Abstract

Voltage-gated sodium (NaV) channel subtypes, including NaV1.7, are promising targets for the treatment of neurological diseases, such as chronic pain. Cone snail-derived µ-conotoxins are small, potent NaV channel inhibitors which represent potential drug leads. Of the 22 µ-conotoxins characterised so far, only a small number, including KIIIA and CnIIIC, have shown inhibition against human NaV1.7. We have recently identified a novel µ-conotoxin, SxIIIC, from Conus striolatus. Here we present the isolation of native peptide, chemical synthesis, characterisation of human NaV channel activity by whole-cell patch-clamp electrophysiology and analysis of the NMR solution structure. SxIIIC displays a unique NaV channel selectivity profile (1.4 > 1.3 > 1.1 ≈ 1.6 ≈ 1.7 > 1.2 >> 1.5 ≈ 1.8) when compared to other µ-conotoxins and represents one of the most potent human NaV1.7 putative pore blockers (IC50 152.2 ± 21.8 nM) to date. NMR analysis reveals the structure of SxIIIC includes the characteristic α-helix seen in other µ-conotoxins. Future investigations into structure-activity relationships of SxIIIC are expected to provide insights into residues important for NaV channel pore blocker selectivity and subsequently important for chronic pain drug development.

Highlights

  • Voltage-gated sodium (NaV ) channels are highly conserved pore-forming proteins permeable to sodium ions and are important for the initiation and propagation of action potentials [1,2]

  • We present the discovery of a novel μ-conotoxin from C. striolatus, named

  • Our research discovered that SxIIIC displays a unique NaV channel subtype selectivity profile when compared to other highly-homologous μ-conotoxins

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Summary

Introduction

Voltage-gated sodium (NaV ) channels are highly conserved pore-forming proteins permeable to sodium ions and are important for the initiation and propagation of action potentials [1,2]. Μ-Conotoxins represent favourable drug leads due to two key characteristics They are rich in cysteines which form disulfide bonds to afford structural integrity and secondly, their small molecular size (typically 16–26 residues) presents an advantage over larger biologics as they are synthesised and amenable to chemical modifications to improve pharmacological traits [15]. They present as favourable molecules over acting small molecule NaV inhibitors including anesthetics [16], as the increased surface area provides greater contacts with the NaV channel pore, which can result in an increased subtype selectivity.

Activity-Guided Isolation of SxIIIC
Peptide Sequencing
Peptide Synthesis
Oxidation
Whole-Cell Patch-Clamp Electrophysiology
NMR Experiments
NMR Structure Calculations
Activity-Guided Isolation of SxIIIC from Conus striolatus
Peptide Synthesis of SxIIIC
SxIIIC Displays a Unique NaV Channel Subtype Selectivity Profile
NMR Solution Structure of SxIIIC
Discussion
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