Constitutive activation of RAS-RAF-MEK-ERK signaling pathway (MAPK pathway) frequently occurs in many cancers harboring RAS or RAF oncogenic mutations. Because of the paradoxical activation induced by a single use of BRAF or MEK inhibitors, dual-target RAF and MEK treatment is thought to be a promising strategy. In this work, we evaluated erianin is a novel inhibitor of CRAF and MEK1/2 kinases, thus suppressing constitutive activation of the MAPK signaling pathway induced by BRAF V600E or RAS mutations. KinaseProfiler enzyme profiling, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), cellular thermal shift assay, computational docking, and molecular dynamics simulations were utilized to screen and identify erianin binding to CRAF and MEK1/2. Kinase assay, luminescent ADP detection assay, and enzyme kinetics assay were investigated to identify the efficiency of erianin in CRAF and MEK1/2 kinase activity. Notably, erianin suppressed BRAF V600E or RAS mutant melanoma and colorectal cancer cell by inhibiting MEK1/2 and CRAF but not BRAF kinase activity. Moreover, erianin attenuated melanoma and colorectal cancer in vivo. Overall, we provide a promising leading compound for BRAF V600E or RAS mutant melanoma and colorectal cancer through dual targeting of CRAF and MEK1/2.

RAS-RAF-MEK-ERK 信号通路（MAPK 通路）的组成性激活经常发生在许多携带 RAS 或 RAF 致癌突变的癌症中。由于单次使用 BRAF 或 MEK 抑制剂会引起矛盾的激活，双靶 RAF 和 MEK 治疗被认为是一种有前途的策略。在这项工作中，我们评估了毛兰素是一种新型 CRAF 和 MEK1/2 激酶抑制剂，从而抑制由 BRAF V600E 或 RAS 突变诱导的 MAPK 信号通路的组成型激活。KinaseProfiler 酶分析、表面等离子共振 (SPR)、等温滴定量热法 (ITC)、细胞热位移分析、计算对接和分子动力学模拟被用于筛选和鉴定与 CRAF 和 MEK1/2 结合的毛兰素。激酶测定、发光ADP检测测定、研究了毛兰素和酶动力学测定以确定毛兰素在 CRAF 和 MEK1/2 激酶活性中的效率。值得注意的是，毛兰素通过抑制 MEK1/2 和 CRAF 而不是 BRAF 激酶活性来抑制 BRAF V600E 或 RAS 突变黑色素瘤和结直肠癌细胞。此外，毛兰素在体内减弱了黑色素瘤和结直肠癌。总的来说，我们通过 CRAF 和 MEK1/2 的双重靶向，为 BRAF V600E 或 RAS 突变黑色素瘤和结直肠癌提供了一种有前途的先导化合物。