Aspartic Acid Mutagenesis of Αo-Conotoxin GeXIVA Isomers Reveals Arginine Residues Crucial for Inhibition of the Α9α10 Nicotinic Acetylcholine Receptor

The two most active disulfide bond isomers of the analgesic alpha O-conotoxin GeXIVA, namely GeXIVA[1, 2] and GeXIVA[1, 4], were subjected to Asp -scanning mutagenesis to determine the key amino acid residues for activity at the rat alpha 9 alpha 10 nicotinic acetylcholine receptor (nAChR). These studies revealed the key role of arginine residues for the activity of GeXIVA isomers towards the alpha 9 alpha 10 nAChR. Based on these results, additional analogues with 2-4 mutations were designed and tested. The analogues [T1A,D14A,V28K]GeXIVA[1, 2] and [D14A,I23A, V28K]GeXIVA[1, 4] were developed and showed sub-nanomolar activity for the alpha 9 alpha 10 nAChR with IC 50 values of 0.79 and 0.38 nM. The latter analogue had exceptional selectivity for the alpha 9 alpha 10 receptor subtype over other nAChR subtypes and can be considered as a drug candidate for further development. Molecular dynamics of receptor-ligand complexes allowed us to make deductions about the possible causes of increases in the affinity of key GeXIVA[1, 4] mutants for the alpha 9 alpha 10 nAChR.