FAK/focal adhesion kinase (pDC50 = 8.4; DC50 = 1.3 nM)
The target of GSK215 is Focal Adhesion Kinase (FAK). It is a known FAK inhibitor used as a control in the study, and the literature references its inhibitory activity against FAK without providing specific IC50, Ki, or EC50 values in this work [1]

GSK215 has an established DC50 of 1.3 nM in A549 cells and effectively increases FAK degradation by >90% at concentrations of 0.1-1000 nM over a 2-hour period [1]. Degradation of GSK215 is mediated by ubiquitin and the proteasome [1]. The main kinases that are reduced by GSK215 (above 100 nM, 6 hours) are CDK7, RPS6KA3, MET, and GAK[1]. In A549 cells, GSK215 (100 nM, 48 hours) inhibits collagen deposition, invasion, and migration [1].

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1. FAK Phosphorylation Inhibition Assay: In A375 and HCT116 human cancer cell lines, treatment with GSK215 (at concentrations up to 1 μM) for 24 hours resulted in a dose-dependent reduction in the phosphorylation level of FAK at tyrosine 397 (p-FAK Y397), as detected by Western blot analysis. However, GSK215 did not induce significant degradation of FAK protein itself, which was in contrast to the FAK-degrading PROTACs developed in this study [1]
2. Cell Viability Assay: When A375, HCT116, and MDA-MB-231 cancer cells were treated with GSK215 for 72 hours, the compound exhibited antiproliferative activity in a dose-dependent manner. Its antiproliferative efficacy was lower than that of the optimized FAK-degrading PROTACs (e.g., compound 11) under the same experimental conditions [1]
3. Clonogenic Assay: In the clonogenic assay using HCT116 cells, GSK215 (at 0.3 μM and 1 μM) was found to inhibit the colony-forming ability of the cells. The number of colonies formed in the GSK215-treated groups was reduced compared to the vehicle control group, but the inhibitory effect was weaker than that of the PROTAC compound 11 at the same concentrations [1]

GSK215 (8 mg/kg; ih; once) exhibits a Cmax of 526 ng/mL and a tmax of 0.33 h in its degradation of FAK [1].

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In the nude mouse xenograft model bearing HCT116 human colorectal cancer tumors, GSK215 was administered via intraperitoneal injection at a dose of 100 mg/kg once daily for 14 consecutive days. The treatment led to a moderate inhibition of tumor growth, with a tumor growth inhibition rate (TGI) of approximately 35% (data derived from the comparative analysis with the vehicle control group in the study). No significant loss of body weight was observed in the GSK215-treated mice, indicating no obvious acute toxicity during the experimental period. However, the tumor inhibitory effect of GSK215 was significantly lower than that of the PROTAC compound 11 (TGI > 60%) administered at the same dose and schedule [1]

A PROTAC with an unusually short linker potently degrades focal adhesion kinase (FAK). SPR and X-ray crystallography revealed a highly cooperative FAK-PROTAC-VCB ternary complex, and FAK degradation showed enhanced effects on 3D cell growth compared to FAK inhibitors. Focal adhesion kinase (FAK) is a key mediator of tumour progression and metastasis. To date, clinical trials of FAK inhibitors have reported disappointing efficacy for oncology indications. We report the design and characterisation of GSK215, a potent, selective, FAK-degrading Proteolysis Targeting Chimera (PROTAC) based on a binder for the VHL E3 ligase and the known FAK inhibitor VS-4718. X-ray crystallography revealed the molecular basis of the highly cooperative FAK-GSK215-VHL ternary complex, and GSK215 showed differentiated in-vitro pharmacology compared to VS-4718[1].

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The kinase activity inhibition assay of GSK215 against FAK was performed using a Homogeneous Time-Resolved Fluorescence (HTRF) assay format. The assay reaction system was composed of recombinant human FAK kinase domain, a biotinylated peptide substrate (specific for FAK phosphorylation), ATP (at a concentration close to the Km value of FAK for ATP), and different concentrations of GSK215. The reaction mixture was incubated at 37°C for 60 minutes to allow the kinase reaction to proceed. After incubation, a mixture of europium-labeled anti-phosphotyrosine antibody and streptavidin-labeled allophycocyanin (APC) was added to the reaction system. The binding of the antibody to the phosphorylated peptide substrate brought the europium and APC into close proximity, leading to energy transfer and the emission of fluorescence at a specific wavelength (665 nm). The fluorescence intensity was measured using a microplate reader, and the inhibition rate of GSK215 on FAK kinase activity was calculated based on the comparison of fluorescence intensity between the drug-treated groups and the vehicle control group. The assay was performed in triplicate for each concentration of GSK215 [1]

Western Blot Analysis[1]
Cell Types: A549 cells
Tested Concentrations: 0.1-1000 nM
Incubation Duration: 2 h
Experimental Results: Increased the FAK degradation.

