D-SAP: A New, Noncytotoxic, and Fully Protease Resistant Cell-Penetrating Peptide

• Published in: ChemMedChem Vol. 3; no. 2; pp. 296 - 301
• Main Authors: Pujals, Sílvia, Fernández-Carneado, Jimena, Ludevid, M. Dolors, Giralt, Ernest
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 15.02.2008; WILEY‐VCH Verlag
• Subjects: Cell Membrane - chemistry; Cell Membrane - metabolism; Cell Membrane Permeability; cell-penetrating peptides; Cells, Cultured; Circular Dichroism; D-amino acids; Drug Carriers - metabolism; drug delivery; Endopeptidases - metabolism; Flow Cytometry; HeLa Cells; Humans; Microscopy, Electron, Transmission; Peptides - chemistry; Peptides - metabolism; proteolytic resistance; Saposins - chemistry; Saposins - metabolism; self-assembly
• ISSN: 1860-7179; 1860-7187
• DOI: 10.1002/cmdc.200700267

Protease resistant cell‐penetrating peptides (CPPs) are promising carriers for drugs unable to cross the cell membrane. As these CPPs are stable in vivo for much longer periods of time compared to other classes of therapeutic peptides, noncytotoxicity is a property sine qua non for their pharmacological development. Described herein is a fully protease resistant CPP that is noncytotoxic at concentrations up to 1 mM. Proteolytic stability was obtained by chiral inversion of the residues of a known self‐assembling CPP—from all L‐amino acids to all D‐amino acids—and then assessed against trypsin and human serum. Circular dichroism studies confirmed the enantiomeric structure of the analogue, and transmission electron microscopy (TEM) studies indicated that the new inverso analogue retains the ability of the original peptide to self‐assemble. The results of uptake experiments indicate that the protease‐stable (that is, D‐amino acid) analogue of the peptide is internalised by cells to the same extent as the protease‐susceptible (that is, L‐amino acid) parent peptide. Also reported herein are the results of studies on the cellular internalisation mechanism of the all‐D analogue, which reveal the steps followed by the peptide upon its entry into the cell. Penetrating particles. Given that most potential biomolecular drugs (for example, DNA, RNA, peptides, and proteins) are very often directed at intracellular targets, intracellular drug delivery is a focal point in drug development. SAP is an efficient and noncytotoxic cell‐penetrating peptide. However, it is labile to proteases. Herein, its enantiomeric version, D‐SAP, is reported to retain the properties of SAP, and is shown to be fully protease resistant.