Abstract

The venom toxin δ-ctenitoxin-Pn2a of the spider Phoneutria nigriventer can cause severe envenomation in humans. Furthermore, the cystine-knot motif of δ-ctenitoxin-Pn2a provides exceptional stability, thereby hampering immune response activation. Here we identified epitope G³⁴YFWIAWYKLANCKK⁴⁸ from δ-ctenitoxin-Pn2a through the Immune Epitope Database Analysis Resource and used it to design antigenic peptides. The Cys residue was replaced by α-aminobutyric acid (Abu) to prevent disulfide bond formation. To increase the immunogenicity of these molecules, branched and N-palmitoylated versions were synthesized. Ac-GYFWIAWYKLAN-Abu-KKG-NH₂ (A), Palm-GYFWIAWYKLAN-Abu-KKG-NH₂ (B) and (Ac-GYFWIAWYKLAN-Abu-KK)₂-KG-NH₂ (C) were prepared by solid-phase synthesis and their identity was confirmed by ESI-MS. They were then studied by RP-HPLC and all the chromatograms obtained showed only one main peak. Cytotoxicity was evaluated on the murine macrophage cell line RAW 264.7 using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay in the presence of increasing doses of each peptide (0.25–10.0 µM). Peptide A did not show cytotoxicity between 0.25 and 10.0 µM, while B and C did at concentrations equal or over 0.5 and 10.0 respectively. The cellular distribution of NF-κB was examined by immunofluorescence after exposing macrophages to 0.5 µM of each peptide. Early activation was observed for all three peptides, thereby indicating that they are promising immunogens for antivenom production. Nevertheless, in vivo tests are still required to assess their immunogenic capacity and whether the antibodies generated can confer protection against the venom.

Keywords: Delta-CNTX-Pn2a; PnTx2-6; Neurotoxin Tx2-6; palmitoylated peptide; envenoming; arachnids; solid-phase peptide synthesis