The structure, dynamics and selectivity profile of a NaV 1.7 potency-optimised huwentoxin-IV variant

Rahnama, Sassan, Deuis, Jennifer R., Cardoso, Fernanda C., Ramanujam, Venkatraman, Lewis, Richard J., Rash, Lachlan D., King, Glenn F., Vetter, Irina and Mobli, Mehdi (2017) The structure, dynamics and selectivity profile of a NaV 1.7 potency-optimised huwentoxin-IV variant. PLoS One, 12 3: . doi:10.1371/journal.pone.0173551


Author Rahnama, Sassan
Deuis, Jennifer R.
Cardoso, Fernanda C.
Ramanujam, Venkatraman
Lewis, Richard J.
Rash, Lachlan D.
King, Glenn F.
Vetter, Irina
Mobli, Mehdi
Title The structure, dynamics and selectivity profile of a NaV 1.7 potency-optimised huwentoxin-IV variant
Formatted title
The structure, dynamics and selectivity profile of a NaV1.7 potency-optimised huwentoxin-IV variant
Journal name PLoS One   Check publisher's open access policy
ISSN 1932-6203
Publication date 2017-03-16
Year available 2017
Sub-type Article (original research)
DOI 10.1371/journal.pone.0173551
Open Access Status DOI
Volume 12
Issue 3
Total pages 19
Place of publication San Francisco, CA, United States
Publisher Public Library of Science
Language eng
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 (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 (IC50 similar to 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 (IC50 = 0.4 +/- 0.1 nM) without increased potency against hNa(V)1.5. 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 NaV 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 m3-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 NaV subtypes.
Formatted 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 NaV 1.7 (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 hNaV 1.7 (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 isotopelabelled recombinant m3 -HwTx-IV in E. coli, which enabled us to characterise the atomicresolution 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 NaV 1.7 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.
Keyword Common Molecular Determinants
Spider Selenocosmia-Huwena
Sodium-Channel Antagonist
Nmr Chemical-Shifts
Rat Formalin Test
Protein Backbone
Neuropathic Pain
Target Na(V)1.7
Torsion Angles
Morphine
Q-Index Code C1
Q-Index Status Provisional Code
Grant ID APP1034958
Institutional Status UQ

 
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