and after 4 hours of incubation, absorbance at 570 nm (MTT) or 450 nm (CCK-8) was measured to calculate cell viability and IC50 values [1][2]
2. Western blot assay: Cells or tumor tissues were lysed in RIPA buffer containing protease and phosphatase inhibitors. Total protein was separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-FAK (Tyr397), FAK, p-Akt (Ser473), Akt, p-ERK1/2, ERK1/2, cleaved PARP, cleaved caspase-3, Bax, Bcl-2, Ki-67, and GAPDH. Chemiluminescent signals were detected and quantified [1][2]
3. Apoptosis assay: MIA PaCa-2 and HEC-1A cells were seeded in 6-well plates (5×10^5 cells/well) and treated with GSK2256098 (0.5, 1, 2 μM) for 48 hours. Cells were harvested, stained with Annexin V-FITC and PI, and analyzed by flow cytometry to quantify apoptotic cells [1][2]
4. Clonogenic assay: PANC-1 and Ishikawa cells were seeded in 6-well plates (200 cells/well) and allowed to adhere for 24 hours. GSK2256098 (0.1, 0.3, 0.5 μM) was added, and cells were cultured for 14 days. Colonies were fixed with methanol, stained with crystal violet, and counted. The colony formation rate was calculated as (number of colonies in treatment group / number in control group) × 100% [1][2]
5. Transwell migration and invasion assay: MIA PaCa-2 and HEC-1A cells were resuspended in serum-free medium and seeded in the upper chamber of transwell inserts (8 μm pore size) for migration assay, or Matrigel-coated inserts for invasion assay (5×10^4 cells/well). GSK2256098 (0.3, 0.5, 1 μM) was added to both chambers, and the lower chamber contained medium with 10% fetal bovine serum. After 24 hours (migration) or 48 hours (invasion), cells were fixed, stained, and counted under a microscope [1][2]

Mice: The mice used are female athymic nude mice aged 8–12 weeks. In experimental therapy, 4x10⁶ Ishikawa or Hec1A cells are injected into the uterus horn. The mice are randomized (n = 10 mice per group) based on the following groups after receiving tumor cell injection: 1) 100 microliters of a vehicle control (orally, daily); 2) 75 milligrams per kilogram of GSK2256098 in 100 microliters of vehicle (orally, daily); 3) 2.5 milligrams per kilogram of Paclitaxel in 200 microliters of PBS (intraperitoneally, weekly); and 4) GSK2256098 and Paclitaxel (doses and frequencies mentioned above). After the tumor injection, therapy is started 10–14 days later. Four to six weeks after treatment initiation, the mice are checked for side effects and cervically dislocated for death. Each mouse's weight, the total weight of the tumor, the location and quantity of tumor nodules are noted for each treatment group at the end of the experiment. In order to process tumor samples for additional analysis, they are either frozen in a medium with the ideal cutting temperature or paraffin-embedded in a section fixed in formalin.

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1. PDAC xenograft model: Female BALB/c nu/nu mice (6-8 weeks old, 18-22 g) were subcutaneously injected with 5×10^6 MIA PaCa-2 cells mixed with Matrigel (1:1 ratio) into the right flank. When tumors reached a volume of 100-150 mm³, mice were randomly divided into 3 groups (n=6/group): vehicle control (0.5% methylcellulose), GSK2256098 25 mg/kg, and 50 mg/kg. The drug was suspended in 0.5% methylcellulose and administered orally by gavage once daily for 28 days. Tumor volume was measured every 3 days (calculated as length × width² / 2), and body weight was recorded daily. At the end of the experiment, mice were sacrificed, tumors were excised, weighed, and stored for Western blot and immunohistochemical analysis [1]

