Where spiders and cone snails meet: development of novel sodium channel probes

Abstract

Voltage-gated sodium (NaV) channels play crucial roles in a range of (patho)physiological processes. Much interest has arisen within the pharmaceutical industry to pursue these channels as analgesic targets following the overwhelming evidence that NaV channel subtypes NaV1.7- NaV1.9 are involved in nociception (Ahern et al., 2016; Luiz and Wood, 2016). More recently, also NaV1.1, NaV1.3 and NaV1.6 have been identified to be involved in pain pathways. Venom-derived disulphide-rich peptide toxins (1580 amino acids), isolated from spiders and cone snails, have been used extensively as probes to investigate these channels and have attracted much interest as drug leads for pharmaceutical development (Schroeder et al., 2012; Saez et al., 2010). However, few peptide drug leads have made it as drugs due to unfavourable physiochemical attributes including poor in vivo pharmacokinetics, rapid proteolytic cleavage and limited oral bioavailability. This project aims to bridge the gap in the development pipeline between drug leads and drugs candidates by downsizing these larger venom-derived NaV inhibitors into smaller, drug-like molecules. As a first step, we searched the venom of the spider Phoneutria nigriventer for promising NaV channel ligands. A first toxin sparking our interest was the toxin PnTx2-1. Based on the in vivo tests in mice and the sequence identity with PnTx2-5 and PnTx2-6, this peptide is classified as a NaV channel toxin. However, it has never been tested on any NaV channel isoform. Therefore, in this study the NaV channel subtype selectivity and species specificity of PnTx2-1 are investigated. We performed an in-depth functional characterization of the NaV channel modulating properties of PnTx2-1. Furthermore, analysis of the activity of PnTx2-1 on NaV1.5 reveals that this Phoneutria toxin modulates the cardiac NaV channel in a bifunctional manner, resulting in an alteration of the inactivation process and a reduction of the sodium peak current. The obtained results allowed to conclude that PnTx2-1 is an interesting insecticidal peptide but 5 it does not represent a promising lead compound for the development of novel NaV channel ligands with therapeutic potential. Another toxin potentially interesting as a NaV channel modulating ligand is the toxin PnTx1. PnTx1 was found to be the most interesting NaV channel toxin, considering its action in NaVs related to pain (Silva et al., 2012a). Following identification of common sequence motifs from NaV inhibitors PnTx1 (Diniz et al., 2006a) (78 residues, 14 cysteine) and -conotoxin KIIIA (Bulaj et al., 2005) (16 residue, 6 cysteine) from cone snail Conus kinoshitai, we have produced the smallest cyclic peptide-based NaV channel inhibitor known to date (10 residues, 2 cysteines) with demonstrated subtype selectivity across NaV channel subtypes including NaV1.7 and NaV1.9 (Luiz and Wood, 2016). The methodology used in this project, involved a two-pronged approach that incorporates sophisticated peptide engineering carrying out several rounds of small cyclic peptide design to aid in the determination of what drives subtype selectivity and molecular interactions of these downsized inhibitors across NaV subtypes. This allowed us to design small, stable and novel NaV probes with high subtype selectivity and potency coupled with improved biopharmaceutical properties, rendering these peptides suitable as analgesic drug candidates.