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A critical assessment of the synthesis and biological activity of p53/Hdm2 stapled peptide inhibitors

Ruetz, Stephan, Chene, Patrick, Stachyra, Therese-Marie, Brock, Roland and Wallbrecher, Rike (2017) A critical assessment of the synthesis and biological activity of p53/Hdm2 stapled peptide inhibitors. British journal of pharmacology, 2017 .


The covalent linkage of amino acid side chains to conformationally constrain -helices has been employed as a powerful mean to enhance 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 cell entry including cytoplasmic targeting. In particular, stapled peptides inhibiting the interaction of p53 with the human double minute 2 (Hdm2) protein were of interest for this approach. Here, we scrutinized to which degree the pharmacokinetic characteristics are a function of the staple and differ from those of a standard cationic cell-penetrating peptide (CPP). Stapled peptides and their linear counterparts were synthesized to verify activity in biochemical and cellular assays. All peptides showed potent sub-nanomolar potency to Hdm2. Assessing uptake for carboxyfluorescein-labeled variants, for short incubation times, there was only little difference in uptake efficiency for the stapled peptides and their linear counterpart and both were taken up less efficiently than the prototypic CPP nonaarginine (R9). Fluorescence was restricted to vesicular structures. Only following long-term incubation, and for SJSA-1 cells expressing the Hdm2 target protein, the stapled peptides and also the linear counterparts, albeit to a lesser degree, showed an enhanced cytoplasmic and nuclear accumulation. For HeLa cells, lacking target expression no such accumulation was observed. These findings demonstrate that the cytosolic and nuclear accumulation are not an intrinsic property of the stapled peptide but result from capture by the target Hdm2 once leaking out of the endo-lysosomal compartment.

Item Type: Article
Date Deposited: 01 Jun 2017 00:45
Last Modified: 01 Jun 2017 00:45


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