LY3009120 (DP-4978) is a potent pan-Raf inhibitor with IC50 of 44 nM, 31-47 nM, and 42 nM for A-raf, B-Raf, and C-Raf in A375 cells, respectively. Phase 1.

B-Raf (V600E):5.8 nM, C-Raf:4.3 nM, B-Raf (WT):15 nM

LY3009120抑制A375细胞和HCT116细胞的生长，其IC50分别为9.2 μM和220 μM。同时，LY3009120也对酪氨酸激酶KDR有抑制作用，其IC50为3.9 μM。[1]

在携带BRAF V600E ST019VR PDX肿瘤的大鼠中，LY3009120 (15或30 mg/kg, p.o.)表现出剂量依赖性的肿瘤生长抑制效果。在裸鼠植入A375异种移植瘤中，单次口服LY3009120 (3到50 mg/kg, p.o.)显示出对磷酸化ERK的剂量依赖性抑制，其中抑制磷酸化ERK 50%所需剂量(EC50)为4.36 mg/kg，达到抑制磷酸化ERK 50%的血浆浓度(EC50)为68.9 ng/mL或165 nM。[1]

Kinase activity measurement using KiNativ assays: Compounds are screened in A375 cell lysates using the ATP-based probe at 5 μM. IC50 values are reported in micromolar units. Cell pellets are resuspended in four volumes of lysis buffer [25 mM Tris pH 7.6, 150 mM NaCl, 1% CHAPS, 1% Tergitol NP-40 type, 1% v/v phosphatase inhibitor cocktail II], sonicated using a tip sonicator, and dounce homogenized. Lysates are cleared by centrifugation at 100,000 g for 30 min. The cleared lysates are filtered through a 0.22 μM syringe filter, and gel filtered into reaction buffer [20 mM Hepes pH 7.8, 150 mM NaCl, 0.1% triton X-100, 1% v/v phosphatase inhibitor cocktail II]. MnCl2 is then added to the lysate to a final concentration of 20 mM prior to inhibitor treatment and probe labeling. Final inhibitor concentrations used for IC50 determinations are 10, 1, 0.1, and 0.01 μM. ATP competition experiments are performed at 1,000, 100, 10, and 1 μM ATP. All inhibitor treatments are performed at room temperature.

Brie?y, cells are grown in DMEM high glucose supplemented with 10% characterized fetal bovine serum and 1% penicillin/streptomycin/L -glutamine at 37℃, 5% CO2, and 95% humidity. Cells are allowed to expand until 70-95% con?uency. A serial dilution of test compound is dispensed into a 384-well black clear bottom plate. 625 cells are added per well in 50 μL of complete growth medium. Plates are incubated for 67 h at 37℃, 5% CO2 , and 95% humidity. Then, 10 μL of a 440 μM solution of resazurin in PBS is added to each well of the plate and plates are incubated for an additional 5 h at 37℃, 5% CO2, and 95% humidity. Plates are read on a Synergy2 reader using an excitation of 540 nm and an emission of 600 nm. Data are analyzed using Prism software to calculate IC 50 values.(Only for Reference)

Animal Models: Female NIH nude rats bearing BRAF V600E ST019VR PDX tumorsFormulation: 20% cyclodextrin, 25 mM phosphate, pH2.0Dosages: 30 mg/kgAdministration: p.o.[1]