Guanidinium side-chain substitutions in the CPP-grafted p53-MDM2 inhibitor St3-R

Structure-guided grafting of cell-penetrating peptide (CPP) motifs into bioactive peptides can couple intracellular delivery with target recognition, but the chemistry of the grafted cationic residues may also reshape the bioactive conformation and trafficking behavior. Here, we examined the properties associated with global guanidinium-bearing residue substitutions within the p53-MDM2 inhibitor St3-R. All Arg residues in St3-R were replaced with homoarginine (St3-hR) or 2-amino-3-guanidinopropionic acid (St3-Agp), and the derivatives were evaluated by circular dichroism, molecular dynamics, surface plasmon resonance, flow cytometric analysis of cell-associated fluorescence under different temperature and inhibitor conditions, and live-cell NanoBRET. St3-hR showed a numerically higher α-helical content (40% versus 35%), a modestly lower mean MDM2 K _D (K _D = 104.3 ± 3.7 versus 113.8 ± 2.7 nM), similar cell-associated fluorescence of its FAM-labeled derivative, and a lower apparent NanoBRET IC₅₀ (6.15 ± 0.06 versus 9.34 ± 0.54 μM) than St3-R. By contrast, St3-Agp displayed lower helicity, greater deviation from the starting structure, lower cell-associated fluorescence of its FAM-labeled derivative, and a higher apparent NanoBRET IC₅₀ (17.90 ± 0.41 μM). At 4 °C, the cell-associated fluorescence of F-St3-Agp was 15.3% of its value at 37 °C, a larger relative fraction than for the other two labeled peptides; this does not establish direct membrane translocation or greater cytosolic delivery. Because St3-Agp was evaluated as an isomerically unresolved preparation, its properties cannot be attributed solely to side-chain shortening. Together, these findings highlight the importance of considering peptide conformation, target-binding affinity, and cellular behavior when optimizing guanidinium-bearing residues in CPP-grafted PPI inhibitors.