VHL; c-Met

Employing the c-Met inhibitor foretinib (Zillhardt et al., 2011) as a recruiting element, researchers developed a PROTAC, Compound 7/SJF-8240 （PROTAC 7), capable of recruiting VHL to, and thereby inducing degradation of c-Met in a dose- and time-dependent fashion. MDA-MB-231 cells treated with increasing concentrations of foretinib-based PROTAC 7 (Figure 3A) or its cognate diastereomer 8 (Figure 3B), in the presence or absence of a HGF pulse, demonstrated that treatment with approximately 10-fold higher concentration of diastereomer is required to completely inhibit agonist-driven AKT phosphorylation. We observed a similar level of inhibition of Akt phosphorylation in GTL16 cells, a c-Met-overexpressing cell line, when grown and treated in full serum with SJF-8240 （PROTAC 7) and diastereomer 8 in (Figure S3A/B). The reduced potency differential between SJF-8240 （PROTAC 7) and diastereomer 8 inhibition of Akt phosphorylation likely results from the steady-state versus agonist-challenged activation of the signalling cascade between the two cell lines (GTL16 and MDA-MB-231, respectively). The foretinib-based PROTAC 7 is also greater than two-fold more potent than its corresponding diastereomer 8 at inhibiting the proliferation of GTL16 cells (Figure 3C), again highlighting the advantages of developing probes capable of protein degradation, especially in oncogene-addicted contexts[1].

Cycloheximide chase assay[1]
MDA-MB-231 cells were plated at 3×105 cells per well in a 6-well dish, allowed to adhere, and switched to serum-free RPMI-1640 for 16 hr. Cells were then pre-treated with cycloheximide at 100 ug/ml for 1 hr prior to addition of either HGF (100 ng/ml), SJF-8240 （PROTAC 7) (500 nM), or vehicle. At the indicated time points, cells were immediately placed on ice, rinsed with PBS, lysed, and boiled.

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Immunofluorescence Microscopy[1]
MDA-MB-231 cells were plated at a density of 1×105 cells/ml onto 12 mm round coverslips, cultured overnight, switched to serum free media for >12 hours and then treated with 500 nM SJF-8240 （PROTAC 7) or 100 ng/ml HGF for the indicated times before washing with PBS. Cells were fixed with 4% formaldehyde for 20 minutes at room temperature, washed with ice-cold PBS, permeabilized and blocked with 0.25% Triton X-100/1% BSA in PBS for 30 minutes. Fixed cells were incubated with c-Met Antibody (1:3000 dilution, Cell Signalling #8198) for 1 hour, washed three times with PBS for 5 minutes, incubated with Alexa Fluor-488 conjugated anti-rabbit antibody (1:1000 dilution, ThermoFisher A-11008) for 1 hour washed three times with PBS for 5 minutes and mounted in vectashield containing DAPI. Imaged on Zeiss Axio Observer Z1 inverted microscope.

Cell Proliferation assays[1]
Following SJF-8240 （PROTAC 7) or diastereomer treatment of cells as indicated, culture medium was supplemented with 330 μg/ml MTS and 25 μM 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 Victor2 platereader. Data analysis and statistics performed using Prism v7.0 software.

