RO5126766 (CH5126766) is a dual RAF/MEK inhibitor with IC50 of 8.2 nM, 19 nM, 56 nM, and 160 nM for BRAF V600E, BRAF, CRAF, and MEK1, respectively. Phase 1.

MEK1:160 nM, B-Raf (V600E):8.2 nM, BRAF:19 nM, Raf:56 nM

在HCT116 KRAS突变型结直肠癌细胞中，CH5126766显著降低了phospho-MEK和phospho-ERK的水平。CH5126766通过与MEK1结合来抑制RAF激酶，使MEK成为RAF的主要负向抑制剂。[1] 在Raf或RAS突变细胞系（包括SK-MEL-28、SK-MEL-2、MIAPaCa-2、SW480、HCT116和PC3细胞）中，CH5126766以分别为65、28、40、46和277 nM的IC50值抑制细胞生长。在携带BRAF V600E或NRAS突变的两个黑色素瘤细胞系中，RO5126766引起G1期细胞周期阻滞，并伴随着CDK抑制剂p27的上调和cyclinD1的下调。[3]

在HCT116 (G13D KRAS) 小鼠异种移植模型中，CH5126766 (25 mg/kg, p.o.) 比标准的MEK抑制剂更有效地抑制ERK信号输出，后者诱导MEK磷酸化且具有强大的抗肿瘤活性。[1] 在HCT116 (K-ras) 和COLO205 (B-raf) 突变型异种移植中，CH5126766 (0.3 mg/kg) 显著减少了[18F]FDG摄取。[2] 在SK-MEL-2异种移植模型中，RO5126766同样抑制了肿瘤生长。[3]

MEK and RAF kinase enzyme assays: The inhibitory activities against CRAF, BRAF, or BRAF V600E enzymes are measured by quantification of phosphorylation of inactive K97R MEK1 [MEK1] by recombinant RAF proteins [BRAF: B-RAF wt, BRAF V600E: B-RAF V600E or CRAF: Raf-1] with Europium-anti-MEK1/2 (pSer218/222) antibody and SureLight allophycocyanine-anti-6his antibody by measuring time-resolved fluorescence (TRF). Alternatively, the inhibitory activities against the RAF enzymes are measured by quantification of phosphorylation of a fluorescein-labeled peptide corresponding to human MEK1 212-224 and human MEK2 217-229 (5-Fl-SGQLIDSMANSFV-NH2, MEKtide) by using the IMAP fluorescence polarization (FP) Screening Express Kit. Inhibition of MEK1 is evaluated by a coupled assay with active MEK1 (MEK1 S218E/S222E) and unactive dephosphorylated ERK2 (MAP kinase 2/Erk 2). The phosphorylation of a fluorescent-labeled peptide substrate (FAM-Erktide, IPTTPITTTYFFFK-5FAM-COOH) by ERK2 is quantified by using the IMAP FP Screening Express Kit.

The number of viable cells is determined using the Cell Counting Kit-8 assay according to the manufacturer's instructions. After the incubation of cells for 72 h with the indicated concentrations of various agents, kit reagent WST-8 is added to the medium and incubated for a further 4 h. The absorbance of samples (450 nm) is determined using a scanning multiwell spectrophotometer that serves as an ELISA reader. Cell numbers and viability are also measured using the ViaCount Assay according to the manufacturer's instructions.(Only for Reference)