Design, Synthesis,\nand Biological Evaluation of Lysine-Stapled\nPeptide Inhibitors of p53-MDM2/MDMX Interactions with Potent Antitumor\nActivity <i>In Vivo</i>

We introduce novel lysine-stapled peptide inhibitors\ntargeting <i>p53</i>-MDM2/MDMX interactions. Leveraging\nthe model peptides\npDI (LTFEHYWAQLTS) and PMI-M3 (LTFLEYWAQLMQ) as starting points, a\nseries of lysine-stapled analogues were designed and synthesized.\nThrough <i>in vitro</i> cell assay screening, two lead compounds,\nSPDI-48-T<sub>1</sub> and SPMI-48-T<sub>3</sub>, were identified for\ntheir excellent antiproliferation activity. Fluorescence polarization\nassays revealed that both compounds exhibited strong binding affinities\nagainst MDM2 and MDMX, ascertained by <i>K</i><sub>d</sub> values within the low micromolar spectrum. Further characterization\nof SPDI-48-T<sub>1</sub> and SPMI-48-T<sub>3</sub> demonstrated that\nSPDI-48-T<sub>1</sub> possessed superior cell permeability and serum\nstability. Notably, SPDI-48-T<sub>1</sub> displayed a dose-dependent\nsuppression of tumor growth in an HCT116 xenograft mouse model. Our\nfindings indicate that SPDI-48-T<sub>1</sub> holds promise as a lead\ncompound for further development as an anticancer agent by modulating <i>p53</i>-MDM2/MDMX interactions. Additionally, this study also\nproved that the lysine stapling strategy may serve as a robust approach\nfor generating peptide ligands targeting other protein–protein\ninteractions.