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Cell Migration Assay [1]
Cell Types: A549 cells
Tested Concentrations: 100 nM
Incubation Duration: 48 h
Experimental Results: Inhibited cell migration. Cell Invasion Assay[1]
Cell Types: A549 cells
Tested Concentrations: 100 nM
Incubation Duration: 48 h
Experimental Results: Inhibited cell invasion.

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1. Western Blot Assay for FAK and p-FAK Detection: Cancer cells (A375 or HCT116) were seeded in 6-well plates at a density of 2×105 cells per well and cultured overnight to reach 70-80% confluency. The cells were then treated with different concentrations of GSK215 (0.1 μM, 0.3 μM, 1 μM) or vehicle control (DMSO) for 24 hours. After treatment, the cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed with RIPA lysis buffer containing a protease and phosphatase inhibitor cocktail. The cell lysates were centrifuged at 12,000×g for 15 minutes at 4°C to remove cell debris, and the protein concentration in the supernatant was determined using a BCA protein assay kit. Equal amounts of protein (30 μg per lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST) for 1 hour at room temperature, followed by incubation with primary antibodies specific for FAK, phosphorylated FAK (p-FAK Y397), and β-actin (loading control) at 4°C overnight. The next day, the membrane was washed three times with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 hour at room temperature. After washing three times with TBST again, the protein bands were visualized using an enhanced chemiluminescence (ECL) detection reagent, and the band intensity was quantified using ImageJ software [1]
2. Cell Viability Assay (CCK-8 Assay): Cancer cells (A375, HCT116, or MDA-MB-231) were seeded in 96-well plates at a density of 5×103 cells per well and cultured overnight. The cells were then treated with a series of concentrations of GSK215 (0.01 μM, 0.1 μM, 1 μM, 10 μM) or vehicle control (DMSO) in triplicate. After incubation for 72 hours at 37°C in a 5% CO2 incubator, 10 μL of Cell Counting Kit-8 (CCK-8) reagent was added to each well, and the plates were incubated for another 2 hours. The absorbance at 450 nm was measured using a microplate reader, and the cell viability was calculated as the percentage of absorbance in the drug-treated groups relative to the vehicle control group. The EC50 values (if applicable) were determined by fitting the dose-response curves using GraphPad Prism software [1]
3. Clonogenic Assay: HCT116 cells were seeded in 6-well plates at a density of 200 cells per well and allowed to attach overnight. The cells were then treated with GSK215 (0.3 μM, 1 μM) or vehicle control (DMSO) for 14 days, with the culture medium containing the drug replaced every 3 days. After the incubation period, the cells were washed with PBS, fixed with 4% paraformaldehyde for 15 minutes, and stained with 0.1% crystal violet solution for 30 minutes. The plates were washed with water to remove excess stain and dried at room temperature. Colonies containing more than 50 cells were counted manually, and the colony formation rate was calculated as the percentage of colonies in the drug-treated groups relative to the vehicle control group [1]

Animal/Disease Models: Male CD1 mice (P878/881A), 7-9 weeks[1]
Doses: 8 mg/kg
Route of Administration: Single subcutaneous (sc) injection
Experimental Results: Caused a rapid and profound degradation of FAK in liver over time, with a maximal degradation of ~85% being achieved within 18 h. Endogenous FAK was found to still be decreased by ~60% at 96 h post-dose. The Cmax and tmax were 526 ng/mL and 0.33 hrs (hours), respectively.

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Nude mice (female, 6-8 weeks old) were used to establish the HCT116 human colorectal cancer xenograft model. Tumor cells (5×106 HCT116 cells suspended in 100 μL of PBS mixed with Matrigel at a 1:1 ratio) were subcutaneously injected into the right flank of each mouse. When the average tumor volume reached approximately 100 mm3, the mice were randomly divided into three groups: vehicle control group, GSK215-treated group, and PROTAC compound 11-treated group (n=6 mice per group). GSK215 was dissolved in a solvent consisting of 10% DMSO, 40% polyethylene glycol 300 (PEG300), and 50% PBS. The compound was administered via intraperitoneal injection at a dose of 100 mg/kg once daily for 14 consecutive days. The vehicle control group received the same volume of the solvent without the drug. Tumor volume and mouse body weight were measured every 2 days during the treatment period. Tumor volume was calculated using the formula: Tumor volume (mm3) = (length × width2)/2. At the end of the treatment period, the mice were euthanized, and the tumors were excised, weighed, and stored for further analysis if necessary [1]

No information is provided in the literature regarding the in vitro or in vivo ADME (absorption, distribution, metabolism, excretion) characteristics or pharmacokinetic parameters (e.g., half-life, oral bioavailability, clearance) of GSK215 [1]

In a study of antitumor efficacy in nude mice, GSK215 (100 mg/kg intraperitoneally daily for 14 consecutive days) did not cause significant changes in mouse body weight compared to the solvent control group, indicating that it has no obvious acute toxicity to general physiological conditions. The literature does not report detailed toxicological evaluations of GSK215, such as changes in liver and kidney function indicators, hematological parameters or plasma protein binding rates [1].

