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

Abstract

Peptide conjugates incorporating the N-based ligands (Me2)PyTACN or (S,S')-BPBP at the N- or the C-terminus of the cell-penetrating peptide were synthesized (PyTACN-BP16 (), BP16-PyTACN (), BPBP-BP16 (), and BP16-BPBP ()). Metal binding peptides bearing at the N-terminus the ligand, an additional Lys and a β-Ala were also prepared (PyTACN-βAK-BP16 () and BPBP-βAK-BP16 ()). Moreover, taking into account the clathrin-dependent endocytic mechanism of , the enzymatic cleavable tetrapeptide Gly-Phe-Leu-Gly was incorporated between the ligand and the N- or C-terminus of (BPBP-GFLG-BP16 () and BP16-GLFG-BPBP ()). 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 (Me2)PyTACN, and (iv) the introduction of the cleavable tetrapeptide significantly enhanced the activity of the BPBP conjugates (IC50 of 4.3 to 11.7 μM ( and ) compared to 26.0 to >50 μM (, and )). The most active peptide was BPBP-GFLG-BP16 () (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.

Reducing Dendrimer Generation and PEG Chain Length Increases Drug Release and Promotes Anticancer Activity of PEGylated Polylysine Dendrimers Conjugated with Doxorubicin via a Cathepsin-Cleavable Peptide Linker

“…These constructs displayed good anticancer activity in rodent models of primary breast and secondary lung cancer but also showed some evidence of “burst” drug release. ,, Peptide-based linkers that are explicitly cleavable by enzymes that are overexpressed within tumors are expected to provide better control over in vivo drug liberation. For example, cathepsin B expression is upregulated in many solid tumors and is correlated with an invasive phenotype, but it is constitutively expressed at only low levels in noncancerous tissues. − The peptides glycine–leucine–phenylalanine–glycine (GLFG) and valine–citrulline (VCit) are specifically cleavable by cathepsin B and have been used previously with a good effect to conjugate chemotherapeutic drugs to antibodies and drug carriers. − In light of these data, we therefore sought to engineer PEGylated polylysine dendrimers, where Dox was conjugated to the dendrimer surface via a VCit linker. Drug liberation from peptide linkers such as this, however, relies on enzyme access, and the dendrimer structure and extent of surface PEGylation might be expected to sterically restrict enzyme access to the peptide and limit drug release and antitumor activity.…”

Chemical Modification of Cytochrome C for Acid-Responsive Intracellular Apoptotic Protein Delivery for Cancer Eradication

“…The cell-penetrating peptide BP16 (Fmoc-GCKKLFKKILKKL-CONH 2 ) [ 9 ], to which cysteine was added at the N-terminus for conjugation purposes, was synthesised using a 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis protocol with commercially available N-α-Fmoc-protected amino acids. The CEM Liberty Blue Automated Microwave Peptide Synthesiser was used for synthesis, with rink amide resin as the solid support.…”

“…The second approach employs cell-penetrating peptides (CPPs) attached onto the surface of nanocarriers to facilitate direct penetration of the membrane, delivering the protein cargo into the cytoplasm or even the nucleus [ 6 ]. Cell-penetrating peptides (CPPs) are short peptides (5–30 amino acids) that possess the unique ability to translocate across the cell membrane while preserving membrane integrity, exhibiting minimal invasiveness, and demonstrating low cytotoxicity [ 7 , 8 , 9 ]. The versatility of CPPs allows them to transport a range of cargo molecules, including peptides, proteins, nucleic acids, small molecules, and nanoparticles [ 10 , 11 , 12 , 13 , 14 , 15 ].…”

Doxorubicin Conjugation and Drug Linker Chemistry Alter the Intravenous and Pulmonary Pharmacokinetics of a PEGylated Generation 4 Polylysine Dendrimer in Rats

“…A series of G4 dendrimers conjugated with PEG570 and doxorubicin (20-30 kDa in size) were synthesised based on previous data showing that this scaffold construction offers both prolonged systemic exposure and good lung absorption 2,3 (see figure in supporting information). Three peptide-based drug linkers that are specifically cleavable by cathepsin B were used to conjugate doxorubicin to this dendrimer scaffold: a pentapeptide (Glu-GLFG 4 ) that results in the liberation of both peptide-modified and unmodified doxorubicin, and two self-emolative systems (diglycolic acid-GLFG-para-amino benzoic acid 4 and diglycolic acid-Valine-Citrulline (VCit)-para-amino benzoic acid 5 ) that facilitate the liberation of unmodified doxorubicin. Cathepsin B-cleavable peptides were used since the extracellular and lysosomal expression of this enzyme is highly upregulated by cancer cells, particularly those at the invasive front of a tumour, but is constitutively expressed at low levels in normal tissue [6][7][8] .…”