title: Metabolic cleavage of cell-penetrating peptides in contact with epithelial models: human calcitonin (hCT)-derived peptides, Tat(47-57) and penetratin(43-58).
abstract: We assessed the metabolic degradation kinetics and cleavage patterns of some selected CPP (cell-penetrating peptides) after incubation with confluent epithelial models. Synthesis of N-terminal CF [5(6)-carboxyfluorescein]-labelled CPP, namely hCT (human calcitonin)-derived sequences, Tat(47-57) and penetratin(43-58), was through Fmoc (fluoren-9-ylmethoxycarbonyl) chemistry. Metabolic degradation kinetics of the tested CPP in contact with three cell-cultured epithelial models, MDCK (Madin-Darby canine kidney), Calu-3 and TR146, was evaluated by reversed-phase HPLC. Identification of the resulting metabolites of CF-hCT(9-32) was through reversed-phase HPLC fractionation and peak allocation by MALDI-TOF-MS (matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry) or direct MALDI-TOF-MS of incubates. Levels of proteolytic activity varied highly between the investigated epithelial models and the CPP. The Calu-3 model exhibited the highest proteolytic activity. The patterns of metabolic cleavage of hCT(9-32) were similar in all three models. Initial cleavage of this peptide occurred at the N-terminal domain, possibly by endopeptidase activity yielding both the N- and the C-terminal counterparts. Further metabolic degradation was by aminopeptidase, endopeptidase and/or carboxypeptidase activities. In conclusion, when in contact with epithelial models, the studied CPP were subject to efficient metabolism, a prerequisite of cargo release on the one hand, but with potential for premature cleavage and loss of the cargo as well on the other. The results, particularly on hCT(9-32), may be used as a template to suggest structural modifications towards improved CPP performance.
authors.affiliation: Drug Formulation & Delivery Group, Department of Chemistry and Applied BioSciences, Swiss Federal Institute of Technology Zurich (ETH Zurich), Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
authors.forename: Rachel
authors.initials: R
authors.lastname: Tréhin
authors.forename: Hanne M
authors.initials: HM
authors.lastname: Nielsen
authors.forename: Heinz-Georg
authors.initials: HG
authors.lastname: Jahnke
authors.forename: Ulrike
authors.initials: U
authors.lastname: Krauss
authors.forename: Annette G
authors.initials: AG
authors.lastname: Beck-Sickinger
authors.forename: Hans P
authors.initials: HP
authors.lastname: Merkle
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001711:Biotransformation; D002116:Calcitonin / Q000378:metabolism*; D002460:Cell Line; D057846:Cell-Penetrating Peptides; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004285:Dogs; D004337:Drug Carriers / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D015696:Gene Products, tat / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism*; D013329:Structure-Activity Relationship; D054322:tat Gene Products, Human Immunodeficiency Virus
chemical_list: D057846:Cell-Penetrating Peptides; D004337:Drug Carriers; D015696:Gene Products, tat; D018398:Homeodomain Proteins; D010446:Peptide Fragments; C497136:calcitonin (9-32), human; C497138:penetratin (43-58), human; D054322:tat Gene Products, Human Immunodeficiency Virus; C497137:tat peptide (47-57), Human immunodeficiency virus 1; D002116:Calcitonin