Abstract
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, α-conotoxins (α-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 α-CTX mutagenesis studies, although doing so with traditional docking methods is difficult due to the lack of α-CTX/nAChR crystal structures. Here, we use homology modeling to predict the structures of α-CTXs bound to two nearly identical nAChR subtypes, α3β2 and α3β4, and use free-energy perturbation (FEP) to re-predict the relative potency and selectivity of α-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 α-CTXs LvIA, LsIA, and GIC, with an overall root mean square error (RMSE) of 1.08 ± 0.15 kcal/mol and an R2 of 0.62, equivalent to experimental uncertainty. We then use AChBP as a template for α3β2 and α3β4 nAChR homology models bound to the α-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 R2 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 R2 of 0.06 on the same test set. Next, we demonstrate that FEP accurately classifies α3β2 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 α3β2 nAChR. Our results demonstrate the FEP is well-suited to accurately predict potency- and selectivity-enhancing mutations of α-CTXs for nAChRs and to identify alternative strategies for developing selective α-CTXs.
Highlights
This study expands the domain of applicability of free-energy perturbation (FEP) to include selectivity calculations for α-CTXs and Nicotinic acetylcholine receptor (nAChR), illustrates in principle how such an approach could be employed in a biologics drug discovery program devoted to this ion channel target and peptide modality, and identifies approaches for engineering selectivity into α-CTXs
We sought to examine the suitability of acetylcholine-binding protein (AChBP)/α-CTX complexes as templates for nAChR/α-CTX homology models
At the α3β2 nAChR, thirty-three of these unstable water sites overlapped with the binding mode of LvIA (Figure 6A,C) versus seventeen at the α3β4 nAChR (Figure 6B,D). These results suggest that LvIA displaces more and higher-energy unstable waters when binding to the α3β2 nAChR than the α3β4 nAChR, which could account for why it is more potent in the former subtype
Summary
We build selectivity into α-CTXs. homology models of the α3β2 and α3β4 nAChRs and retrospectively test the accuracy of FEP and MM-GB/SA in predicting the potency and selectivity of a set of point mutations of LvIA in these subtypes [26]. This study expands the domain of applicability of FEP to include selectivity calculations for α-CTXs and nAChRs, illustrates in principle how such an approach could be employed in a biologics drug discovery program devoted to this ion channel target and peptide modality, and identifies approaches for engineering selectivity into α-CTXs. D) binding (blue) interface of LvIA and images. Lines connecting cysteines labeled with Roman numerals indicate disulfide bonds
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