title: Enzyme-triggered delivery of chlorambucil from conjugates based on the cell-penetrating peptide BP16.
abstract: The undecapeptide KKLFKKILKKL-NH2 (BP16) is a non-toxic cell-penetrating peptide (CPP) that is mainly internalized into cancer cells through a clathrin dependent endocytic mechanism and localizes in late endosomes. Moreover, this CPP is able to enhance the cellular uptake of chlorambucil (CLB) improving its cytotoxicity. In this work, we further explored the cell-penetrating properties of BP16 and those of its arginine analogue BP308. We investigated the influence on the cytotoxicity and on the cellular uptake of conjugating CLB at the N- or the C-terminal end of these undecapeptides. The effect of incorporating the cathepsin B-cleavable sequence Gly-Phe-Leu-Gly in CLB-BP16 and CLB-BP308 conjugates was also evaluated. The activity of CLB was significantly improved when conjugated at the N- or the C-terminus of BP16, or at the N-terminus of BP308. While CLB alone was not active (IC50 of 73.7 to >100 μM), the resulting conjugates displayed cytotoxic activity against CAPAN-1, MCF-7, PC-3, 1BR3G and SKMEL-28 cell lines with IC50 values ranging from 8.7 to 25.5 μM. These results were consistent with the internalization properties observed for the corresponding 5(6)-carboxyfluorescein-labeled conjugates. The presence of the tetrapeptide Gly-Phe-Leu-Gly at either the N- or the C-terminus of CLB-BP16 conjugates further increased the efficacy of CLB (IC50 of 3.6 to 16.2 μM), which could be attributed to its selective release in the lysosomal compartment. Enzymatic assays with cathepsin B showed the release of CLB-Gly-OH from these sequences within a short time. Therefore, the combination of BP16 with an enzymatic cleavable sequence can be used as a drug delivery system for the effective uptake and release of drugs in cancer cells.
authors.affiliation: QBIS-CAT Research Group, Institut de Química Computacional i Catàlisi (IQCC) and Departament de Química, Universitat de Girona, Campus Montilivi, E-17071 Girona, Catalonia, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Soler
authors.forename: Marta
authors.initials: M
authors.lastname: González-Bártulos
authors.forename: Eduard
authors.initials: E
authors.lastname: Figueras
authors.forename: Xavi
authors.initials: X
authors.lastname: Ribas
authors.forename: Miquel
authors.initials: M
authors.lastname: Costas
authors.forename: Anna
authors.initials: A
authors.lastname: Massaguer
authors.forename: Marta
authors.initials: M
authors.lastname: Planas
authors.forename: Lidia
authors.initials: L
authors.lastname: Feliu
mesh_terms: D000595:Amino Acid Sequence; D001692:Biological Transport; D002401:Cathepsin B / Q000378:metabolism*; D045744:Cell Line, Tumor; D057846:Cell-Penetrating Peptides / Q000737:chemistry* / Q000378:metabolism; D002699:Chlorambucil / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D004337:Drug Carriers / Q000737:chemistry* / Q000378:metabolism; D015195:Drug Design; D065546:Drug Liberation; D006801:Humans; D008969:Molecular Sequence Data; D059748:Proteolysis
chemical_list: D057846:Cell-Penetrating Peptides; D004337:Drug Carriers; D002699:Chlorambucil; D002401:Cathepsin B