Encorafenib (LGX818) is an orally available mutated BRaf V600E inhibitor(IC50=0.3 nM) with potential antineoplastic activity.

B-Raf (V600E):0.3 nM

在A375（BRAFV600E）人类黑色素瘤细胞系中，Encorafenib抑制磷酸化ERK（EC50 = 3 nM），从而强效抑制增殖（EC50 = 4 nM）。在针对100种激酶的检测中未观察到显著活性（IC50 > 900 nM），且Encorafenib不抑制表达野生型BRAF的400多种细胞系的增殖。Encorafenib的高效力部分归因于其从BRAFV600E解离的极其缓慢速率，这一点在其他RAF抑制剂中未见。生化实验中，解离半衰期超过24小时，这意味着在化合物清洗后，细胞中的靶点抑制作用能持续。[1]

Encorafenib通过口服，在低至6 mg/kg剂量下便可强效（75%）并持续（>24小时）降低磷酸化MEK水平，即便是在化合物从血液循环清除后的单剂量PK/PD研究中，对人源黑色素瘤异种移植模型（BRAFV600E）仍然有效。在免疫缺陷的小鼠和大鼠体内，Encorafenib在低至1 mg/kg的剂量下引起多种BRAF突变型人肿瘤异种移植模型的肿瘤退缩。与体外数据一致，Encorafenib对BRAF野生型肿瘤在高达300 mg/kg bid的剂量下无活性，但具有良好的耐受性和曝光量的线性增加。在更具疾病相关性的自发性转移性黑色素瘤和黑色素瘤脑转移模型中也实现了疗效。Encorafenib是一种强效且选择性的RAF激酶抑制剂，具有独特的生化特性，有助于其出色的药理学特性。[1]

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.