The structure, dynamics and selectivity profile of a NaV1.7 potency-optimised huwentoxin-IV variant

PLoS One

Sassan Rahnama¹  Richard J. Lewis¹  Jennifer R. Deuis²  Mehdi Mobli²  Fernanda C. Cardoso²  Venkatraman Ramanujam²  Glenn F. King²  Lachlan D. Rash²  Irina Vetter³ 

[1] Centre for Advanced Imaging, The University of Queensland, St Lucia, QLD, Australia;Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, Australia;School of Biomedical Sciences, The University of Queensland, St Lucia, QLD, Australia

关键词: Toxins;    Nuclear Overhauser effect spectroscopy;    NMR relaxation;    NMR spectroscopy;    Matrix-assisted laser desorption ionization time-of-flight mass spectrometry;    Animal behavior;    Cell fusion;    Reversed-phase high performance liquid chromatography;   

DOI  :  10.1371/journal.pone.0173551

学科分类：医学（综合）

来源: Public Library of Science

【 摘 要 】

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.

【 授权许可】

CC BY