Encorafenib (LGX818) is an oral, highly selective inhibitor of the BRAF V600E mutation with an IC50 of 0.3 nM, exhibiting antitumor activity. Encorafenib can be used for studying tumor signaling pathway regulation and mechanisms of drug resistance.

B-Raf (V600E):0.3 nM

The Raf kinase activity reaction is started by the addition of 10 μL per well of 2×ATP diluted in assay buffer. After 3 hours (bRaf(V600E)) or 1 hour (c-Raf), the reactions are stopped with the addition of 10 μL of stop reagent (60 mM EDTA). Phosphorylated product is measured using a rabbit anti-p-MEK antibody and the Alpha Screen IgG (ProteinA) detection Kit, by the addition of 30 μL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in bead buffer (50 mM Tris, pH 7.5, 0.01% Tween20). The additions are carried out under dark conditions to protect the detection beads from light. A lid is placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence is read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) is calculated by non-linear regression using XL Fit data analysis software

LGX818 is dissolved in DMSO. A375 is a melanoma cell line that harbors the B-Raf V600E mutation. A375-luc cells engineered to express luciferase is plated to 384-well white clear bottom plates as 1,500 cells/50 μL/well in DMEM containing 10% FBS. Test compounds, dissolved in 100% DMSO at appropriate concentrations, are transferred to the cells by a robotic Pin Tool (100 mL). The cells are incubated for 2 days at 25°C, then 25 μL of BrightGloTM is added to each well and the plates are read by luminescence. The concentration of each compound for 50% inhibition (IC50) is calculated by non-linear regression using XL Fit data analysis software. wild type and V600E B-Raf.

LGX818

H2O: < 1 mg/mL (insoluble or slightly soluble)

Ethanol: 93 mg/mL (172.22 mM), Sonication is recommended.

DMSO: 257 mg/mL (475.92 mM), Sonication is recommended.

10% DMSO+40% PEG300+5% Tween 80+45% Saline: 9.3 mg/mL (17.22 mM), Suspension.