Encorafenib

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

Methods: Encorafenib (0.5 μM) + Cetuximab (0.25 μM) and DAG (10 μM) were added to sensitive RKO and HT29 cells and incubated for 48 hours; cell viability was assessed using the CCK-8 assay, and apoptosis was detected by flow cytometry.
Results: Co-administration of DAG significantly reduced the cell growth inhibition and apoptosis induced by Encorafenib/Cetuximab. [1]
Methods: NRAS-mutant melanoma cell lines SKMel147, PDX129, and PDX62.1 were treated with Encorafenib (1 μM) and Binimetinib (1 μM) for 72 hours. Cell cycle analysis was performed via flow cytometry (PI staining), and the SubG1 apoptosis peak was quantified.
Results: Encorafenib monotherapy induced significant apoptosis. [2]
Methods: Neonatal rat ventricular myocytes (NRVMs) were treated with Encorafenib (0.1 μM) for 24 hours, and cell size was assessed by immunofluorescence staining.
Results: Encorafenib treatment significantly increased the surface area of cardiomyocytes, indicating that it promotes cardiomyocyte hypertrophy.[3]

Methods: Xenograft models (tumor-bearing nude mice) derived from BRAFV600E-mutant mCRC patients received oral Encorafenib (20 mg/kg, once daily) and intraperitoneal injections of Cetuximab (20 mg/kg, twice weekly) and PF-06471553 (MOGAT3 inhibitor) for 4 weeks.
Results: The triple therapy (Encorafenib + Cetuximab + Pf) significantly inhibited resistant tumor growth, reduced DAG levels, and increased apoptosis. [1]
Methods: NSG mice were subcutaneously implanted with three NRAS-mutant melanoma cell lines: SKMel147, PDX129, and PDX62.1. Treatment with oral Encorafenib (6 mg/kg, once daily) and Binimetinib (8 mg/kg, twice daily) was administered for up to 28 days.
Results: Encorafenib significantly inhibited tumor growth.[2]

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.

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.