The structure, dynamics and selectivity profile of a NaV1.7 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_V1.7), which is a genetically validated analgesic target. The peptide was promising as it showed high potency at Na_V1.7 (IC₅₀ ~26 nM) and selectivity over the cardiac Na_V subtype (Na_V1.5). 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 hNa_V1.7 (IC₅₀ = 0.4 ± 0.1 nM) without increased potency against hNa_V1.5. The activity of m₃-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 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 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_V1.7 target engagement of the peptide in vivo using a rodent pain model, where m₃-HwTx-IV dose-dependently suppressed spontaneous pain induced by the Na_V1.7 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. [ABSTRACT FROM AUTHOR]