A unique mid-sequence linker used to multimerize the lipid-phosphatidylserine (PS) binding peptide-peptoid hybrid PPS1

Abstract

Ligand multimerizations enhance the binding affinity towards cell surface biomarkers through their avidity effects. Typical linkers connect individual monomeric ligand moieties from one end (e.g., C- or N-terminus of a peptide) and exclusively target protein receptors. The lipid phosphatidylserine (PS) is normally present on the cytoplasmic side of the eukaryotic cell membrane, but in tumors and tumor endothelial cells, this negatively charged PS flips to the outer layer. We recently reported a PS binding peptide-peptoid hybrid (PPS1) that has distinct positively charged and hydrophobic residue-containing regions. The PPS1 monomer is inactive, and upon C-terminal dimerization (PPS1D1), it triggers cytotoxicity. In the current study, a unique series of PPS1 multimeric derivatives were synthesized by switching the linker from the C-terminus to an internal position. The unimportant fourth residue (N-lys) from the C-terminus was utilized to build the linker. The synthesis strategy was developed employing variations of (I) the linker size, (II) the number of positively charged residues, and (III) the number of hydrophobic regions. Cytotoxicity of these new derivatives on HCC4017 lung cancer cells showed that a minimum of two hydrophobic regions was important to retain the activity and that the shortest linker length was optimal for activity.

Keywords: Multimers; Peptoids; Sequence modifications.

Figure 1

(A) Chemical structure and pictorial representation of PPS1D1 homodimers connected to each other at the C-terminus. (B) Pictorial representation of current strategy, involving positively charged peptide residues separated from hydrophobic peptoids via the mid-sequence linker instead of the C-terminal linker.

Figure 2

Transfer of the linker to the middle (A) Chemical structure of PPS1-4P2H1. (B) MTS cell viability assay results in HCC4017 cells treated with PPS1D1, PPS1, PC462D1, and PPS1-4P2H1 respectively.

Figure 3

The effects of the mid-sequence linker length. (A) and (B) Chemical structures of PPS1-4P2H2 and PPS1-4P2H3 having mid-sequence linkers of variable lengths (C) The MTS cell viability assay results in HCC4017 cells treated with PPS1D1, PPS1, PC462D1, PPS1-4P2H2, and PPS1-4P2H3 respectively.

Figure 4

The effect of reducing number of hydrophobic regions. (A) Chemical structure of compound PPS1-4P1H with one hydrophobic region. (B) MTS cell viability assay results on HCC4017 cells treated with PPS1D1, PPS1, PC462D1, and PPS1-4P1H respectively.

Figure 5

The effect of increased number of hydrophobic regions and changes to the positively charged region. (A) and (B) the chemical structures of PPS1-2P3H and PPS1-4P3H, respectively. (C) MTS cell viability assay results for HCC4017 cells treated with PPS1D1, PPS1, PC462D1, PPS1-2P3H, and PPS1-4P3H. (D) Cell viability calcein AM activity determination for HCC4017 cells treated with PPS1D1, PPS1, PC462D1, PPS1-2P3H, and PPS1-4P3H respectively.

Scheme 1. Synthesis route for PPS1-4P2H1 containing the mid-sequence linker

Scheme 2

Synthesis scheme for PPS1-4P3H having two positive regions and three hydrophobic regions in the moiety.