Alanine Scan of alpha-conotoxin RegIIA reveals a selective alpha 3 beta 4 nicotinic acetylcholine receptor antagonist

Activation of the alpha 3 beta 4 nicotinic acetylcholine receptor (nAChR) subtype has recently been implicated in the pathophysiology of various conditions, including development and progression of lung cancer and in nicotine addiction. As selective alpha 3 beta 4 nAChR antagonists, alpha-conotoxins are valuable tools to evaluate the functional roles of this receptor subtype. We previously reported the discovery of a new beta 4/7-conotoxin, RegIIA. RegIIA was isolated from Conus regius and inhibits acetylcholine (ACh)-evoked currents mediated by alpha 3 beta 4, alpha 3 beta 2, and alpha 7 nAChR subtypes. The current study used alanine scanning mutagenesis to understand the selectivity profile of RegIIA at the alpha 3 beta 4 nAChR subtype. [N11A] and [N12A] RegIIA analogs exhibited 3-fold more selectivity for the alpha 3 beta 4 than the alpha 3 beta 2 nAChR subtype. We also report synthesis of [N11A, N12A] RegIIA, a selective alpha 3 beta 4 nAChR antagonist (IC50 of 370 nM) that could potentially be used in the treatment of lung cancer and nicotine addiction. Molecular dynamics simulations of RegIIA and [N11A, N12A] RegIIA bound to alpha 3 beta 4 and alpha 3 beta 2 suggest that destabilization of toxin contacts with residues at the principal and complementary faces of alpha 3 beta 2 (alpha 3-Tyr(92), Ser(149), Tyr(189), Cys(192), and Tyr(196); beta 2-Trp(57), Arg(81), and Phe(119)) may form the molecular basis for the selectivity shift