Identification of Short Hydrophobic Cell-Penetrating Peptides for Cytosolic Peptide Delivery by Rational Design
Schmidt, S., Adjobo-Hermans, M.J.W., Kohze, R., Enderle, T., Brock, R., Milletti, F.
Cell-penetrating peptides (CPPs) enhance the cellular uptake of membrane-impermeable molecules. Most CPPs are highly cationic,
potentially increasing the risk of toxic side effects and leading to accumulation in organs such as the liver. As a consequence, there is an unmet need for less cationic
CPPs. However, design principles for effective CPPs are still missing. Here, we demonstrate a design principle based on a classification of peptides according to
accumulated side-chain polarity and hydrophobicity. We show that in comparison to randomly selected peptides, CPPs cover a distinct parameter space. We designed
peptides of only six to nine amino acids with a maximum of three positive charges covering this property space. All peptides were tested for cellular uptake and subcellular
distribution. Following an initial round of screening we enriched the collection with short and hydrophobic peptides and introduced D-amino acid substitutions and lactam
bridges which increased cell uptake, in particular for long-term incubation. Using a GFP complementation assay, for the most active peptides we demonstrate cytosolic
delivery of a biologically active cargo peptide.