RAF709

Catalog No.S8690

RAF709 Chemical Structure

Molecular Weight(MW): 542.55

RAF709 is a potent inhibitor of B/C RAF kinase with almost equivalent IC50 values of 0.4 nM for B-RAF and C-RAF, showing a high level of selectivity, demonstrating greater than 99% on-target binding to BRAF, BRAFV600E, and CRAF at 1 μM and very few off-targets with DDR1 (>99%), DDR2 (86%), FRK (92%), and PDGFRb (96%), the only kinases with binding >80% at 1 μM.

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Biological Activity

Description RAF709 is a potent inhibitor of B/C RAF kinase with almost equivalent IC50 values of 0.4 nM for B-RAF and C-RAF, showing a high level of selectivity, demonstrating greater than 99% on-target binding to BRAF, BRAFV600E, and CRAF at 1 μM and very few off-targets with DDR1 (>99%), DDR2 (86%), FRK (92%), and PDGFRb (96%), the only kinases with binding >80% at 1 μM.
Targets
C-Raf [1]
(Cell-free assay)
BRAF(V600E) [2]
(Cell-free assay)
B-Raf [2]
(Cell-free assay)
0.4 nM 1 nM 1.5 nM
In vitro

RAF709 appears to have very slow dissociation kinetics (T1/2 > 6.5 h) using the rapid dilution method to measure its dissociation rate constant. In cellular assays, the dose−response of pMEK and pERK are measured in Calu-6 cells with EC50 of 0.02 and 0.1 μM with minimal paradoxical activation and inhibition of proliferation with EC50 of 0.95 μM. RAF709 stabilizes BRAF−CRAF dimers with an EC50 of 0.8 μM. Of the 456 kinases tested, RAF709 shows a high level of selectivity, demonstrating greater than 99% on-target binding to BRAF, BRAFV600E, and CRAF at 1 μM and very few off-targets with DDR1 (>99%), DDR2 (86%), FRK (92%), and PDGFRb (96%), the only kinases with binding >80% at 1 μM[1]. RAF709 shows equal activity against both RAF monomers and dimers. In in vitro biochemical assays, RAF709 exhibits potent inhibitory activity targeting BRAF, BRAFV600E, and CRAF with IC50 values ranging between 0.3 to 1.5 nmol/L. RAF709 treatment leads to a dose-dependent induction of B/CRAF heterodimerization in HCT116, but inhibits MEK and ERK phosphorylation, in line with the ability of RAF709 to effectively inhibit the RAF dimers. RAF709 selectively inhibits oncogenic signaling and proliferation in tumor cells with BRAF, NRAS, or KRAS mutations with minimal paradoxical activation[2].

In vivo RAF709 is well tolerated and efficacious in KRAS mutant xenograft models. It is reasonably stable in plasma after a 3 h incubation at 37℃ across species [plasma stability (%remaining): rat 85%, mouse 82%, dog 95%, human 101%], and plasma protein binding is measured to be 98% across species. In pharmacokinetic experiments, RAF709 has moderate clearance in mouse (35 mL/min/kg) and dog (14 mL/min/kg) and high clearance in rat (50 mL/min/kg). Cmax in mouse (1 μM), dog (0.5 μM), and rat (0.5 μM) reach pharmacologically active concentrations, and acceptable oral availability is observed in mouse (68%), rat (24%), and dog (48%). In the Calu-6 xenograft nude mouse model, treatment with RAF709 results in dose-dependent antitumor activity with 10 mg/kg being subefficacious (%T/C = 92%), 30 mg/kg resulted in measurable antitumor activity (% T/C = 46%), and 200 mg/kg resulted in mean tumor regression of 92%, while the same high dose is not efficacious in the PC3, KRAS WT mode[1].

Protocol

Kinase Assay:

[1]

+ Expand

CRAF kinase assay:

The CRAF kinase assay was carried out using 10 nM kinase-dead MEK1 protein substrate (carrying a K97R mutation), 3 μM ATP, and 10 pM CRAF Y340E/Y341E. The reaction buffer contained 50 mM Tris pH 7.5, 10 mM MgCl2, 0.05% BSA, 50 mM NaCl, 0.01% Tween-20, and 1 mM DTT. The reactions were carried out at room temperature in a volume of 10 μL in white 384-shallow-well plates for 40 min and stopped by adding 5 μL/well quench solution (50 mM Tris pH 7.5, 50 mM EDTA). Terminated reactions received 5 μL/well detection reagents consisting of 50 mM Tris pH 7.5, 0.01% Tween-20, 1:1000 diluted antiphospho MEK1/2 S217/S221 antibody, 0.01 mg/mL each of AlphaScreen Protein A-coated acceptor beads, and streptavidin-coated donor bead. Plates were read in an EnVision plate reader after overnight incubation at room temperature. In compound inhibition studies, compounds were tested over a concentration range of 25 μM to 1.74 × 10−6 μM in 16-point, 3-fold format. DMSO was at a final concentration of 0.5%. Compounds were preincubated with CRAF for 30 min before adding substrates to start the reaction. Inhibition data were fit to a four-parameter logistic equation to calculate the IC50 of the compounds.
Cell Research:

[2]

+ Expand
  • Cell lines: HCT116 cells
  • Concentrations: 0.1, 1, 10 μM
  • Incubation Time: 1 hour
  • Method:

    HCT116 cells are treated with DMSO or RAF709 at indicated concentrations for 1 hour; 1 mmol/L dabrafenib treatment is included for comparison. BRAF/CRAF dimerization is assessed by immunoprecipitating BRAF or CRAF, followed by Western blot analysis of BRAF and CRAF. Levels of pMEK and pERK in whole-cell lysates (WCL) are determined by Western blot analysis. GAPDH level is included as a loading control.


    (Only for Reference)
Animal Research:

[1]

+ Expand
  • Animal Models: Calu-6 model (tumor bearing mice)
  • Formulation: 20% Captisol
  • Dosages: 10, 30, or 200 mg/kg
  • Administration: oral administration
    (Only for Reference)

Solubility (25°C)

In vitro DMSO 100 mg/mL (184.31 mM)
Ethanol 100 mg/mL (184.31 mM)
Water 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 Information

Molecular Weight 542.55
Formula

C28H29F3N4O4

CAS No. 1628838-42-5
Storage powder
in solvent
Synonyms N/A

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Raf Signaling Pathway Map

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Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID