A critical assessment of the synthesis and biological activity of p53/human double minute 2-stapled peptide inhibitors

Helix stapling enhances the activity of peptides that interact with a target protein in a helical conformation. These staples are also supposed to change the pharmacokinetics of the molecules and promote cytoplasmic targeting. We assessed the extent to which the pharmacokinetic characteristics are a function of the staple for a peptide inhibiting the interaction of p53 with the human double minute 2 (Hdm2) protein and differ from those of the standard cationic cell-penetrating peptide nona-arginine. Experimental approach: Stapled peptides and linear counterparts were synthesized in free and fluorescently labelled forms. Activity was determined in biochemical time-resolved Förster resonance energy transfer experiments and cellular high-content assays. Cellular uptake and intracellular trafficking were visualized by confocal microscopy. Key

Helix stapling enhances the activity of peptides that interact with a target protein in a helical conformation. These staples are also supposed to change the pharmacokinetics of the molecules and promote cytoplasmic targeting. We assessed the extent to which the pharmacokinetic characteristics are a function of the staple for a peptide inhibiting the interaction of p53 with the human double minute 2 (Hdm2) protein and differ from those of the standard cationic cell-penetrating peptide nona-arginine. Experimental approach: Stapled peptides and linear counterparts were synthesized in free and fluorescently labelled forms. Activity was determined in biochemical time-resolved Förster resonance energy transfer experiments and cellular high-content assays. Cellular uptake and intracellular trafficking were visualized by confocal microscopy. Key

Peptides showed sub-nanomolar potency. For short-time incubation, uptake efficiencies for the stapled and linear peptides were similar and both were taken up less efficiently than nona-arginine. Only for SJSA-1 cells expressing the Hdm2 target protein, the stapled peptides showed an enhanced cytoplasmic and nuclear accumulation after long-term incubation. This was also observed for the linear counterparts, albeit to a lesser degree. For HeLa cells, which lack target expression, no such accumulation was observed.