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 Selenocosmia huwena. It has 6 cysteine residues that form three disulfide bonds, which fold into a stable inhibitor cystine knot (ICK) motif. This neurotoxin is one of the most potent human NaV1.7 (hNaV1.7) inhibitors described to date [1]. NaV1.7 is a subtype of voltage-gated sodium channel that is involved in the initiation and propagation of pain signals, and a validated analgesic target. HwTx-IV inhibits hNaV1.7 with an IC₅₀ of ~20 nM but inhibits the cardiac subtype NaV1.5 with far less potency [2].

In this study, a rationally designed [1] highly potent triple-mutant HWTX-IV analog (E1G,E4G,Y33W) was produced recombinantly via expression in the periplasm of E. coli [3]. This mutant has significantly increased potency on hNaV1.7 (IC₅₀ = 0.4 ± 0.1 nM) without increased potency against hNaV1.5, although its activity against other subtypes is not known. Here the activity of the recombinant peptide on all human NaV channel subtypes was measured via two-electrode voltage-clamp electrophysiology using Xenopus oocytes and automated patch-clamp electrophysiology using a HEK293 cell line stably expressing hNaV1.7. In parallel, the analgesic efficacy of the peptide was investigated using a rodent pain model. Recombinant expression enabled production of uniformly ¹⁵N/¹³C-labelled peptide for structure determination using multidimensional heteronuclear NMR spectroscopy. The knowledge gained from this work will lay the foundation for ligand-channel interaction studies and provide a platform for rational development of novel analgesics for treatment of chronic pain.