Hydrophobic and Polarized Aromatic Residues Promote Internalization of Arg-Rich Cell-Penetrating Peptides through Ionpair-π Interactions

Abstract

Cell penetrating peptides (CPPs) are small sequences that can cross cell membranes. Arg and Trp are highly prevalent amino acids in natural and synthetic efficient CPP sequences. In particular, Trp is essential and cannot be substituted by other hydrophobic or aromatic amino acids. The aim of the present study is to decipher the role of Trp in synthetic Arg/Trp CPP sequences. To do so, a small peptide library in which this residue was substituted by other natural or nonnatural amino acids was designed. Internalization of these peptides in cells was evaluated, and it appeared that combining aromaticity and hydrophobicity in the presence of Arg residues leads to enhanced internalization. The study of the interaction of these peptides with model lipid membranes revealed that the modulation of hydrophobicity promoted insertion in bilayers but had little impact on the binding affinity. On the other hand, more hydrophobic substitutes of Trp led to more favorable binding enthalpies to heparin. With density functional theory (DFT) analysis, we suggest that ion-pair···π interactions between the aromatic ring and the ion pair formed by the positively charged Arg and the negatively charged cell surface groups can be established and could be at the origin of the unique internalization properties of Trp-containing Arg-rich CPPs.

Keywords: DFT; cell‐penetrating peptides; glycosaminoglycans; tryptophan.

Figure 1

Peptide library. A) Peptide sequence, X being different natural or nonnatural L‐amino acids. B) Peptide list with the structure of the side chain on X, color‐coded from the most hydrophilic (green) to the most hydrophobic (red) sequence, and chemical properties.

Figure 2

Quantification of peptide internalization for an extracellular concentration of 10 µM and 1 million A) CHO‐K1 cells and B) CHO‐pgsA 745. Values were averaged from three independent experiments performed in triplicate. Error bars represent the standard error of the mean. Peptides were classified as poorly internalized (“Low” grey shading) when the amount internalized was less than half of that of 1, highly internalized (“High” grey shading) when more than twice the amount of 1, and moderately internalized (“Medium” grey shading) in between.

Figure 3

Interactions of R₆X₃ with anionic lipids studied by ITC A) and DSC B,C). A) Thermodynamic parameters obtained by ITC are averaged over two independent experiments. B) DSC thermograms are given for a peptide‐to‐lipid ratio (P/L) of 1/50; the thermogram obtained for pure DMPG is shown in grey; the thermograms were offset for easier comparisons. C) Thermodynamic parameters associated with the main transition on a heating scan. T_M’ indicates the second maximum of the transition peak when splitting was observed.

Figure 4

Interactions of peptides with HI studied by ITC. A) Thermodynamic parameters were obtained by titrating HI into the different peptides. Values are averaged over two experiments. B) Relationship between amounts internalized and binding enthalpies.

Figure 5

MEP surfaces of the side chain of W A), 1ForW B), 3BT C), and Nal D). Energies at selected points in kcal/mol.

Figure 6

Theoretical modes of GAG, POPG, and CPPs used for simulations.

Figure 7

Optimized geometries of 2′···POPG_red A), 2′···GAG_dim B), 6′···POPG_red C), and 6′···GAG_dim D) with indication of the ion‐pair (top) and ion‐pair···π interactions (bottom). The C‐atoms of the peptide motifs are colored blue, and those of POPG_red and GAG_dim are colored green. The ion‐pair distances are i) 3.406Å (O···C) in 2′···POPG_red complex and ii) 3.785Å (O···C) and 3.727Å (C···C) in the 2′···GAG_dim complex.

Figure 8

Optimized geometries of 9′···POPG_red A), 9′···GAG_dim B), 11′···POPG_red C), and 11′···GAG_dim D) with indication of the ion‐pair···π interactions. The C‐atoms of the peptide motifs are colored blue, and those of POPG_red and GAG_dim are colored green.

Figure 9

Model ion‐pair···π complexes analyzed in this work along with the interaction energies and interplane distances (d).