N‑Terminal Capping of the αO-Conotoxin Analogue GeX‑2 Improves the Serum Stability and Selectivity toward the Human α9α10 Nicotinic Acetylcholine Receptor

α9α10 nicotinic acetylcholine receptors (nAChRs)
are
a promising nonopioid analgesic target, with α9α10 nAChR
antagonists showing efficacy against chemotherapy-induced hyperalgesia
and allodynia. GeX-2, a potent analgesic conotoxin antagonist of α9α10
nAChRs, has limited serum stability. This study improved GeX-2 stability
by capping its N-terminal with fatty acids or polyethylene glycol
chains, which enhanced its serum stability but eliminated activity
at G protein-coupled γ-aminobutyric acid type B (GABAB) receptor-coupled CaV2.2 channels while preserving activity
at α9α10 nAChRs. In vivo, α9α10 nAChRs antagonism
alone did not alleviate neuropathic pain, highlighting the importance
of GABAB receptor-coupled CaV2.2 channels in
GeX-2’s antinociceptive effects in the chronic constriction
injury rat model. The GeX-2 analogue, with an N-terminal methyl group,
showed improved activity and selectivity for α9α10 nAChRs,
increased serum half-life, and strong analgesic effects in oxaliplatin-induced
cold allodynia models. AlphaFold3 and molecular dynamics simulations
provided insights into the binding modes and the effects of N-terminal
capping, which informed future peptide therapeutic developments.