Delivering aminopyridine ligands into cancer cells through conjugation to the cell-penetrating peptide BP16

Abstract

Peptide conjugates incorporating the N-based ligands ^Me2PyTACN or (S,S′)-BPBP at the N- or the C-terminus of the cell-penetrating peptide BP16 were synthesized (PyTACN–BP16 (BP341), BP16-PyTACN (BP342), BPBP–BP16 (BP343), and BP16-BPBP (BP344)). Metal binding peptides bearing at the N-terminus the ligand, an additional Lys and a β-Ala were also prepared (PyTACN-βAK–BP16 (BP345) and BPBP-βAK–BP16 (BP346)). Moreover, taking into account the clathrin-dependent endocytic mechanism of BP16, the enzymatic cleavable tetrapeptide Gly-Phe-Leu-Gly was incorporated between the ligand and the N- or C-terminus of BP16 (BPBP-GFLG-BP16 (BP347) and BP16-GLFG-BPBP (BP348)). Analysis of the cytotoxicity of all the peptide conjugates showed that: (i) the position of the ligand influenced the IC₅₀ values, (ii) the incorporation of the βAla-Lys dipeptide rendered non active sequences, (iii) peptide conjugates derived from the (S,S′)-BPBP ligand were more active than those bearing ^Me2PyTACN, and (iv) the introduction of the cleavable tetrapeptide significantly enhanced the activity of the BPBP conjugates (IC₅₀ of 4.3 to 11.7 μM (BP347 and BP348) compared to 26.0 to >50 μM (BP343, BP344 and BP346)). The most active peptide was BPBP-GFLG-BP16 (BP347) (IC₅₀ of 4.3 to 5.0 μM). This high activity was attributed to its high internalization in MCF-7 cells, as shown by flow cytometry, and to the subsequent release of the ligand by the intracellular cleavage of the enzyme-labile spacer, as observed in cathepsin B enzymatic assays. Therefore, these results pave the way for the design of novel peptide conjugates to be used in pro-oxidant anticancer therapies.