Abstract
Huwentoxin-IV (HwTx-IV), a peptide discovered in the venom of the Chinese bird spider Cyriopagopus schmidti, has been reported to be a potent antinociceptive compound due to its action on the genetically-validated NaV1.7 pain target. Using this peptide for antinociceptive applications in vivo suffers from one major drawback, namely its negative impact on the neuromuscular system. Although studied only recently, this effect appears to be due to an interaction between the peptide and the NaV1.6 channel subtype located at the presynaptic level. The aim of this work was to investigate how HwTx-IV could be modified in order to alter the original human (h) NaV1.7/NaV1.6 selectivity ratio of 23. Nineteen HwTx-IV analogues were chemically synthesized and tested for their blocking effects on the Na+ currents flowing through these two channel subtypes stably expressed in cell lines. Dose-response curves for these analogues were generated, thanks to the use of an automated patch-clamp system. Several key amino acid positions were targeted owing to the information provided by earlier structure-activity relationship (SAR) studies. Among the analogues tested, the potency of HwTx-IV E4K was significantly improved for hNaV1.6, leading to a decreased hNaV1.7/hNaV1.6 selectivity ratio (close to 1). Similar decreased selectivity ratios, but with increased potency for both subtypes, were observed for HwTx-IV analogues that combine a substitution at position 4 with a modification of amino acid 1 or 26 (HwTx-IV E1G/E4G and HwTx-IV E4K/R26Q). In contrast, increased selectivity ratios (>46) were obtained if the E4K mutation was combined to an additional double substitution (R 26A/Y33W) or simply by further substituting the C-terminal amidation of the peptide by a carboxylated motif, linked to a marked loss of potency on hNaV1.6 in this latter case. These results demonstrate that it is possible to significantly modulate the selectivity ratio for these two channel subtypes in order to improve the potency of a given analogue for hNaV1.6 and/or hNaV1.7 subtypes. In addition, selective analogues for hNaV1.7, possessing better safety profiles, were produced to limit neuromuscular impairments.
Highlights
For over 10 years, the NaV1.7 channel subtype is considered as an attractive pain target owing to several compelling genetic evidences
It was remarkable that none of the analogues posed problems for the folding of the peptide (RP-HPLC data and yield calculations, not shown) in spite of the fact that this peptide folds according to an Inhibitory Cystine Knot (ICK) motif and that some analogues harbor up to three substitutions
Some of the earlier structure-activity relationship (SAR) investigations were performed with recombinant HwTx-IV peptides that are lacking C-terminal amidation (Minassian et al, 2013; Sermadiras et al, 2013; Rahnama et al, 2017; Neff et al, 2020), an amidation that is crucial for potency onto hNaV1.7 (Revell et al, 2013)
Summary
For over 10 years, the NaV1.7 channel subtype is considered as an attractive pain target owing to several compelling genetic evidences. Gain-offunction mutations of the same gene lead to erythromelalgia and paroxysmal extreme pain disorder (Estacion et al, 2008) Because of these clinical evidences, the hNaV1.7 channel has been the target of choice for high-throughput screening campaigns to identify blockers as pain therapeutics. Owing to chemical spaces larger than those of small molecules, they possess better affinities and selectivity profiles even when the target of interest shares high sequence identity with other ion channel subtypes This was illustrated by peptides issued from spider venoms, often possessing an Inhibitory Cystine Knot (ICK) fold, that were shown to be able of distinguishing closely related subtypes of NaV channels (for reviews, see (Gonçalves et al, 2018a; Cardoso and Lewis, 2019; Dongol et al, 2019)). This ability is facilitated by the interaction of these peptides with the different voltage-sensor domains, known to be the most divergent in sequence in contrast to pore region
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