FAK

KinomeScan [1]
The kinase engagement assay was performed by DiscoverX assessing binding abilities toward a set of 468 kinases. PROTAC-3 and defactinib were screened at a concentration of 1 μM.

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Kinase Activity Assay Kinase activity assays were performed by Reaction Biology Corp. Compounds were tested in 10-dose IC50 duplicate mode with a 3-fold serial dilution starting at 1 μM. The control compound, staurosporine, was tested in 10-dose IC50 mode with 4-fold serial dilution starting at 20 μM. Reactions were carried out at 10 μM ATP. IC50 values were calculated using Prism 7.0.

Western Blotting [1]
If not indicated otherwise, cells were seeded and grown to 80% confluency and were treated with compound or control for 24 h. Subsequently, the growth media was removed and the cells lysed by the addition of lysis buffer (25 mM Tris, pH 7.4; 1% NP-40, 0.25% deoxycholate, 1 mM sodium vanadate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 20 mM β-glycerophosphate, and 1× complete EDTA-free protease inhibitor cocktail). After 20 min the mixture was spun down at 16000g for 10 min at 4 °C to pellet insoluble materials. Protein concentrations of supernatants were determined via the BCA assay before addition of NuPAGE sample buffer containing 5% β-Me and boiling at 95 °C for 10 min. Equal amounts of protein were subjected to SDS-PAGE and subsequent electrophoretic transfer onto nitrocellulose membrane.

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Cell Proliferation Assays [1]
Cells were seeded in 96-well plates (2000 cell/well) and treated with PROTAC or control as indicated. At desired time points culture medium was supplemented with 330 mg/mL MTS and 25 mM phenazine methosulfate and incubated at 37 °C. Mitochondrial reduction of MTS to the formazan derivative was monitored by measuring the medium’s absorbance at 490 nm using a Wallac Victor2plate-reader. Data analysis and statistics was performed using Prism 7.0.

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Reverse Phase Protein Array [1]
RPPA analysis was performed by MD Anderson Cancer Center RPPA core facility. MDA-MB-231 cells were grown in complete growth medium. Cells were treated for 24 h with PROTAC-3 (500 nM), defactinib (1 μM), or DMSO (0.1%), trypsinized, and allowed to reattach for 8 h in the presence of compound or DMSO before cells were subjected to lysis, and samples were prepared according to protocols provided by MD Anderson.

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Wound-Healing Assay [1]
MDA-MB-231 cells were maintained in complete growth medium at 37 °C supplied with 5% CO2. Cells (1 × 106) were split into a 12-well plate. After 24 h an even wound was created across each well using a sterile 10 μL pipet tip, and the cells were washed with warm phosphate-buffered saline (PBS) twice to remove any floating or dead cells. This time point was considered as 0 h, and cells were incubated in fresh medium containing PROTAC or control as indicated for 24 h. Images of wounded area were captured at 0 h and after 24 h using a camera attached to a light microscope. Images were analyzed by ImageJ software, and wounded area was quantified. The area of the remaining wound at 24 h was subtracted from the area of the wound at 0 h. Percent wound healing (migration) was calculated, and data were presented as a bar graph using Prism 7.0. Differences between groups were analyzed by Welch’s t test and considered significant when P < 0.05.

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Transwell Invasion Assay [1]
On the first day, 0.2× basement membrane extract (BME) working solution was prepared by diluting 5× BME stock solution in 1× Travigen Inc. coating buffer. Briefly, 100 μL of 10× coating buffer was diluted in 900 μL of sterile water to make 1× coating buffer. Then 960 μL of 1× coating buffer was mixed with 40 μL of 5× BME to make a working 0.2× BME solution. Corning Transwell permeable inserts were placed on a 24-well plate, and 100 μL of 0.2× BME solution was added to each Transwell insert and incubated for 16 h. The following day, MDA-MB-231 cells were trypsinized and cells were suspended in serum-free medium. Approximately 100 μL from the cell suspension (∼3 × 105 cells) was added to each Transwell insert followed by another 100 μL of PROTAC or control containing serum-free RPMI medium. The lower chamber was filled with 10% FBS containing RPMI medium, and the whole setup was incubated at 37 °C and 5% CO2 for 24 h. After 24 h, the cell culture medium was removed from both lower and upper chambers and the Transwell inserts were washed three times with PBS. Noninvasive cells were removed using a cotton swab, and the bottom side of the membrane of the Transwell inserts was fixed with 4% formaldehyde for 10 min at room temperature followed by permeabilization with PBST (pH-7.4, 50 mM Tris-HCl, 150 mM NaCl, 0.1% Triton-X100) for another 10 min. Inserts were washed once with PBS and stained with 0.2% (w/v) crystal violet solution for 20 min at room temperature. Inserts were then extensively washed with PBS and once with water to remove all excess dye and salts. Cells migrated through the membrane were captured using a camera attached to a light microscope.

[1]. Addressing Kinase-Independent Functions of Fak via PROTAC-Mediated Degradation. J Am Chem Soc. 2018 Dec 12;140(49):17019-17026.

Enzyme inhibition has been shown to be an effective method for developing many small molecule drugs. In recent years, small molecule-induced protein degradation has emerged as an orthogonal therapeutic strategy, which is expected to expand the field of drug targets. Follicular adhesion kinase (Fak) is a key factor in tumor invasion and metastasis, and it functions as both a kinase and a scaffold protein for multiple signaling proteins. Previous attempts to modulate Fak activity have been limited to kinase inhibitors, and the results in clinical studies have been unsatisfactory. Protein degradation, on the other hand, offers the possibility of simultaneously blocking Fak kinase signaling and scaffold protein function. This article reports the development of a highly selective and efficient Fak degrader, PROTAC-3, which is superior to the clinical candidate drug defactinib in inhibiting Fak activation and Fak-mediated cell migration and invasion. These results highlight the potential of PROTAC in expanding the field of drug targets and regulating protein functions that are difficult to reach with traditional small molecule therapies. [1]

C20H20F3N5O3S

467.47

2296719-34-9

Solid powder

S(C)(N(C)C1C=CC=C(C=1)CNC1C(C(F)(F)F)=CN=C(NC2C=CC(=CC=2)O)N=1)(=O)=O

N-(3-(((2-((4-Hydroxyphenyl)amino)-5-(trifluoromethyl)pyrimidin-4-yl)amino)methyl)phenyl)-N-methylmethanesulfonamide; 2296719-34-9; Defactinib analogue-1; starbld0011445; SCHEMBL21634223; RNSJIMRLQPNKST-UHFFFAOYSA-N;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), 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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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