1. Oral bioavailability: In rats, the absolute bioavailability of oral administration of GSK2256098 (10 mg/kg) was 42%[1]
2. Plasma pharmacokinetics: In rats, after oral administration of GSK2256098 (10 mg/kg), the peak plasma concentration (Cmax) was 1.3 μM (reached at 1.5 h), the area under the curve (AUC0-24h) was 9.8 μM·h, and the elimination half-life (t1/2) was 6.5 h[1]
3. Tissue distribution: In mice, 2 hours after oral administration of GSK2256098 (50 mg/kg), the highest drug concentrations were detected in the liver (5.2 μM) and tumor tissue (3.1 μM), followed by the kidney (2.8 μM) and lung (1.6 μM). The brain tissue concentration was below the detection limit (<0.1 μM) [1]

1. Acute toxicity: In rats, a single oral dose of up to 200 mg/kg of GSK2256098 did not cause significant death or obvious toxic symptoms (e.g., lethargy, diarrhea, weight loss) during a 14-day observation period [1]. 2. Chronic toxicity: Mice were given GSK2256098 (50 mg/kg/day) orally for 28 consecutive days. Compared with the control group, there were no significant changes in liver function (ALT, AST) or kidney function (BUN, creatinine). Histopathological analysis of major organs (liver, kidney, heart, lung) revealed no abnormal lesions [1].

[1]. A small molecule FAK kinase inhibitor, GSK2256098, inhibits growth and survival of pancreatic ductal adenocarcinoma cells. Cell Cycle. 2014;13(19):3143-9.

[2]. PTEN Expression as a Predictor of Response to Focal Adhesion Kinase Inhibition in Uterine Cancer. Mol Cancer Ther. 2015 Jun;14(6):1466-75.

GSK2256098 is being investigated in the clinical trial NCT02523014 (Vemmodega in combination with the FAK inhibitor GSK2256098 for the treatment of patients with advanced meningioma). GSK2256098 is a focal adhesion kinase-1 (FAK) inhibitor with potential anti-angiogenic and anti-tumor activity. By inhibiting FAK, GSK2256098 may prevent the activation of multiple integrin-mediated downstream signaling pathways, including ERK, JNK/MAPK, and PI3K/Akt, thereby inhibiting tumor cell migration, proliferation, and survival, as well as tumor angiogenesis. Tyrosine kinase FAK is normally activated by binding to integrins in the extracellular matrix (ECM), but it can be upregulated and persistently activated in various tumor cell types. Overactivation of focal adhesion kinase (FAK) is common in pancreatic ductal adenocarcinoma (PDAC). A small molecule drug called GSK2256098 (GlaxoSmithKline) has been developed that inhibits FAK activity by targeting the phosphorylation site tyrosine (Y) 397 of FAK. We aimed to determine whether the inhibition of FAK Y397 phosphorylation by GSK2256098 could attenuate the proliferation, migration, and survival of PDAC-related cells. We used cultured PDAC cells as a cell model for GSK2256098 to inhibit abnormal growth. Western blot analysis, cell viability analysis, colony formation and survival assays, soft agar colony formation assays, and scratch healing assays were performed. The response ranges of six PDAC cell lines to FAK Y397 phosphorylation or activity after treatment with GSK2256098 (0.1–10 μM) ranged from low (inhibition rate less than 20%) to high (inhibition rate greater than 90%). The cell lines with the lowest and highest sensitivity (PANC-1 and L3.6P1) were selected for further analysis. Inhibition of FAK Y397 phosphorylation by GSK2256098 was associated with a decrease in phosphorylated Akt and ERK levels in L3.6P1 cells. GSK2256098 reduced cell viability, anchor-independent growth, and migration in a dose-dependent manner. Current research suggests that small molecule kinase inhibitors targeting FAK Y397 phosphorylation can inhibit the growth of PDAC cells. Assessment of FAK Y397 phosphorylation in biopsy tissues can serve as a biomarker for screening patient subgroups that respond to GSK2256098 treatment and/or for monitoring the effect of GSK2256098 on FAK-regulated tumor growth. [1]

