PLX-4720 is a potent and selective B-Raf (V600E) inhibitor designed to block the ATP-binding site of oncogenic B-Raf with IC50 of 13 nM.

Csk:1500 nM, CSF1R:3300 nM, Aurora A:3400 nM, KDR:2300 nM, BRK:130 nM, C-Raf1 (340D/Y341D):6.7 nM (cell free), FRK:1300 nM, GK (human):2800 nM, HGK:2800 nM, FAK:1700 nM, B-Raf (V600E):13 nM (cell free), Src:1700 nM, B-Raf:160 nM (cell free), FGFR:1900 nM

In vitro Raf kinase activities: The in vitro kinase activities of wild type Raf and mutants are determined by measuring phosphorylation of biotinylated-MEK protein using Perkin-Elmer's AlphaScreen Technology. For each enzyme (0.1 ng), 20-μL reactions are carried out in 20 mM Hepes (pH 7.0), 10 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 100 nM biotin-MEK protein, various ATP concentrations, and increasing concentrations of PLX-4720 at room temperature. Reactions are stopped at 2, 5, 8, 10, 20, and 30 minutes with 5 μL of a solution containing 20 mM Hepes (pH 7.0), 200 mM NaCl, 80 mM EDTA, and 0.3% BSA. The stop solution also includes phospho-MEK Antibody, Streptavidin-coated Donor beads and Protein A Acceptor beads. The antibody and beads are preincubated in stop solution in the dark at room temperature for 30 minutes. The final dilution of antibody is 1/2,000, and the final concentration of each bead is 10 μg/mL. The assay plates are incubated at room temperature for one hour then are read on a PerkinElmer AlphaQuest reader [1].

Cells are treated with various concentrations PLX-4720 for 24, 48, and 72 hours. Cell proliferation is measured by using the CellTiter-Glo Luminescent Cell Viability Assay or MTT assay. For cell cycle analysis, supernatant and cells are collected, pelleted, and fixed with 70% ethanol. Before staining with propidium iodide (10 μg/mL), cells are incubated for 1 hour at 37 °C in 0.5 mg/mL RNase I to rid samples of residual RNA contamination. Samples are then analyzed by using the EPICS XL apparatus. For the assessment of apoptosis, media and cells are harvested and pelleted before staining with annexin-FITC and propidium iodide. Samples are subsequently analyzed by using the EPICS XL apparatus [1].

Female athymic mice (NCr nu/nu) were implanted s.c. on day 0 with 30–60 mg COLO205 tumor fragments. Treatments began on day 11, when the mean estimated tumor mass was 104 mg (range, 95–113 mg). All animals were dosed with vehicle (5% DMSO, 1% methylcellulose) or PLX4720 suspended in vehicle by gavage daily for 14 consecutive days. Tumor burden (mg) was estimated from caliper measurements [1].