The structure, dynamics and selectivity profile of a Na(V)1.7 potency-optimised huwentoxin-IV variant

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 (hNa(V)1.7), which is a genetically validated analgesic target. The peptide was promising as it showed high potency at Na(V)1.7 (IC(50) ~26 nM) and selectivity over the cardiac Na(V) subtype (Na(V)1.5). Mutagenesis studies aimed at optimising the potency of the peptide resulted in the development of a triple-mutant of HwTx-IV (E1G, E4G, Y33W, m(3)-HwTx-IV) with significantly increased potency against hNa(V)1.7 (IC(50) = 0.4 ± 0.1 nM) without increased potency against hNa(V)1.5. The activity of m(3)-HwTx-IV against other Na(V) 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 m(3)-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 Na(V) subtype selectivity of the recombinant peptide was characterised, revealing potent inhibition of neuronal Na(V) subtypes 1.1, 1.2, 1.3, 1.6 and 1.7. In parallel to the in vitro studies, we investigated Na(V)1.7 target engagement of the peptide in vivo using a rodent pain model, where m(3)-HwTx-IV dose-dependently suppressed spontaneous pain induced by the Na(V)1.7 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 Na(V) subtypes.