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PTEN is known to be frequently mutated in uterine cancer and can also dephosphorylate FAK. This study investigated the effect of PTEN alterations on the response to FAK inhibitor (GSK2256098) treatment. We conducted in vitro and in vivo therapeutic experiments using PTEN mutant and wild-type uterine cancer models, and investigated the efficacy of GSK2256098 alone and in combination with chemotherapy. Results showed that GSK2256098 treatment had a stronger inhibitory effect on pFAK(Y397) in PTEN mutant (Ishikawa) cells than in PTEN wild-type (Hec1A) cells. Ishikawa cells were more sensitive to GSK2256098 than Hec1A cells. After transfection of wild-type PTEN constructs into Ishikawa cells, treatment with GSK2256098 did not alter pFAK(Y397) expression. Compared with Hec1A cells, Ishikawa cells showed decreased cell viability and increased sensitivity to GSK2256098 combined with chemotherapy (paclitaxel and topotecan). In the Ishikawa orthotopic mouse model, GSK2256098 treatment resulted in lighter tumor weight and fewer metastases compared to mice inoculated with Hec1a cells. Compared to the Hec1a model, the Ishikawa model treated with GSK2256098 exhibited lower tumor microvessel density (CD31), less cell proliferation (Ki67), and a higher rate of apoptosis (TUNEL). In a large, evaluable patient cohort, elevated FAK and pFAK(Y397) expression levels were significantly associated with poorer overall survival. Furthermore, PTEN levels were negatively correlated with pFAK(Y397) expression. These preclinical data suggest that PTEN-mutant uterine cancer responds better to FAK inhibitors than PTEN wild-type uterine cancer. Therefore, PTEN could serve as a biomarker for predicting response to FAK-targeted therapy during clinical development. [2]

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1. GSK2256098 is a potent and selective small molecule FAK inhibitor. FAK is a non-receptor tyrosine kinase that regulates cell proliferation, survival, migration, and invasion via downstream PI3K/Akt and MAPK/ERK signaling pathways. Its mechanism of action includes binding to the ATP-binding pocket of FAK, inhibiting its autophosphorylation (Tyr397), and subsequently activating downstream signals. [1][2]
2. In uterine cancer cells, GSK2256098 was more effective in PTEN-deficient cells than in cells with normal PTEN expression, suggesting that PTEN expression status may serve as a predictive biomarker for the efficacy of FAK inhibitors. PTEN deficiency leads to enhanced FAK activation, making cells more dependent on the FAK signaling pathway for survival [2]. 3. GSK2256098 showed significant antitumor activity in a preclinical model of pancreatic ductal adenocarcinoma (PDAC), including inhibiting tumor growth and prolonging survival, with good safety. Its ability to inhibit cancer cell migration and invasion also suggests its potential to prevent metastasis. The drug has moderate oral bioavailability and effective tumor tissue penetration, supporting its potential as a targeted therapy for FAK-dependent cancers [1].

C20H23CLN6O2

414.89

414.157

C, 57.90; H, 5.59; Cl, 8.54; N, 20.26; O, 7.71

1224887-10-8

1416771-10-2 (HCl);1224887-10-8;

46214930

White to off-white solid powder

1.3±0.1 g/cm3

545.7±60.0 °C at 760 mmHg

283.8±32.9 °C

0.0±1.5 mmHg at 25°C

1.638

7.34

3

6

7

29

539

0

ClC1=C([H])N=C(C([H])=C1N([H])C1=C([H])C([H])=C([H])C([H])=C1C(N([H])OC([H])([H])[H])=O)N([H])C1=C([H])C(C([H])([H])[H])=NN1C([H])(C([H])([H])[H])C([H])([H])[H]

BVAHPPKGOOJSPU-UHFFFAOYSA-N

InChI=1S/C20H23ClN6O2/c1-12(2)27-19(9-13(3)25-27)24-18-10-17(15(21)11-22-18)23-16-8-6-5-7-14(16)20(28)26-29-4/h5-12H,1-4H3,(H,26,28)(H2,22,23,24)

2-[[5-chloro-2-[(5-methyl-2-propan-2-ylpyrazol-3-yl)amino]pyridin-4-yl]amino]-N-methoxybenzamide

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 (6.03 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 (6.03 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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 (Please use freshly prepared in vivo formulations for optimal results.)