From identification to functional characterization of cyriotoxin‐1a, an antinociceptive toxin from the spider Cyriopagopus schioedtei

Abstract

Background and Purpose:The Na V 1.7 channel is highly expressed in dorsal root ganglia of the sensory nervous system and plays a central role in the pain signalling process. We investigated a library prepared from original venoms of 117 different animals to identify new selective inhibitors of this target.Experimental Approach: We used high throughput screening of a large venom collection using automated patch-clamp experiments on human voltage-gated sodium channel subtypes and then in vitro and in vivo electrophysiological experiments to characterize the active peptides that have been purified, sequenced, and chemically synthesized. Analgesic effects were evaluated in vivo in mice models.Key Results: We identified cyriotoxin-1a (CyrTx-1a), a novel peptide isolated from Cyriopagopus schioedtei spider venom, as a candidate for further characterization. This 33 amino acids toxin belongs to the inhibitor cystine knot structural family and inhibits hNa V 1.1-1.3 and 1.6-1.7 channels in the low nanomolar range, compared to the micromolar range for hNa V 1.4-1.5 and 1.8 channels. CyrTx-1a was 920 times more efficient at inhibiting tetrodotoxin (TTX)-sensitive than TTX-resistant sodium currents recorded from adult mouse dorsal root ganglia neurons and in vivo electrophysiological experiments showed that CyrTx-1a was approximately 170 times less efficient than huwentoxin-IV at altering mouse skeletal neuromuscular excitability properties. CyrTx-1a exhibited an analgesic effect in mice by increasing reaction time in the hot-plate assay. Conclusions and Implications:The pharmacological profile of CyrTx-1a paves the way for further molecular engineering aimed to optimize the potential antinociceptive properties of this peptide.

Structure–Function and Therapeutic Potential of Spider Venom-Derived Cysteine Knot Peptides Targeting Sodium Channels

“…The NaSpTx3 peptides Gr1b and Gr2c reversed acute and inflammatory pain intrathecally (Lampe, 1998), while ProTx-II and its optimized analog JNJ63955918 reversed neuropathic and inflammatory pain when administered intrathecally or locally (Tanaka et al, 2015; Flinspach et al, 2017). The NaSpTx1 peptides HwTx-IV and HNTX-IV also reversed neuropathic and inflammatory pains intraperitonially (Liu et al, 2014a,b), while ides Ca2a and cyriotoxin-1a reversed inflammatory and thermal pain following intraperitoneal or intraplantar administration, respectively (Zhang et al, 2018a; Goncalves et al, 2019).…”

Transfection methods for high-throughput cellular assays of voltage-gated calcium and sodium channels involved in pain

“…These platforms harness a range of commercially available stable cell lines expressing VGICs and receptors, that are validated in these HT assays [ 44 – 46 ]. On the other hand, only one report has studied chemically-transfected cells in HT automated patch clamp using FuGENE [ 40 ]. In these automated systems, the unguided selection of cells leads to a number of whole-cells displaying no currents and/or poor membrane integrity, hence broad and efficient and transfection across cells is essential to achieve reasonable success rates.…”

Evaluation of the Spider (Phlogiellus genus) Phlotoxin 1 and Synthetic Variants as Antinociceptive Drug Candidates

“…Among the eight peptide variant forms tested on HEK-293 cells overexpressing the hNa V 1.7 subtype, W24A-, K25A-, and Y26A-PhlTx1 were shown to completely lose affinity for this type of channel subtype, indicating that the C-terminal residues Trp 24 , Lys 25 , and Tyr 26 are essential for hNa V 1.7 inhibition. It has already been mentioned in the literature that toxins belonging to the NaSpTx family 1 display positively charged amino acids Lys 25 , His 26 , Lys 27 , Lys 30 , surrounded by hydrophobic Phe 5 and Trp 28 clustered on one molecule face, which may be involved in the peptide interactions with TTX-sensitive Na V channels [13,15,37,38]. However, PhlTx1 possesses only two of the four positively charged amino acids (K25 and R30 instead of K30), while the hydrophobic amino acids are almost preserved (Q5, instead of F5, and W28).…”

“…The effects of synthetic PhlTx1 and some of its variants were investigated on the cell lines overexpressing hNa V 1.1–1.8, hCa V 1.2, and hK V 11.1 subtypes, using the automated QPatch HTX patch-clamp system (Sophion Bioscience, Denmark), allowing current recordings in whole-cell configuration and both signal acquisition and data analyses, as previously reported [15]. On the day of their use, HEK-293 and CHO cells were transferred into Eppendorf tubes containing a FreeStyle TM 293 expression medium (Gibco) which were then placed in the automated electrophysiology platform.…”

“…Hence, numerous high-throughput screenings of venom banks from various animals (spiders, scorpions, snakes, lizards, cone snails, etc.) have been performed on the Na V 1.7 subtype, leading to the discovery of potential analgesic candidates mainly from spider venoms [13,14,15]. In fact, approximately 20 spider toxins, comprising between 26 and 35 amino acids and sharing the same inhibitor cystine knot (ICK) architectural motif, are known to inhibit with high affinity the Na V 1.7 subtype (reviewed by [12,16]).…”