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Cell surface biotinylation degradation assay[1]
A protocol was adapted from Joffre et. al to measure the removal of c-Met from the cell surface of MDA-MB-231 cells (Joffre et al., 2011). Cells were plated in full serum, allowed to adhere, and switched to serum-free RPMI-1640 for 16 hr. After this time, cells were placed on ice and rinsed with ice-cold 1X PBS-CM (0.1 mM CaCl2, 1 mM MgCl2) twice and incubated with PBS-CM for 5 min at 4°C. PBS-CM was aspirated, at which point cells were labelled with a cell membrane impermeant reagent, EZ-link Sulfo-NHS-SS-biotin at 0.5 mg/ml for 30 min at 4°C with gentle rocking. This step enabled covalent labelling of all cell surface proteins. All of the following were carried out at 4°C to prevent trafficking of said proteins. Cells were subsequently rinsed with ice-cold PBS-CM twice and excess biotin was quenched with Tris-glycine buffer (100 mM Tris pH 8.0, 150 mM NaCl, 0.1 mM CaCl2, 1 mM MgCl2 10 mM glycine, 1% BSA) for 15 min at 4°C with gentle rocking. Cells were then rinsed with ice-cold PBS-CM three times before being chased with warm serum-free RPMI-1640 medium containing either HGF (100 ng/ml) or SJF-8240 （PROTAC 7) (500 nM) and placed in a humidified incubator at 37°C for the indicated amount of time, at which point the cells were lysed with lysis buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 10% glycerol, 1% NP-40, 1 mM EDTA) supplemented with 1X protease inhibitors. Lysates were spun down at 14,000 × g at 4°C for 10 min and protein content was measured by BCA assay. Protein lysate was normalized and aliquoted onto pre-equilibrated NeutrAvidin agarose beads for 2 hrs at 4°C, with gentle rotation. Beads were washed three times with wash buffer (100 mM Tris, pH 7.5, 300 mM NaCl, and 1% Triton X-100) and resuspended in 2X elution buffer (62.5 mM Tris, pH 6.8, 3% SDS, 10% glycerol, 0.02% bromophenol blue, 160 mM DTT). Protein was eluted off of the beads by heating at 95°C for 5 min and the supernatant was run on an SDS-PAGE gel and evaluated for the presence of cell surface c-Met protein. Whole-cell lysate refers to the lysate loaded onto NeutrAvidin beads, thereby representing the total c-Met protein.

Cell Chem Biol.2018 Jan 18;25(1):67-77.e3.

Proteolysis targeting chimera (PROTAC) technology has emerged over the last two decades as a powerful tool for targeted degradation of endogenous proteins. Herein we describe the development of PROTACs for receptor tyrosine kinases, a protein family yet to be targeted for induced protein degradation. The use of VHL-recruiting PROTACs against this protein family reveals several advantages of degradation over inhibition alone: direct comparisons of fully functional, target-degrading PROTACs with target-inhibiting variants that contain an inactivated E3 ligase-recruiting ligand show that degradation leads to more potent inhibition of cell proliferation and a more durable and sustained downstream signaling response, and thus addresses the kinome rewiring challenge seen with many receptor tyrosine kinase inhibitors. Combined, these findings demonstrate the ability to target receptor tyrosine kinases for degradation using the PROTAC technology and outline the advantages of this degradation-based approach.[1]

C58H65F2N7O11S

1106.24

1105.443

2230821-68-6

135175904

White to light yellow solid powder

7.9

5

16

26

79

1990

3

CC1=C(SC=N1)C2=CC=C(C=C2)CNC(=O)[C@@H]3C[C@H](CN3C(=O)[C@H](C(C)(C)C)NC(=O)CCOCCCOCCCOC4=CC5=NC=CC(=C5C=C4OC)OC6=C(C=C(C=C6)NC(=O)C7(CC7)C(=O)NC8=CC=C(C=C8)F)F)O

SCUZPTNDZHEHLB-ZWUDBOSPSA-N

InChI=1S/C58H65F2N7O11S/c1-35-51(79-34-63-35)37-10-8-36(9-11-37)32-62-53(70)45-29-41(68)33-67(45)54(71)52(57(2,3)4)66-50(69)19-27-76-24-6-23-75-25-7-26-77-49-31-44-42(30-48(49)74-5)46(18-22-61-44)78-47-17-16-40(28-43(47)60)65-56(73)58(20-21-58)55(72)64-39-14-12-38(59)13-15-39/h8-18,22,28,30-31,34,41,45,52,68H,6-7,19-21,23-27,29,32-33H2,1-5H3,(H,62,70)(H,64,72)(H,65,73)(H,66,69)/t41-,45+,52-/m1/s1

N-(3-fluoro-4-((7-(3-(3-(3-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropoxy)propoxy)propoxy)-6-methoxyquinolin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dic

Foretinib-Based PROTAC-7; 2230821-68-6; SJF 8240; SJF-8240; CHEMBL4577916; SCHEMBL20326275; PD142878; Foretinib-Based PROTAC7; Foretinib-Based PROTAC 7

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)

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.)