Design, synthesis, and antitumor activity of stapled peptide inhibitors targeting the RAS-RAF interactions

RAS-RAF interactions play a vital role in the RAS-RAF-MEK-ERK signaling pathway, significantly regulating cell proliferation, differentiation, and survival. Some small molecule inhibitors targeting various components of this pathway, such as MRTX849 and AMG 510, have been introduced for clinical application. However, peptide-based drugs encounter several challenges, such as poor cell permeability, low biological stability, and rapid in vivo clearance, which hinder their application. Herein, based on co-crystal complex structures and RAS-RAF interaction hotspots, we identified four linear peptides-Raf-0 to Raf-2 and CRD-0-derived from the α-helical regions of the RAS-binding domain (RBD) and the cysteine-rich domain (CRD) of CRAF. Raf-1 was selected for further modification using a hydrocarbon stapling strategy, capping it with stearic acid at the N-terminal due to its highest binding affinity in the SPR assay. As a result, Sraf-2-1 and Sraf-7-1 bound to KRAS^G12C with K_d values of 3.56 μM and 2.62 μM, respectively, demonstrating robust anticancer activity in the CCK8 assay. Additionally, Sraf-2-1 and Sraf-7-1 reduced AKT phosphorylation, induced cancer cell apoptosis in a concentration-dependent manner, and effectively inhibited cancer cell migration, showing improved α-helix stability and cell permeability. In summary, our findings indicate that the hydrocarbon stapling strategy and stearic acid tagging enhanced the therapeutic potential of peptide inhibitors, offering methods for targeting RAS in cancer therapy.