Cyclisation increases the stability of the sea anemone peptide APETx2 but decreases its activity at acid-sensing ion channel 3

Abstract

APETx2 is a peptide isolated from the sea anemone Anthopleura elegantissima. It is the most potent and selective inhibitor of acid-sensing ion channel 3 (ASIC3) and it is currently in preclinical studies as a novel analgesic for the treatment of chronic inflammatory pain. As a peptide it faces many challenges in the drug development process, including the potential lack of stability often associated with therapeutic peptides. In this study we determined the susceptibility of wild-type APETx2 to trypsin and pepsin and tested the applicability of backbone cyclisation as a strategy to improve its resistance to enzymatic degradation. Cyclisation with either a six-, seven- or eight-residue linker vastly improved the protease resistance of APETx2 but substantially decreased its potency against ASIC3. This suggests that either the N- or C-terminus of APETx2 is involved in its interaction with the channel, which we confirmed by making N- and C-terminal truncations. Truncation of either terminus, but especially the N-terminus, has detrimental effects on the ability of APETx2 to inhibit ASIC3. The current work indicates that cyclisation is unlikely to be a suitable strategy for stabilising APETx2, unless linkers can be engineered that do not interfere with binding to ASIC3.

Keywords: APETx2; ASIC3; cyclisation; peptide; sea anemone; stability; truncation.

Figure 1

Wild-type APETx2 and truncation analogues were made in a two-step approach whereby the two peptide chains were synthesised separately then joined via NCL. The cyclic analogues were synthesised as single peptide and cyclised using NCL.

Figure 2

(A) SOFAST-HMQC spectrum of APETx2_wt (black) overlaid with that of APETx2_1–39 (red). A crosspeak is present for each H-N bond (i.e., for each non-proline residue), including non-labile sidechain NH groups (Trp Nε-Hε, Asn Nδ-Hδ2, Gln Nε-Hε and Arg Nε-Hε). (B) Backbone ¹⁵N chemical shifts for APETx2_wt (black), APETx2_1–39 (red) and APETx2_3–42 (cyan).

Figure 3

(A) Overlaid HMQC spectra of APETx2_wt (black) and cAPETx2_6RL (green). (B) Overlay of the backbone ¹⁵N chemical shifts of APETx2_wt (black) and cyclic APETx2 with either a 6-residue (green), 7-residue (maroon), or 8‑residue (blue) linker.

Figure 4

Decay curves showing the stability of APETx2_wt (black), cAPETx2_6RL (green), cAPETx2_7RL (brown), and cAPETx2_8RL (blue) against proteolytic cleavage by (A) trypsin and (B) pepsin.

Figure 5

Comparison of the activity of wild-type APETx2 with cyclic and truncation analogues. Concentration-effect curves are shown for inhibition of rASIC3 currents by APETx2 wt (black), APETx2_1–39 (red) and APETx2_3–42 (cyan) (n = 6). The activity of the cyclic APETx2 analogues is shown only at 1 µM (n = 6).

Figure 6

(A) Surface representation of APETx2 highlighting residues potentially involved in its interaction with ASIC3: Cluster 1 (residues A3, S5, N8, K10, T39 and A41) is coloured red and Cluster 2 (residues Y16, R17, P18, R31 and T36) is coloured cyan. The N- and C-termini are coloured yellow and violet, respectively. Arg17 is coloured dark blue. (B) Surface representation of homology model of cAPETx2_6RL showing the six-residue linker in green. Potential residues interacting with ASIC3 are highlighted as for (A).