PeIA-5466: A Novel Peptide Antagonist Containing Non-natural Amino Acids That Selectively Targets α3β2 Nicotinic Acetylcholine Receptors

Pharmacologically distinguishing alpha 3 beta 2 nicotinic acetylcholine receptors (nAChRs) from closely related subtypes, particularly alpha 6 beta 2, has been challenging due to the lack of subtype-selective ligands. We created analogs of alpha-conotoxin (alpha-Ctx) PeIA to identify ligand receptor interactions that could be exploited to selectively increase potency and selectivity for alpha 3 beta 2 nAChRs. A series of PeIA analogs were synthesized by replacing amino acid residues in the second disulfide loop with standard or nonstandard residues and assessing their activity on alpha 3 beta 2 and alpha 6/alpha 3 beta 2 beta 3 nAChRs heterologously expressed in Xenopus laevis oocytes. Asparagine(11) was found to occupy a pivotal position, and when replaced with negatively charged amino acids, selectivity for alpha 3 beta 2 over alpha 6/alpha 3 beta 2 beta 3 nAChRs was substantially increased. Second generation peptides were then designed to further improve both potency and selectivity. One peptide, PeIA-5466, was similar to 300-fold more potent on alpha 3 beta 2 than alpha 6/alpha 3 beta 2 beta 3 and is the most alpha 3 beta 2-selective antagonist heretofore reported.