Cloning, synthesis, and characterization of αO-conotoxin GeXIVA, a potent α9α10 nicotinic acetylcholine receptor antagonist

• Published in: Proceedings of the National Academy of Sciences - PNAS 2015, Vol.112 (30), p.E4026-E4035
• Main Authors: Luo, Sulan, Dongting Zhangsun, Peta J. Harvey, Quentin Kaas, Yong Wu, Xiaopeng Zhu, Yuanyan Hu, Xiaodan Li, Victor I. Tsetlin, Sean Christensen, Haylie K. Romero, Melissa McIntyre, Cheryl Dowell, James C. Baxter, Keith S. Elmslie, David J. Craik, J. Michael McIntosh
• Format: Article
• Language: English
• Subjects: analgesics; antagonists; breast neoplasms; cholinergic receptors; medicinal properties; nicotinic; NMR; pain; peptides; α9α10 nAChR; αO-conotoxin GeXIVA
• ISSN: 0027-8424; 1091-6490

We identified a previously unidentified conotoxin gene from Conus generalis whose precursor signal sequence has high similarity to the O1-gene conotoxin superfamily. The predicted mature peptide, αO-conotoxin GeXIVA (GeXIVA), has four Cys residues, and its three disulfide isomers were synthesized. Previously pharmacologically characterized O1-superfamily peptides, exemplified by the US Food and Drug Administration-approved pain medication, ziconotide, contain six Cys residues and are calcium, sodium, or potassium channel antagonists. However, GeXIVA did not inhibit calcium channels but antagonized nicotinic AChRs (nAChRs), most potently on the α9α10 nAChR subtype (IC ₅₀ = 4.6 nM). Toxin blockade was voltage-dependent, and kinetic analysis of toxin dissociation indicated that the binding site of GeXIVA does not overlap with the binding site of the competitive antagonist α-conotoxin RgIA. Surprisingly, the most active disulfide isomer of GeXIVA is the bead isomer, comprising, according to NMR analysis, two well-resolved but uncoupled disulfide-restrained loops. The ribbon isomer is almost as potent but has a more rigid structure built around a short 3 ₁₀-helix. In contrast to most α-conotoxins, the globular isomer is the least potent and has a flexible, multiconformational nature. GeXIVA reduced mechanical hyperalgesia in the rat chronic constriction injury model of neuropathic pain but had no effect on motor performance, warranting its further investigation as a possible therapeutic agent. The α9α10 nicotinic AChR (nAChR) subtype is a recently identified target for the development of breast cancer chemotherapeutics and analgesics, particularly to treat neuropathic pain. Structure/function analyses of antagonists of this subtype are therefore essential for the development of specific therapeutic compounds. The Conus genus is a rich source of pharmacologically active peptides, and we report here that the αO-conotoxin GeXIVA is a potent and selective antagonist of the α9α10 nAChR subtype. GeXIVA displays unique structural properties among other Conus peptides and represents a previously unidentified template for molecules active against neuropathic pain.