Huwentoxin IV

The structure, dynamics and selectivity profile of a NaV1.7 potency-optimised huwentoxin-IV variant.[Pubmed:28301520]

PLoS One. 2017 Mar 16;12(3):e0173551.

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.

Analgesic effects of Huwentoxin-IV on animal models of inflammatory and neuropathic pain.[Pubmed:24188048]

Protein Pept Lett. 2014;21(2):153-8.

Huwentoxin-IV (HWTX-IV), a peptide with 35 amino acid residues, was discovered in the venom of spider Ornithoctonus huwena. The peptide had an inhibitory effect on a tetrodotoxin-sensitive (TTX-S) sodium channel with highly sensitive to Nav1.7, an attractive target for pain release therapy. In this study we further demonstrated the analgesic effects of HWTX-IV using mouse and rat as an inflammatory pain model and/or a neuropathic pain models. In the both cases, the analgesic effects of the peptide were dose-dependent, and statistically significant. In the inflammatory model, 100 microg/kg of HWTX-IV produced an efficient reversal of hyperalgesia up to 63.6% after injection of formalin in rats with the efficiency equivalent to that of morphine at 50 microg/kg, and 200 microg/kg of HWTX-IV produced protective effect up to 55.6% after injection of acetic acid with the efficiency equivalent to that of morphine at 100 microg/kg. In the spinal nerve model, the peptide produced the longer and higher reversal effect on allodynia than Mexiletine. These results demonstrated that HWTX-IV released efficiently the acute inflammatory pain and chronic neuropathic pain in these animals, suggesting that HWTX-IV was a potential and efficient candidate for further clinical drug development against inflammatory and neuropathic pain.

Recombinant expression and in vitro characterisation of active Huwentoxin-IV.[Pubmed:24324842]

PLoS One. 2013 Dec 6;8(12):e83202.

Huwentoxin-IV (HwTx-IV) is a 35-residue neurotoxin peptide with potential application as a novel analgesic. It is a member of the inhibitory cystine knot (ICK) peptide family, characterised by a compact globular structure maintained by three intramolecular disulfide bonds. Here we describe a novel strategy for producing non-tagged, fully folded ICK-toxin in a bacterial system. HwTx-IV was expressed as a cleavable fusion to small ubiquitin-related modifier (SUMO) in the cytoplasm of the SHuffle T7 Express lysY Escherichia coli strain, which allows cytosolic disulfide bond formation. Purification by IMAC with selective elution of monomeric SUMO fusion followed by proteolytic cleavage and polishing chromatographic steps yielded pure homogeneous toxin. Recombinant HwTx-IV is produced with a C-terminal acid, whereas the native peptide is C-terminally amidated. HwTx-IV(acid) inhibited Nav1.7 in a dose dependent manner (IC50 = 463-727 nM). In comparison to HwTx-IV(amide) (IC50 = 11 +/- 3 nM), the carboxylate was ~50 fold less potent on Nav1.7, which highlights the impact of the C-terminus. As the amide bond of an additional amino acid may mimic the carboxamide, we expressed the glycine-extended analogue HwTx-IV(G36)(acid) in the SUMO/SHuffle system. The peptide was approximately three fold more potent on Nav1.7 in comparison to HwTx-IV(acid) (IC50 = 190 nM). In conclusion, we have established a novel system for expression and purification of fully folded and active HwTx-IV(acid) in bacteria, which could be applicable to other structurally complex and cysteine rich peptides. Furthermore, we discovered that glycine extension of HwTx-IV(acid) restores some of the potency of the native carboxamide. This finding may also apply to other C-terminally amidated peptides produced recombinantly.

Detection and identification of huwentoxin-IV interacting proteins by biotin-avidin chemistry combined with mass spectrometry.[Pubmed:24803923]

J Venom Anim Toxins Incl Trop Dis. 2014 Apr 28;20:18.

