Abstract
Venom-derived peptides have attracted much attention as potential lead molecules for pharmaceutical development. A well-known example is Huwentoxin-IV (HwTx-IV), a peptide toxin isolated from the venom of the Chinese bird-eating spider Haplopelma schmitdi. HwTx-IV was identified as a potent blocker of a human voltage-gated sodium channel (hNaV1.7), which is a genetically validated analgesic target. The peptide was promising as it showed high potency at NaV1.7 (IC50 ~26 nM) and selectivity over the cardiac NaV subtype (NaV1.5). Mutagenesis studies aimed at optimising the potency of the peptide resulted in the development of a triple-mutant of HwTx-IV (E1G, E4G, Y33W, m3-HwTx-IV) with significantly increased potency against hNaV1.7 (IC50 = 0.4 ± 0.1 nM) without increased potency against hNaV1.5. The activity of m3-HwTx-IV against other NaV subtypes was, however, not investigated. Similarly, the structure of the mutant peptide was not characterised, limiting the interpretation of the observed increase in potency. In this study we produced isotope-labelled recombinant m3-HwTx-IV in E. coli, which enabled us to characterise the atomic-resolution structure and dynamics of the peptide by NMR spectroscopy. The results show that the structure of the peptide is not perturbed by the mutations, whilst the relaxation studies reveal that residues in the active site of the peptide undergo conformational exchange. Additionally, the NaV subtype selectivity of the recombinant peptide was characterised, revealing potent inhibition of neuronal NaV subtypes 1.1, 1.2, 1.3, 1.6 and 1.7. In parallel to the in vitro studies, we investigated NaV1.7 target engagement of the peptide in vivo using a rodent pain model, where m3-HwTx-IV dose-dependently suppressed spontaneous pain induced by the NaV1.7 activator OD1. Thus, our results provide further insight into the structure and dynamics of this class of peptides that may prove useful in guiding the development of inhibitors with improved selectivity for analgesic NaV subtypes.
Highlights
Voltage-gated ion channels are ubiquitous signalling molecules that respond to changes in the cell membrane polarisation caused by ion concentration gradients between the intra- and extracellular environment [1]
Mutations in SCN9A, the gene encoding NaV1.7, which lead to a loss of function, result in congenital insensitivity to pain (CIP) with anosmia as the only other sensory deficit, whilst gain-of-function mutations are associated with extreme sensitivity to pain [3, 4]
The fusion protein was purified from the soluble cell fraction using Ni-NTA affinity chromatography (Fig 1C). m3-HwTx-IV was cleaved from the His6-MBP fusion tag using tobacco etch virus (TEV) protease, semi-preparative RP-HPLC (Fig 1D) was employed to yield a single dominant disulfide-bond isoform with a purity of >98% as assessed by analytical RP-HPLC and MALDI-TOF MS (Fig 1E)
Summary
Voltage-gated ion channels are ubiquitous signalling molecules that respond to changes in the cell membrane polarisation caused by ion concentration gradients between the intra- and extracellular environment [1]. They are important in the initiation and propagation of electrical signals in excitable cells. Dysfunction of these transmembrane proteins can lead to a range of severe channelopathies including epilepsy, cardiac arrhythmias and various pain disorders. Off-target effects at other homologous NaVs (1.1, 1.2, 1.4, 1.5 and 1.6) are likely to lead to severe side-effects including seizures, arrhythmias and impaired motor function [5]
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