RAF265 (CHIR-265)

Catalog No.S2161

RAF265 (CHIR-265) Chemical Structure

Molecular Weight(MW): 518.41

RAF265 (CHIR-265) is a potent selective inhibitor of C-Raf/B-Raf/B-Raf V600E with IC50 of 3-60 nM, and exhibits potent inhibition on VEGFR2 phosphorylation with EC50 of 30 nM in cell-free assays. Phase 2.

Size Price Stock Quantity  
In DMSO USD 238 In stock
USD 170 In stock
USD 320 In stock
USD 970 In stock
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2 Customer Reviews

  • Raf265 inhibited the kinase activity of B-Raf but not of Raf-1 in Pkd2cKO cholangiocytes. Cells were treated for 30 min with different concentrations of Raf265. B-Raf and Raf-1 were immunoprecipated as described in the methods section and a kinase assay in vitro was performed using MEK as a substrate. The kinase activity of Raf was assessed by immunoblot analysis and quantified as optical density of pMEK with respect to untreated cells. In WT cholangiocytes (A) Raf265 inhibited both B-Raf and Raf-1 kinase activity. In Pkd2cKO cholangiocytes (B), Raf265 inhibited only B-Raf while a biphasic effect was found in Raf-1 with a significant increase at doses from 0.001 to 1 uM and a significant inhibition at 10 uM. Blots are representative of four different experiments. (*p<0.05 vs controls; **p<0.001 vs controls).

    Hepatology 2012 56(6), 2363-74. RAF265 (CHIR-265) purchased from Selleck.

    Immunoblots showing levels of phospho-MEK (p-MEK), total MEK (t-MEK), phospho-ERK1/2 (p-ERK1/2) and total ERK1/2 (t-ERK1/2) in A375 cells transduced with a retrovirus expressing BRAFV600E, BRAFV600E/L505H, BRAFV600E/F516G or BRAFV600E/T529N and treated with increasing doses of RAF265. α-tubulin (TUBA) was monitored as a loading control.

    Pigment Cell Melanoma Res 2014 27(1), 124-33. RAF265 (CHIR-265) purchased from Selleck.

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Biological Activity

Description RAF265 (CHIR-265) is a potent selective inhibitor of C-Raf/B-Raf/B-Raf V600E with IC50 of 3-60 nM, and exhibits potent inhibition on VEGFR2 phosphorylation with EC50 of 30 nM in cell-free assays. Phase 2.
Targets
VEGFR2 [1]
(Cell-free assay)
B-Raf [1]
(Cell-free assay)
30 nM(EC50) 3 nM-60 nM
In vitro

RAF265 inhibits C-Raf, wild type B-Raf and mutant (V600E) B-Raf. RAF265 effectively block phosphorylation of Raf's downstream substrates MEK and ERK in cells and also kill melanoma and colorectal cancer cell lines harboring B-Raf mutations independent of PTEN mutation status. Raf kinase inhibition by RAF265 in mutant B-Raf melanoma cell lines causes cell cycle arrest and induces apoptosis, mimicking the effect of Raf RNAi in these cells. RAF265 also potently inhibits the phosphorylation of VEGFR2 and proliferation of VEGF-stimulated hMVEC. [1] In HT29 and MDAMB231 cells, RAF265 shows inhibitory activity with IC20 of 1 to 3 μM and IC50 of 5 to 10 μM, respectively. While RAF265 leads to a significant decrease in clonogenic survival in all tested cell lines, which means that RAF265 induces a dominant effect on clonogenic survival. Addition of RAF265 to RAD001 in HCT116 cells could lead to moderately decreased AKT, S6 protein, and 4EBP1 phosphorylation. [2] Raf265 markedly reduces the protein level of Bcl-2 and great inhibitory in CM- and NCI-H727 cells, while having no effect on the TRAIL susceptibility of BON1 and GOT1 cells. [3] Protein kinase D3 (PRKD3) that when knocked down could enhance cell killing by RAF265 in A2058 melanoma cells, which prevent reactivation of MAPK signaling, induce PARP cleavage, increase caspase activity, interrupt cell-cycle progression, and inhibit colony formation. [4]

