Rational development of analgesics for the treatment of chronic pain: dissecting the molecular details of the interaction between gating modifier peptide modulators and human voltage-gated sodium channels

Abstract

Neurotoxins are of interest as lead molecules for the development of pharmaceuticals and insecticides. Huwentoxin-IV (HwTx-IV) is a 35-residue peptide isolated from the venom of Chinese bird spider Cyriopagopus schmidti. It has six cysteine residues that form three disulfide bonds, which form a stable inhibitor cystine knot (ICK) motif. This neurotoxin is one of the most potent inhibitors of the human voltage-gated sodium channel (hNaV1.7) described to date. Humans have nine NaV channel subtypes with different roles and tissue distribution. NaV1.1–1.3 are found primarily in the central nervous system (CNS), NaV1.6 is found in both the CNS and nodes of Ranvier in the peripheral nervous system (PNS), NaV1.4 and NaV1.5 are found in skeletal and cardiac muscles, respectively, and NaV1.7–1.9 are found primarily in peripheral sensory neurons where they are involved in pain sensing. NaV1.7 plays a key role in the propagation of pain signals, and it is a validated analgesic target. HwTx-IV potently inhibits hNaV1.7 with an IC50 of ~20 nM but it inhibits the off-target cardiac subtype NaV1.5 with far lower potency.In this study, a rationally designed triple-mutant HwTx-IV analogue (E1G,E4G,Y33W) was produced recombinantly via expression in the periplasm of Escherichia coli. The mutant peptide (m3-HwTx-IV) has significantly increased potency against hNaV1.7 (IC50 = 0.4 ± 0.1 nM) without increased potency against hNaV1.5. Neither its activity against other subtypes nor its structure has been characterised. Bacterial expression enabled production of uniformly 15N/13C-labelled recombinant m3-HwTx-IV for atomic-resolution structure determination using multidimensional heteronuclear NMR spectroscopy. Additionally, the activity of the recombinant peptide on various NaV channel subtypes was measured via automated patch-clamp electrophysiology using human (HEK293 and CHO) cell lines stably expressing the NaV channels. In parallel the analgesic efficacy of the NaV1.7 potency-optimised peptide was investigated using a rodent pain model.m3-HwTx-IV and similar spider-venom peptides from the same family inhibit NaV channel activation by binding to the domain II voltage sensor (VSDII) of the channel. In this project, I applied NMR-based methods to probe the atomic details of the interaction between gating-modifier venom peptides and the isolated VSDII of several human NaV subtypes, including hNaV1.7, hNaV1.6, and hNaV1.1. Key channel-interacting residues were identified by titration of the isolated VSDs with isotope-labelled gating-modifier peptides using solution NMR. The knowledge gained from this work, in combination with other studies such as the recently elucidated cryo-EM structures of NaV1.7, provides a platform for structure-guided rational engineering of subtype-selective inhibitors of human NaV channels as potential therapeutics for the treatment of chronic pain.