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.

B-Raf (V600E):13 nM (cell free), B-Raf:160 nM (cell free), C-Raf1 (340D/Y341D):6.7 nM (cell free)

方法：1205Lu 和 C8161 细胞用 PLX4720（0.1,1,10μM，24小时） 并用膜联蛋白 V/FITC 和碘化丙啶 （PI） 染色以分析细胞凋亡。
结果：PLX4720诱导1205Lu细胞周期停滞并引起细胞凋亡，且呈现浓度依赖性。[1]
方法：8505c、TPC-1 和 NT 细胞用PLX4720（1μM或10μM）处理72小时后，加入溴脱氧尿苷（BrdU，10μM）1小时，随后进行细胞周期分析、BrdU测定和凋亡测定。
结果：8505c细胞用 1 μM PLX4720处理导致 1 小时后磷酸化 ERK-1/ERK-2 蛋白水平降低 >90%；即使在 72 小时后，细胞增殖也没有显着差异，而用 10 μM PLX4720处理 8505c 细胞 1 小时或 72 小时可降低磷酸化 ERK-1/ERK-2 BrdU摄取降低；在 TPC-1 细胞中，用 1 μM PLX4720处理 1 小时导致磷酸化 ERK-1/ERK-2 蛋白水平升高（约 80%）;用 10 μM 处理 1 小时PLX4720磷酸化 ERK-1/ERK-2 降低约 45%；1 μM 和 10 μM PLX4720 都引起较低的细胞增殖；只有 10 μM PLX4720 在处理 72 小时后减少了 TPC-1 细胞的迁移；10 μM PLX4720处理的NT细胞显示出显著降低的细胞增殖。[3]

方法：利用携带BRAF COLO205细胞（BRAFV600E系列突变）的裸鼠服用PLX4720(5 ,20,1000mg/kg,每日一次,口服),观察小鼠体内肿瘤生长情况。
结果：较低剂量的 5 mg/kg PLX4720对肿瘤生长的影响非常有限；20mg/kg的PLX4720治疗荷瘤小鼠导致肿瘤生长的大量阻滞；PLX4720治疗耐受性良好，将PLX4720剂量增加至1,000mg / kg导致血浆水平增加（高达600μM），而没有任何不良反应的证据。[1]

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].