A synthetic combinatorial strategy for developing α-conotoxin analogs as potent α₇ nicotinic acetylcholine receptor antagonists

α-Conotoxins are peptide neurotoxins isolated from venomous cone snails that display exquisite selectivity for different subtypes of nicotinic acetylcholine receptors (nAChR). They are valuable research tools that have profound implications in the discovery of new drugs for a myriad of neuropharmacological conditions. They are characterized by a conserved two-disulfide bond framework, which gives rise to two intervening loops of extensively mutated amino acids that determine their selectivity for different nAChR subtypes. We have used a multistep synthetic combinatorial approach using α-conotoxin ImI to develop potent and selective α₇ nAChR antagonists. A positional scan synthetic combinatorial library was constructed based on the three residues of the n-loop of α-conotoxin ImI to give a total of 10,648 possible combinations that were screened for functional activity in anα₇ nAChR Fluo-4/Ca²⁺ assay, allowing amino acids that confer antagonistic activity for this receptor to be identified. A second series of individual α-conotoxin analogs based on the combinations of defined active amino acid residues from positional scan synthetic combinatorial library screening data were synthesized. Several analogs exhibited significantly improved antagonist activity for the α₇ nAChR compared with WT-ImI. Binding interactions between the analogs and the α₇ nAChR were explored using a homology model of the amino-terminal domain based on a crystal structure of an acetylcholine-binding protein. Finally, a third series of refined analogs was synthesized based on modeling studies, which led to several analogs with refined pharmacological properties. Of the 96 individual α-conotoxin analogs synthesized, three displayed ≥10-fold increases in antagonist potency compared with WT-ImI.