Cell Data
Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
human Malme-3M cells M1vUe2Z2dmO2aX;uJIF{e2G7 MmLUTY5pcWKrdHnvckBw\iCELWLBSkBXPjByRTDteZRidnRiaX6gbJVu[W5iTXHscYUuO01iY3XscJMh[XO|ZYPz[YQh[XNicHjvd5Bpd3K7bHH0bY9vKG:oIFXST{whUUN3ME2wMlA1KM7:TR?= Mkj6NlY{QTZ4OEG=
A375 cells M32xcXBzd2yrZnXyZZRqd25iYYPzZZk> MnT5RY51cXC{b3zp[oVz[XSrdnWgZYN1cX[rdImgZYdicW6|dDDoeY1idiCDM{e1JINmdGy|IHjhdoJwemmwZzDCMXJCTiCYNkCwSUBufXSjboSsJGlEPTB;MD6wOEDPxE1? Mny2NlY{QTZ4OEG=
WM1799 cells NGrM[YRRem:uaX\ldoF1cW:wIHHzd4F6 NYO5XnJSSW62aYDyc4xq\mW{YYTpeoUh[WO2aY\peJkh[WejaX7zeEBpfW2jbjDXUVE4QTliY3XscJMhcGG{Yn;ybY5oKEJvUlHGJHY3ODCHIH31eIFvfCxiSVO1NF0xNjB2IN88US=> MmXONlY{QTZ4OEG=
SK-MEL-28 cells NYfSZlFETnWwY4Tpc44h[XO|YYm= MlvvTY5pcWKrdHnvckBw\iCELWLBSkBXPjByRTDteZRidnRiaX6gbJVu[W5iU1utUWVNNTJ6IHPlcIx{KGG|c3Xzd4VlKGG|IIDoc5NxcG:{eXzheIlwdiCxZjDFVmstKEmFNUC9NE4yPCEQvF2= NYnlTVV5OjZ|OU[2PFE>

... Click to View More Cell Line Experimental Data

In vivo RAF265 shows 71% to 72% TVI% (tumor volume inhibition percentage) in HCT116 xenografts at 12 mg/kg. While the combination of RAF265 and RAD001 shows enhanced antitumor activity with increased T10 (time to achieve a relative tumor volume of 10 times the initial tumor volume) and tumor growth delay. The combination of RAD001 and RAF265 also significantly enhances the activation of caspase-3 in HCT116 and MDAMB231 but not in A549 xenografts. [2] RAF265 inhibits FDG (2-deoxy-2-[18F]fluoro-d-glucose) accumulation and decreases the tumor volumes in A375M xenografts by orally dosed of 100 mg/kg. [5]

Protocol

Kinase Assay:[1]
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Assay Protocol:

Raf and Mek are combined at 2 × final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2. 0.1 mM EDTA and 1 mM DTT) and dispensed 15 μL per well in polypropylene assay plates. Background levels are determined in wells containing Mek and DMSO without Raf. To the Raf/Mek containing wells is added 3 μL of 10 × of RAF265 diluted in 100% DMSO. The raf kinase activity reaction is started by the addition of 12 μL per well of 2.5 × 33P-ATP diluted in assay buffer. After 45-60 minutes, the reactions are stopped with the addition of 70 μL of stop reagent (30 mM EDTA). Filtration plates are pre-wetted for 5 min with 70% ethanol, and then rinsed by filtration with wash buffer. Samples (90 μL) from the reaction wells are then transferred to the filtration plates. The filtration plates are washed 6 × with wash buffer using Millipore filtration apparatus. The plates are dried and 100 μL per well of scintillation fluid is added. The CPM is then determined using a Wallac Microbeta 1450 reader.
Cell Research:[2]
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  • Cell lines: Human A549 and H460 lung, HT29 and HCT 116 colon, and MDAMB231 breast cancer cell lines
  • Concentrations: 0.1 - 10 μM
  • Incubation Time: 48 hours
  • Method: The MTT assay and Bliss additivism model are used to assess the effect of RAF265 on cell viability. In each well of a 96-well plate, 1 × 104 cells are grown in 200 μL of medium. After 24 hours, RAF265 is added to achieve a final concentration of 0.1 to 10 μM. After 48 hours of treatment, 20 μL of 5 mg/mL MTT solution in PBS is added to each well. After 4 hours, supernatant is removed and formazan crystals are discarded in 200 μL of DMSO. Absorbance is then measured at 595 nm using an absorbance plate reader. Data are expressed as the percentage of viable cells.
    (Only for Reference)
Animal Research:[2]
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  • Animal Models: A549, H460, HCT116, or MDAMB231 cells are injected s.c. into the flank region of 6-wk-old female athymic mice.
  • Formulation: Dissolved in polyethylene glycol-400 (PEG-400) to a concentration of 25 mg/mL. [5]
  • Dosages: 12 mg/kg
  • Administration: Orally administered daily
    (Only for Reference)

Solubility (25°C)

In vitro DMSO 100 mg/mL (192.89 mM)
Ethanol 33 mg/mL (63.65 mM)
Water Insoluble
In vivo Add solvents individually and in order:
30% PEG400+0.5% Tween80+5% propylene glycol
30 mg/mL

* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.

Chemical Information

Molecular Weight 518.41
Formula

C24H16F6N6O

CAS No. 927880-90-8
Storage powder
Synonyms N/A

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Clinical Trial Information

NCT Number Recruitment Conditions Sponsor/Collaborators Start Date Phases
NCT01352273 Completed Advanced Solid Tumors Array BioPharma June 2011 Phase 1
NCT00304525 Completed Metastatic Melanoma Novartis Pharmaceuticals|Novartis April 2006 Phase 2

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Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID