α-Conotoxins are well-known probes for the characterization of the various subtypes of nicotinic acetylcholine receptors (nAChRs). Identifying new α-conotoxins with different pharmacological profiles can provide further insights into the physiological or pathological roles of the numerous nAChR isoforms found at the neuromuscular junction, the central and peripheral nervous systems, and other cells such as immune cells. This study focuses on the synthesis and characterization of two novel α-conotoxins obtained from two species endemic to the Marquesas Islands, namely Conus gauguini and Conus adamsonii. Both species prey on fish, and their venom is considered a rich source of bioactive peptides that can target a wide range of pharmacological receptors in vertebrates. Here, we demonstrate the versatile use of a one-pot disulfide bond synthesis to achieve the α-conotoxin fold [Cys 1-3; 2-4] for GaIA and AdIA, using the 2-nitrobenzyl (NBzl) protecting group of cysteines for effective regioselective oxidation. The potency and selectivity of GaIA and AdIA against rat nicotinic acetylcholine receptors were investigated electrophysiologically and revealed potent inhibitory activities. GaIA was most active at the muscle nAChR (IC₅₀ = 38 nM), whereas AdIA was most potent at the neuronal α6/3 β2β3 subtype (IC₅₀ = 177 nM). Overall, this study contributes to a better understanding of the structure–activity relationships of α-conotoxins, which may help in the design of more selective tools.

Understanding the determinants of α-conotoxin (α-CTX) selectivity for different nicotinic acetylcholine receptor (nAChR) subtypes is a requisite for the design of tool compounds to study nAChRs. However, selectivity optimization of these small, disulfide rich peptides is difficult not only because of an absence of α-CTX/nAChR co-structures, but also because it is challenging to predict how a mutation to an α-CTX will alter its potency and selectivity. As a prototypical system to investigate selectivity, we employed the α-CTX LvIA that is 18-fold selective for the α3β2 nAChR over the closely related α3β4 nAChR subtype that is a target for nicotine addiction. Using two-electrode voltage clamp electrophysiology, we identified LvIA[D11R] that is 2-fold selective for the α3β4 nAChR, reversing its subtype preference. This effect is specific to the charge and not shape of LvIA[D11R], as substitution with citrulline retains selectivity for the α3β2 nAChR. Furthermore, LvIA[D11K] shows a stronger reversal, with 4-fold selectivity for the α3β4 nAChR. Motivated by these findings, using site-directed mutagenesis it was found that β2[K79A], but not β2[K78A], largely restores the antagonism of basic mutants at position 11. Finally, to understand the structural basis of this effect we used AlphaFold2 to generate models of LvIA in complex with both nAChR subtypes. Both models confirm the plausibility of an electrostatic mechanism to explain the data and also reproduce a broad range of potency and selectivity structure-activity relationships for LvIA mutants, as measured using free-energy perturbation simulations. Our work highlights how electrostatic interactions can drive α-CTX selectivity and may prove useful as a strategy for optimizing the selectivity of LvIA and other ⍺-CTXs.