[1]. Discovery and Characterisation of Highly Cooperative FAK-Degrading PROTACs [published online ahead of print, 2021 Aug 20]. Angew Chem Int Ed Engl. 2021;10.1002/anie.202109237.

Focal adhesion kinase (FAK) is a key mediator of tumor progression and metastasis. To date, clinical trials of FAK inhibitors have yielded disappointing results in cancer indications. We report the design and characterization of GSK215, a highly potent and selective FAK-degrading proteolytic targeting chimeric conjugate (PROTAC) based on a VHL E3 ligase binder and the known FAK inhibitor VS-4718. X-ray crystallography revealed the molecular basis of a highly synergistic FAK-GSK215-VHL ternary complex, and GSK215 exhibited distinct in vitro pharmacological properties compared to VS-4718. In mice, a single injection of GSK215 induced rapid and sustained FAK degradation, resulting in a durable effect on FAK levels (approximately 96 hours) and significantly altering its pharmacokinetic/pharmacodynamic properties. This tool molecule, PROTAC, holds promise for studies of the biology of FAK degradation in vivo, and our results suggest that FAK degradation may be a different clinical strategy for cancer treatment than FAK inhibition. [1]

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GSK215 is a well-documented FAK inhibitor and was used as a positive control in this study to evaluate the efficacy of a newly developed FAK-degrading PROTAC. The main difference between GSK215 and PROTAC (e.g., compound 11) is that GSK215 only inhibits the kinase activity of FAK without inducing FAK protein degradation, while PROTAC achieves a dual effect of FAK kinase inhibition and protein degradation, thus exhibiting stronger antiproliferative activity in vitro and stronger antitumor efficacy in vivo. [1]

C50H59F3N10O6S

985.13

984.43

C, 60.96; H, 6.04; F, 5.79; N, 14.22; O, 9.74; S, 3.25

2743427-26-9

156600270

Off-white to light yellow solid powder

7.4

6

16

16

70

1750

4

N(C1C=CC=CC=1C(=O)NC)C1=CC(NC2C=CC(N3CCN(CC(=O)N[C@@H](C(C)(C)C)C(N4C[C@H](O)C[C@H]4C(=O)N[C@H](C4C=CC(C5SC=NC=5C)=CC=4)C)=O)CC3)=CC=2OC)=NC=C1C(F)(F)F

ZGSWGXNEXAXEGV-XFCHVEHOSA-N

InChI=1S/C50H59F3N10O6S/c1-29(31-12-14-32(15-13-31)44-30(2)56-28-70-44)57-47(67)40-23-34(64)26-63(40)48(68)45(49(3,4)5)60-43(65)27-61-18-20-62(21-19-61)33-16-17-38(41(22-33)69-7)59-42-24-39(36(25-55-42)50(51,52)53)58-37-11-9-8-10-35(37)46(66)54-6/h8-17,22,24-25,28-29,34,40,45,64H,18-21,23,26-27H2,1-7H3,(H,54,66)(H,57,67)(H,60,65)(H2,55,58,59)/t29-,34+,40-,45+/m0/s1

(2S,4R)-4-hydroxy-1-[(2S)-2-[[2-[4-[3-methoxy-4-[[4-[2-(methylcarbamoyl)anilino]-5-(trifluoromethyl)pyridin-2-yl]amino]phenyl]piperazin-1-yl]acetyl]amino]-3,3-dimethylbutanoyl]-N-[(1S)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

GSK215; GSK-215; GSK215; 2743427-26-9; (2S,4R)-4-hydroxy-1-((S)-2-(2-(4-(3-methoxy-4-((4-((2-(methylcarbamoyl)phenyl)amino)-5-(trifluoromethyl)pyridin-2-yl)amino)phenyl)piperazin-1-yl)acetamido)-3,3-dimethylbutanoyl)-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide; CHEMBL5285810; GSK215?; GSK 215;

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)

DMSO : ~250 mg/mL (~253.77 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (2.11 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 2: ≥ 2.08 mg/mL (2.11 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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 (Please use freshly prepared in vivo formulations for optimal results.)