BACKGROUND: Numerous spider toxins are of interest as tools for neurophysiological research or as lead molecules for the development of pharmaceuticals and insecticides. Direct detection and identification of the interacting proteins of a spider toxin are helpful for its action-mechanism analysis and practical application. The present study employed a combinative strategy for the analysis of interacting proteins of huwentoxin-IV (HWTX-IV), a peptidic neurotoxin from the venom of the spider Selenocosmia huwena. RESULTS: HWTX-IV was first lightly labeled with biotin under the optimized mild experimental conditions and the toxin labeled with a single biotin group (monobiotinylated HWTX-IV) was demonstrated by electrophysiological experiments to retain its original bioactivity and was used in combination with far-western blotting to detect its interacting proteins. Comparative experiments indicated that some membrane proteins from rat neuromuscular junction preparations bind to monobiotinylated HWTX-IV after being transferred onto a PVDF membrane from the SDS-gel. With capillary high performance liquid chromatography-tandem mass spectrometry, several membrane proteins with which HWTX-IV potentially interacted were identified from the preparations and then bioinformatically analyzed. CONCLUSIONS: This work has provided not only a new insight into the action mechanism of HWTX-IV but also a reference technology for the relevant researches.

Common molecular determinants of tarantula huwentoxin-IV inhibition of Na+ channel voltage sensors in domains II and IV.[Pubmed:21659528]

J Biol Chem. 2011 Aug 5;286(31):27301-10.

The voltage sensors of domains II and IV of sodium channels are important determinants of activation and inactivation, respectively. Animal toxins that alter electrophysiological excitability of muscles and neurons often modify sodium channel activation by selectively interacting with domain II and inactivation by selectively interacting with domain IV. This suggests that there may be substantial differences between the toxin-binding sites in these two important domains. Here we explore the ability of the tarantula huwentoxin-IV (HWTX-IV) to inhibit the activity of the domain II and IV voltage sensors. HWTX-IV is specific for domain II, and we identify five residues in the S1-S2 (Glu-753) and S3-S4 (Glu-811, Leu-814, Asp-816, and Glu-818) regions of domain II that are crucial for inhibition of activation by HWTX-IV. These data indicate that a single residue in the S3-S4 linker (Glu-818 in hNav1.7) is crucial for allowing HWTX-IV to interact with the other key residues and trap the voltage sensor in the closed configuration. Mutagenesis analysis indicates that the five corresponding residues in domain IV are all critical for endowing HWTX-IV with the ability to inhibit fast inactivation. Our data suggest that the toxin-binding motif in domain II is conserved in domain IV. Increasing our understanding of the molecular determinants of toxin interactions with voltage-gated sodium channels may permit development of enhanced isoform-specific voltage-gating modifiers.

Tarantula huwentoxin-IV inhibits neuronal sodium channels by binding to receptor site 4 and trapping the domain ii voltage sensor in the closed configuration.[Pubmed:18628201]

J Biol Chem. 2008 Oct 3;283(40):27300-13.

Peptide toxins with high affinity, divergent pharmacological functions, and isoform-specific selectivity are powerful tools for investigating the structure-function relationships of voltage-gated sodium channels (VGSCs). Although a number of interesting inhibitors have been reported from tarantula venoms, little is known about the mechanism for their interaction with VGSCs. We show that huwentoxin-IV (HWTX-IV), a 35-residue peptide from tarantula Ornithoctonus huwena venom, preferentially inhibits neuronal VGSC subtypes rNav1.2, rNav1.3, and hNav1.7 compared with muscle subtypes rNav1.4 and hNav1.5. Of the five VGSCs examined, hNav1.7 was most sensitive to HWTX-IV (IC(50) approximately 26 nM). Following application of 1 microm HWTX-IV, hNav1.7 currents could only be elicited with extreme depolarizations (>+100 mV). Recovery of hNav1.7 channels from HWTX-IV inhibition could be induced by extreme depolarizations or moderate depolarizations lasting several minutes. Site-directed mutagenesis analysis indicated that the toxin docked at neurotoxin receptor site 4 located at the extracellular S3-S4 linker of domain II. Mutations E818Q and D816N in hNav1.7 decreased toxin affinity for hNav1.7 by approximately 300-fold, whereas the reverse mutations in rNav1.4 (N655D/Q657E) and the corresponding mutations in hNav1.5 (R812D/S814E) greatly increased the sensitivity of the muscle VGSCs to HWTX-IV. Our data identify a novel mechanism for sodium channel inhibition by tarantula toxins involving binding to neurotoxin receptor site 4. In contrast to scorpion beta-toxins that trap the IIS4 voltage sensor in an outward configuration, we propose that HWTX-IV traps the voltage sensor of domain II in the inward, closed configuration.