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Comprehensive engineering of the tarantula venom peptide huwentoxin-IV to inhibit the human voltage-gated sodium channel hNa_v1.7.

ABSTRACT: Pain is a significant public health burden in the United States, and current treatment approaches rely heavily on opioids, which often have limited efficacy and can lead to addiction. In humans, functional loss of the voltage-gated sodium channel Na_v1.7 leads to pain insensitivity without deficits in the central nervous system. Accordingly, discovery of a selective Na_v1.7 antagonist should provide an analgesic without abuse liability and an improved side-effect profile. Huwentoxin-IV, a component of tarantula venom, potently blocks sodium channels and is an attractive scaffold for engineering a Na_v1.7-selective molecule. To define the functional impact of alterations in huwentoxin-IV sequence, we produced a library of 373 point mutants and tested them for Na_v1.7 and Na_v1.2 activity. We then combined favorable individual changes to produce combinatorial mutants that showed further improvements in Na_v1.7 potency (E1N, E4D, Y33W, Q34S-Na_v1.7 pIC₅₀ = 8.1 ± 0.08) and increased selectivity over other Na_v isoforms (E1N, R26K, Q34S, G36I, Na_v1.7 pIC₅₀ = 7.2 ± 0.1, Na_v1.2 pIC₅₀ = 6.1 ± 0.18, Na_v1.3 pIC₅₀ = 6.4 ± 1.0), Na_v1.4 is inactive at 3 ?m, and Na_v1.5 is inactive at 10 ?m We also substituted noncoded amino acids at select positions in huwentoxin-IV. Based on these results, we identify key determinants of huwentoxin's Na_v1.7 inhibition and propose a model for huwentoxin-IV's interaction with Na_v1.7. These findings uncover fundamental features of huwentoxin involved in Na_v1.7 blockade, provide a foundation for additional optimization of this molecule, and offer a basis for the development of a safe and effective analgesic.

SUBMITTER: Neff RA

PROVIDER: S-EPMC6996889 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Comprehensive engineering of the tarantula venom peptide huwentoxin-IV to inhibit the human voltage-gated sodium channel hNav1.7.

Neff Robert A RA Flinspach Mack M Gibbs Alan A Shih Amy Y AY Minassian Natali A NA Liu Yi Y Fellows Ross R Libiger Ondrej O Young Stephanie S Pennington Michael W MW Hunter Michael J MJ Wickenden Alan D AD

The Journal of biological chemistry 20191223 5

Pain is a significant public health burden in the United States, and current treatment approaches rely heavily on opioids, which often have limited efficacy and can lead to addiction. In humans, functional loss of the voltage-gated sodium channel Nav1.7 leads to pain insensitivity without deficits in the central nervous system. Accordingly, discovery of a selective Nav1.7 antagonist should provide an analgesic without abuse liability and an improved side-effect profile. Huw ...[more]

PMID: 31871053

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Project description: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.

Project description:The human TE671 cell line was originally used as a model of medulloblastoma but has since been reassigned as rhabdomyosarcoma. Despite the characterised endogenous expression of voltage-sensitive sodium currents in these cells, the specific voltage-gated sodium channel (VGSC) subtype underlying these currents remains unknown. To profile the VGSC subtype in undifferentiated TE671 cells, endpoint and quantitative reverse transcription-PCR (qRT-PCR), western blot and whole-cell patch clamp electrophysiology were performed. qRT-PCR profiling revealed that expression of the SCN9A gene was ∼215-fold greater than the SCN4A gene and over 400-fold greater than any of the other VGSC genes, while western blot confirmed that the dominant SCN9A RNA was translated to a protein with a molecular mass of ∼250 kDa. Elicited sodium currents had a mean amplitude of 2.6 ± 0.7 nA with activation and fast inactivation V50 values of -31.9 ± 1.1 and -69.6 ± 1.0 mV, respectively. The currents were completely and reversibly blocked by tetrodotoxin at concentrations greater than 100 nm (IC50 = 22.3 nm). They were also very susceptible to the NaV 1.7 specific blockers Huwentoxin-IV and Protoxin-II with IC50 values of 14.6 nm and 0.8 nm, respectively, characteristic of those previously determined for NaV 1.7. Combined, the results revealed the non-canonical and highly dominant expression of NaV 1.7 in the human TE671 rhabdomyosarcoma cell line. We show that the TE671 cell line is an easy to maintain and cost-effective model for the study of NaV 1.7, a major target for the development of analgesic drugs and more generally for the study of pain. KEY POINTS: Undifferentiated TE671 cells produce a voltage-sensitive sodium current when depolarised. The voltage-gated sodium channel isoform expressed in undifferentiated TE671 cells was previously unknown. Through qRT-PCR, western blot and toxin pharmacology, it is shown that undifferentiated TE671 cells dominantly (>99.5%) express the NaV 1.7 isoform that is strongly associated with pain. The TE671 cell line is, therefore, a very easy to maintain and cost-effective model to study NaV 1.7-targeting drugs.

Dataset Information

Comprehensive engineering of the tarantula venom peptide huwentoxin-IV to inhibit the human voltage-gated sodium channel hNa_v1.7.

Publications

Comprehensive engineering of the tarantula venom peptide huwentoxin-IV to inhibit the human voltage-gated sodium channel hNa<sub>v</sub>1.7.

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