Potency- and Selectivity-Enhancing Mutations of Conotoxins for Nicotinic Acetylcholine Receptors Can Be Predicted Using Accurate Free-Energy Calculations

Nicotinic acetylcholine receptor (nAChR) subtypes are key drug targets, but it is challenging to pharmacologically differentiate between them because of their highly similar sequence identities. Furthermore, alpha-conotoxins (alpha-CTXs) are naturally selective and competitive antagonists for nAChRs and hold great potential for treating nAChR disorders. Identifying selectivity-enhancing mutations is the chief aim of most alpha-CTX mutagenesis studies, although doing so with traditional docking methods is difficult due to the lack of alpha-CTX/nAChR crystal structures. Here, we use homology modeling to predict the structures of alpha-CTXs bound to two nearly identical nAChR subtypes, alpha 3 beta 2 and alpha 3 beta 4, and use free-energy perturbation (FEP) to re-predict the relative potency and selectivity of alpha-CTX mutants at these subtypes. First, we use three available crystal structures of the nAChR homologue, acetylcholine-binding protein (AChBP), and re-predict the relative affinities of twenty point mutations made to the alpha-CTXs LvIA, LsIA, and GIC, with an overall root mean square error (RMSE) of 1.08 +/- 0.15 kcal/mol and an R-2 of 0.62, equivalent to experimental uncertainty. We then use AChBP as a template for alpha 3 beta 2 and alpha 3 beta 4 nAChR homology models bound to the alpha-CTX LvIA and re-predict the potencies of eleven point mutations at both subtypes, with an overall RMSE of 0.85 +/- 0.08 kcal/mol and an R-2 of 0.49. This is significantly better than the widely used molecular mechanics-generalized born/surface area (MM-GB/SA) method, which gives an RMSE of 1.96 +/- 0.24 kcal/mol and an R-2 of 0.06 on the same test set. Next, we demonstrate that FEP accurately classifies alpha 3 beta 2 nAChR selective LvIA mutants while MM-GB/SA does not. Finally, we use FEP to perform an exhaustive amino acid mutational scan of LvIA and predict fifty-two mutations of LvIA to have greater than 100X selectivity for the alpha 3 beta 2 nAChR. Our results demonstrate the FEP is well-suited to accurately predict potency- and selectivity-enhancing mutations of alpha-CTXs for nAChRs and to identify alternative strategies for developing selective alpha-CTXs.

Automated summary

Nicotinic acetylcholine receptor (nAChR) subtypes are challenging to differentiate pharmacologically due to highly similar sequences; alpha-conotoxins (alpha-CTXs) are natural selective antagonists with potential for nAChR disorders; homology modeling and free-energy perturbation (FEP) predict mutations to enhance selectivity and potency of alpha-CTXs for nAChRs effectively.