Design, synthesis and antitumor activity of Ascaphin-8 derived stapled peptides based on halogen-sulfhydryl click chemical reactions

Abstract

Ascaphin-8 (GFKDLLKGAAKALVKTVLF-NH₂), isolated from the norepinephrine-stimulated skin secretion of the North American-tailed frog Ascaphus truei, is a C-terminal α-helical antimicrobial peptide with potential antitumor activity. However, linear peptides are difficult to be applied directly as drugs because of their inherent defects, such as low hydrolytic enzyme tolerance and poor structural stability. In this study, we designed and synthesized a series of stapled peptides based on Ascaphin-8 via thiol-halogen click chemistry. Most of the stapled peptide derivatives showed enhanced antitumor activity. Among them, A8-2-o and A8-4-Dp had the most improved structural stability, stronger hydrolytic enzyme tolerance and highest biological activity. This research may provide a reference for the stapled modification of other similar natural antimicrobial peptides.

Fig. 1. Structures of Ascaphin-8 and stapled derivatives. The key residues are colored blue. The Cys residues are colored red and two Cys were cross-linked by dibromo substituted benzene.

Scheme 1. Synthesis route of A8-1-p. The following protecting groups for amino acids side chains were used: tert-butoxycarbonyl (Boc; for Lys), tert-butyl ester (OtBu, for Asp) and tert-butyl (tBu; for Thr).

Fig. 2. CD spectra of Ascaphin-8 and its derived peptide.

Fig. 3. (A) Detection of haemolysis of rabbit erythrocytes by aromatic thioether staple peptide. (B) Detection of anti-enzymatic hydrolysis ability of Ascaphin-8, A8-2-o and A8-4-Dp. (C) Effects of Ascaphin-8, A8-2-o, and A8-4-Dp on lateral migration and growth of MCF-7 cells. (D) Calculation of crack area reduction. (E) Effects of Ascaphin-8, A8-2-o, and A8-4-Dp on vertical migration capacity of MCF-7 cells, (F) calculation of cell numbers that passed through the polycarbonate membrane. Scale: 200 μm (data are expressed as mean ± standard deviation; n = 3; compared with the control group, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 4. (A) Detection of apoptosis effects of Ascaphin-8, A8-2-o, and A8-4-Dp (10 μM) on MCF-7 cells. (B) Fluorescent images of life-and-death staining of MCF-7 cells treated with Ascaphin-8, A8-2-o and A8-4-Dp.