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RAF265 (CHIR-265) 927880-90-8

Product Description

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Molecular Weight:

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. Phase 2.


Biological Activity


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. 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. 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. 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.


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. 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.


Protocol(Only for Reference)


Kinase Assay: [1]

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.
In Vitro Raf Screen The activity of various isoforms of Raf serine/threonine kinases can be measured by providing ATP, MEK substrate, and assaying the transfer of phosphate moiety to the MEK residue. Recombinant isoforms of Raf are obtained by purification from sf9 insect ce


Cell Assay: [2]

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.


Animal Study: [2]

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
Solubility 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.


Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)


Species Baboon Dog Monkey Rabbit Guinea pig Rat Hamster Mouse
Weight (kg) 12 10 3 1.8 0.4 0.15 0.08 0.02
Body Surface Area (m2) 0.6 0.5 0.24 0.15 0.05 0.025 0.02 0.007
Km factor 20 20 12 12 8 6 5 3

Animal A (mg/kg) = Animal B (mg/kg) multiplied by Animal B Km
Animal A Km



For example, to modify the dose of resveratrol used for a mouse (22.4 mg/kg) to a dose based on the BSA for a rat, multiply 22.4 mg/kg by the Km factor for a mouse and then divide by the Km factor for a rat. This calculation results in a rat equivalent dose for resveratrol of 11.2 mg/kg.

Rat dose (mg/kg) = mouse dose (22.4 mg/kg) × mouse Km(3) = 11.2 mg/kg
rat Km(6)





Chemical Information




Molecular Weight (MW) 518.41
Formula

C24H16F6N6O

CAS No. 927880-90-8

Storage 3 years -20℃Powder
6 months-80℃in solvent (DMSO, water, etc.)
Synonyms



Solubility (25°C) * In vitro DMSO 100 mg/mL (192.89 mM)
Water <1 mg/mL (
Ethanol 33 mg/mL (63.65 mM)
In vivo 30% PEG400/0.5% Tween80/5% propylene glycol 30 mg/mL
* <1 mg/ml means slightly soluble or insoluble.
* 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 Name 1-methyl-5-(2-(5-(trifluoromethyl)-1H-imidazol-2-yl)pyridin-4-yloxy)-N-(4-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-2-amine







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