Design, Synthesis, and Biological Evaluation of Lysine-Stapled Peptide Inhibitors of p53-MDM2/MDMX Interactions with Potent Antitumor Activity <i>In Vivo</i>

We introduce novel lysine-stapled peptide inhibitors targeting <i>p53</i>-MDM2/MDMX interactions. Leveraging the model peptides pDI (LTFEHYWAQLTS) and PMI-M3 (LTFLEYWAQLMQ) as starting points, a series of lysine-stapled analogues were designed and synthesized. Through <i>in vitro</i> cell assay screening, two lead compounds, SPDI-48-T₁ and SPMI-48-T₃, were identified for their excellent antiproliferation activity. Fluorescence polarization assays revealed that both compounds exhibited strong binding affinities against MDM2 and MDMX, ascertained by <i>K</i>_d values within the low micromolar spectrum. Further characterization of SPDI-48-T₁ and SPMI-48-T₃ demonstrated that SPDI-48-T₁ possessed superior cell permeability and serum stability. Notably, SPDI-48-T₁ displayed a dose-dependent suppression of tumor growth in an HCT116 xenograft mouse model. Our findings indicate that SPDI-48-T₁ holds promise as a lead compound for further development as an anticancer agent by modulating <i>p53</i>-MDM2/MDMX interactions. Additionally, this study also proved that the lysine stapling strategy may serve as a robust approach for generating peptide ligands targeting other protein-protein interactions.