Structural Superimposition

Abstact

The chi-conotoxins are venom-derived peptides that specifically target the noradrenaline transporter (also known as norepinephrine transporter, NET). Regulation of noradrenergic signaling by NET affects neurophysiological processes, including pain. Therefore, the chi-conotoxin MrIA and its synthetic analogs have been previously investigated for their analgesic activity. Here we describe the synthesis and pharmacological characterization of chi-AoIA, a peptide that selectively inhibits NET with a higher potency compared to MrIA in in vitro radiotracer flux assays. Furthermore, we resolved the structure of the human NET:chi-AoIA complex by cryogenic electron microscopy, which revealed an atypical binding mode consisting of both the central binding site and the outer vestibule of the transporter. Lastly, chi-AoIA displays antinociceptive efficacy in a model of inflammatory pain after subcutaneous administration in mice. Our results demonstrate the efficacy of chi-AoIA as a highly selective ligand of NET and provide a mechanistic basis for its potential development as a nonopioid analgesic.