Alanine Scan of α-Conotoxin RegIIA Reveals a Selective α3β4 Nicotinic Acetylcholine Receptor Antagonist

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Activation of the α3β4 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 α3β4 nAChR antagonists, α-conotoxins are valuable tools to evaluate the functional roles of this receptor subtype. We previously reported the discovery of a new α4/7-conotoxin, RegIIA. RegIIA was isolated from Conus regius and inhibits acetylcholine (ACh)-evoked currents mediated by α3β4, α3β2, and α7 nAChR subtypes. The current study used alanine scanning mutagenesis to understand the selectivity profile of RegIIA at the α3β4 nAChR subtype. [N11A] and [N12A] RegIIA analogs exhibited 3-fold more selectivity for the α3β4 than the α3β2 nAChR subtype. We also report synthesis of [N11A,N12A]RegIIA, a selective α3β4 nAChR antagonist (IC 50 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 α3β4 and α3β2 suggest that destabilization of toxin contacts with residues at the principal and complementary faces of α3β2 (α3-Tyr 92 , Ser 149 , Tyr 189 , Cys 192 , and Tyr 196 ; β2-Trp 57 , Arg 81 , and Phe 119 ) may form the molecular basis for the selectivity shift.The molecular mechanism by which α-conotoxin RegIIA inhibits α3β4, α3β2, and α7 nAChRs is unknown. Results Alanine scanning mutagenesis and molecular dynamic simulations of RegIIA revealed Asn 11 and Asn 12 confer improved selectivity at α3β4 nAChR. Conclusion We synthesized the [N11A,N12A]RegIIA analog that selectively inhibits α3β4. Significance These findings could be used to develop α3β4-selective drugs to treat lung cancer.