title: The structure, dynamics and selectivity profile of a NaV1.7 potency-optimised huwentoxin-IV variant.
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.
authors.affiliation: Centre for Advanced Imaging, The University of Queensland, St Lucia, QLD, Australia.|Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, Australia.
authors.forename: Sassan
authors.initials: S
authors.lastname: Rahnama
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, Australia.
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Deuis
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, Australia.
authors.forename: Fernanda C
authors.initials: FC
authors.lastname: Cardoso
authors.affiliation: Centre for Advanced Imaging, The University of Queensland, St Lucia, QLD, Australia.
authors.forename: Venkatraman
authors.initials: V
authors.lastname: Ramanujam
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, Australia.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Lewis
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, Australia.|School of Biomedical Sciences, The University of Queensland, St Lucia, QLD, Australia.
authors.forename: Lachlan D
authors.initials: LD
authors.lastname: Rash
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, Australia.
authors.forename: Glenn F
authors.initials: GF
authors.lastname: King
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, Australia.
authors.forename: Irina
authors.initials: I
authors.lastname: Vetter
authors.affiliation: Centre for Advanced Imaging, The University of Queensland, St Lucia, QLD, Australia.
authors.forename: Mehdi
authors.identifier: http://orcid.org/0000-0003-2420-4262
authors.initials: M
authors.lastname: Mobli
mesh_terms: D000818:Animals; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D062556:NAV1.7 Voltage-Gated Sodium Channel / Q000187:drug effects*; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000187:drug effects; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013111:Spider Venoms / Q000737:chemistry* / Q000494:pharmacology*
chemical_list: D062556:NAV1.7 Voltage-Gated Sodium Channel; D011994:Recombinant Proteins; C486866:SCN9A protein, human; D013111:Spider Venoms; C470632:huwentoxin IV, Selenocosmia huwena