Potency-Enhancing Mutations of Gating Modifier Toxins for the Voltage-Gated Sodium Channel NaV1.7 Can Be Predicted Using Accurate Free-Energy Calculations.

Dana Katz,Abba E Leffler,Dan Sindhikara,Michael Dimattia

doi:10.3390/toxins13030193

Abstract

Gating modifier toxins (GMTs) isolated from venomous organisms such as Protoxin-II (ProTx-II) and Huwentoxin-IV (HwTx-IV) that inhibit the voltage-gated sodium channel NaV1.7 by binding to its voltage-sensing domain II (VSDII) have been extensively investigated as non-opioid analgesics. However, reliably predicting how a mutation to a GMT will affect its potency for NaV1.7 has been challenging. Here, we hypothesize that structure-based computational methods can be used to predict such changes. We employ free-energy perturbation (FEP), a physics-based simulation method for predicting the relative binding free energy (RBFE) between molecules, and the cryo electron microscopy (cryo-EM) structures of ProTx-II and HwTx-IV bound to VSDII of NaV1.7 to re-predict the relative potencies of forty-seven point mutants of these GMTs for NaV1.7. First, FEP predicted these relative potencies with an overall root mean square error (RMSE) of 1.0 ± 0.1 kcal/mol and an R2 value of 0.66, equivalent to experimental uncertainty and an improvement over the widely used molecular-mechanics/generalized born-surface area (MM-GB/SA) RBFE method that had an RMSE of 3.9 ± 0.8 kcal/mol. Second, inclusion of an explicit membrane model was needed for the GMTs to maintain stable binding poses during the FEP simulations. Third, MM-GB/SA and FEP were used to identify fifteen non-standard tryptophan mutants at ProTx-II[W24] predicted in silico to have a at least a 1 kcal/mol gain in potency. These predicted potency gains are likely due to the displacement of high-energy waters as identified by the WaterMap algorithm for calculating the positions and thermodynamic properties of water molecules in protein binding sites. Our results expand the domain of applicability of FEP and set the stage for its prospective use in biologics drug discovery programs involving GMTs and NaV1.7.

Highlights

The principal finding from this study is that free-energy perturbation (FEP) can predict the change in potency of Gating modifier toxins (GMTs) mutants targeting voltage-sensing domain II (VSDII) of NaV 1.7 with an root mean square error (RMSE) of 1.0 kcal/mol and an R2 value of 0.7 (Figure 4 and Table 1)
The performance of FEP is impressive given the significant sources of uncertainty in the study, such as the large size of GMTs, the relatively low resolution of the GMTs in the cryoEM structures, and the fact that functional as opposed to binding data were employed for benchmarking
Can be used to accurately predict how a mutation to a GMT that targets VSDII of Nav1.7 will affect its potency for the channel, setting the stage for its prospective application in biologics drug discovery programs

Summary

Introduction

Voltage gated sodium channels (VGSCs) are transmembrane proteins with an essential role in electrical signaling, nerve conduction, skeletal and cardiac muscle contraction, secretion, and neurotransmission [1]. These ion channels are composed of a voltagesensing alpha subunit that forms an ion-conducting pore and one or two beta subunits involved in channel expression and kinetics [2]. NaV 1.9) have been identified in humans and their dysregulation is associated with distinct disease phenotypes [3]. The alpha subunit is composed of four homologous domains (DI to DIV) each consisting of six transmembrane helical segments termed S1 to S6 [2]

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Results

Conclusion