title: Identification of Short Hydrophobic Cell-Penetrating Peptides for Cytosolic Peptide Delivery by Rational Design.
abstract: 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.
authors.affiliation: Department of Biochemistry (286), Radboud Institute for Molecular Life Sciences, Radboud University Medical Center , Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands.
authors.forename: Samuel
authors.initials: S
authors.lastname: Schmidt
authors.affiliation: Department of Biochemistry (286), Radboud Institute for Molecular Life Sciences, Radboud University Medical Center , Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands.
authors.forename: Merel J W
authors.initials: MJ
authors.lastname: Adjobo-Hermans
authors.affiliation: Department of Biochemistry (286), Radboud Institute for Molecular Life Sciences, Radboud University Medical Center , Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands.
authors.forename: Robin
authors.initials: R
authors.lastname: Kohze
authors.affiliation: Roche Pharmaceutical Research and Early Development, Roche Innovation Center Basel , Grenzacherstrasse 124, 4070 Basel, Switzerland.
authors.forename: Thilo
authors.initials: T
authors.lastname: Enderle
authors.affiliation: Department of Biochemistry (286), Radboud Institute for Molecular Life Sciences, Radboud University Medical Center , Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands.
authors.forename: Roland
authors.identifier: 0000-0003-1395-6127
authors.initials: R
authors.lastname: Brock
authors.affiliation: Roche Pharmaceutical Research and Early Development, Roche Innovation Center New York , 430 East 29th Street, 10016 New York, New York, United States.
authors.forename: Francesca
authors.initials: F
authors.lastname: Milletti
mesh_terms: D000595:Amino Acid Sequence; D057846:Cell-Penetrating Peptides / Q000737:chemistry*; D003600:Cytosol / Q000378:metabolism*; D004337:Drug Carriers / Q000737:chemistry*; D015195:Drug Design; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D021381:Protein Transport
chemical_list: D057846:Cell-Penetrating Peptides; D004337:Drug Carriers; D010455:Peptides