A single stable topological stereoisomer of a two disulfide conotoxin mimetic demonstrates full potency on the human norepinephrine transporter

Conotoxin χ Mr1a which is a 13 residue conotoxin which has two disulfide bonds in the ribbon fold is found in the venom of the predatory marine snail, C. marmoreus. It noncompetitively inhibits the human norepinephrine transporter (hNET) leading to reduced allodynia in animal models of neuropathic pain. Rational design was used to prune conotoxin χMr1a while maintaining the armacophore that led to the development of an equipotent mimetic (IC 50 : 760 nM). Surprisingly, oxidative folding of the reduced linear peptide led to four rather than the expected three isoforms. Detailed investigation led to the discovery of two stereotopological isoforms one of which in the globular fold was >1000 fold more potent than the other isoform both in vitro and in vivo in an allodynia mouse mode. Stereotopological isoforms have never been reported for conotoxins and represent an additional level of structural complexity with implications for rational design, chemical synthesis and structural diversity of cysteine rich peptides.