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.

The NaV1.7 Channel Subtype as an Antinociceptive Target for Spider Toxins in Adult Dorsal Root Ganglia Neurons

“…Moreover, the interest to find analgesic toxins highly selective for Na V 1.1 and 1.6 subtypes, or to improve the toxin selectivity for these two subtypes, decreased during the past years because of the consequent inhibition of action potential transmission via axonal nodes of Ranvier which leads to central and peripheral (at the level of neuromuscular junctions) side-effects. Hence, the HwTx-IV mutant m3-HwTx-IV, presenting an additional hydrophobic patch (Gly1-Gly4-Trp33) has a reinforced inhibitory potency for the Na V 1.7 subtype while improving Na V 1.1, 1.2 and 1.6 subtype selectivity ( Rahnama et al, 2017 ). Moreover, the ProTx-II mutant JNJ63955918/GP-ProTX-II (W7Q-W30L) presents a 14-fold decreased potency for the Na V 1.7 subtype but an improved selectivity against Na V 1.1, 1.2, 1.4, and 1.6 subtypes, thus avoiding side-effects such as seizures, arrhythmias and impaired motor functioning ( Flinspach et al, 2017 ; Goncalves et al, 2018 ).…”

“…This test has the advantages of being less invasive and more sensitive than neuropathic and inflammatory tests. Indeed, only a low amount of toxin candidate to be evaluated is necessary to relieve pain, as exemplified by GpTx-I, Cd1a, m3-HwTx-IV and Pn3a ( Deuis et al, 2016 ; Cardoso et al, 2017 ; Rahnama et al, 2017 ; Sousa et al, 2017 ). However, the question of lack of physiological relevance of this test may be raised.…”

Direct evidence for high affinity blockade of NaV1.6 channel subtype by huwentoxin-IV spider peptide, using multiscale functional approaches

“…The peptide was thus promising as a potential lead molecule for the development of novel pain therapeutics. However, until now, very few information was reported about the efficiency of HwTx-IV to interact with the Na V 1.6 channel subtype, a TTX-sensitive (TTX-S) Na V channel subtype present in the brain, in DRG neurons and in Ranvier nodes of motoneurons, as well as the resulting in vivo side-effects resulting from this interaction (Rahnama et al, 2017).…”

A complicated complex: Ion channels, voltage sensing, cell membranes and peptide inhibitors

“…The data in Table 3 is notable because the subtype selectivity of HwTx-IV for Na V s is well characterised and it is known to bind 1.7, 1.6, 1.1, 1.2 and 1.3 channels only [51], as predicted from the above table. The activity of the toxin on Ca V 1.3 has not been described in the literature to the best knowledge of the authors, but our analysis suggests that the peptide will bind strongly to this channel as well as several of the Ca V subtypes.…”