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The inhibition of Nav1.7 is a promising strategy for the development of analgesic treatments. Spider venom‐derived peptide toxins are recognized as significant sources of Nav1.7 inhibitors. However, their development has been impeded by limited selectivity. In this study, eight peptide toxins from three distinct spider venom Nav channel families demonstrated robust inhibition of hNav1.7, rKv4.2, and rKv4.3 (rKv4.2/4.3) currents, exhibiting a similar mode of action. The analysis of structure and function relationship revealed a significant overlap in the pharmacophore responsible for inhibiting hNav1.7 and rKv4.2 by HNTX‐III, although Lys25 seems to play a more pivotal role in the inhibition of rKv4.2/4.3. Pharmacophore‐guided rational design is employed for the development of an mGpTx1 analogue, mGpTx1‐SA, which retains its inhibition of hNav1.7 while significantly reducing its inhibition of rKv4.2/4.3 and eliminating cardiotoxicity. Moreover, mGpTx1‐SA demonstrates potent analgesic effects in both inflammatory and neuropathic pain models, accompanied by an improved in vivo safety profile. The results suggest that off‐target inhibition of rKv4.2/4.3 by specific spider peptide toxins targeting hNav1.7 may arise from a conserved binding motif. This insight promises to facilitate the design of hNav1.7‐specific analgesics, aimed at minimizing rKv4.2/4.3 inhibition and associated toxicity, thereby enhancing their suitability for therapeutic applications. [ABSTRACT FROM AUTHOR]

Spider venoms contain a vast array of bioactive peptides targeting ion channels. A large number of peptides have high potency and selectivity toward sodium channels. Nav1.7 contributes to action potential generation and propagation and participates in pain signaling pathway. In this study, we describe the identification of μ-TRTX-Ca2a (Ca2a), a novel 35-residue peptide from the venom of Vietnam spider Cyriopagopus albostriatus (C. albostriatus) that potently inhibits Nav1.7 (IC50 = 98.1 ± 3.3 nM) with high selectivity against skeletal muscle isoform Nav1.4 (IC50 > 10 μM) and cardiac muscle isoform Nav1.5 (IC50 > 10 μM). Ca2a did not significantly alter the voltage-dependent activation or fast inactivation of Nav1.7, but it hyperpolarized the slow inactivation. Site-directed mutagenesis analysis indicated that Ca2a bound with Nav1.7 at the extracellular S3–S4 linker of domain II. Meanwhile, Ca2a dose-dependently attenuated pain behaviors in rodent models of formalin-induced paw licking, hot plate test, and acetic acid-induced writhing. This study indicates that Ca2a is a potential lead molecule for drug development of novel analgesics. [ABSTRACT FROM AUTHOR]

Spider venom is rich in peptide toxins that could be used to explore the structure and function of voltage-gated sodium channels (Na V s). This study has characterized a 44-amino acid peptide toxin, δ-hexatoxin-MrIX (δ-HXTX-MrIX), from the venom of the spider Macrothele raveni. δ-hexatoxin-MrIX potently inhibited the fast inactivation of Na V s in mouse cerebellar granule cells (CGCs) with an EC 50 of 35.3 ± 5.9 nM. The toxin shifted both the steady-state activation and the steady-state inactivation curves of CGC Na V s to the hyperpolarized direction. δ-hexatoxin-MrIX also acted on Na V 1.3 and Na V 1.4 channels heterologously expressed in HEK293T cells, as well as on Na V s in acutely isolated cockroach DUM neurons. However, the Na V 1.5, Na V 1.7 and Na V 1.8 channels were resistant to δ-hexatoxin-MrIX. The toxin inhibited the fast inactivation of Na V 1.3 and Na V 1.4 with high affinity (EC 50 values of 82.0 ± 3.0 nM and 24.0 ± 4.7 nM, respectively), but the saturating dose of toxin showed distinct efficacy on these two types of channels. δ-hexatoxin-MrIX is a peptide toxin acting on CGC Na V s and could be used as a pharmacological tool to explore the role of Na V s in granule cell maturation during cerebellum development. [ABSTRACT FROM AUTHOR]

Human voltage-gated sodium channel Na v 1.7 (hNa v 1.7) is involved in the generation and conduction of neuropathic and nociceptive pain signals. Compelling genetic and preclinical studies have validated that hNa v 1.7 is a therapeutic target for the treatment of pain; however, there is a dearth of currently available compounds capable of targeting hNav1.7 with high potency and specificity. Hainantoxin-III (HNTX-III) is a 33-residue polypeptide from the venom of the spider Ornithoctonus hainana. It is a selective antagonist of neuronal tetrodotoxin-sensitive voltage-gated sodium channels. Here, we report the engineering of improved potency and Na v selectivity of hNa v 1.7 inhibition peptides derived from the HNTX-III scaffold. Alanine scanning mutagenesis showed key residues for HNTX-III interacting with hNa v 1.7. Site-directed mutagenesis analysis indicated key residues on hNa v 1.7 interacting with HNTX-III. Molecular docking was conducted to clarify the binding interface between HNTX-III and Nav1.7 and guide the molecular engineering process. Ultimately, we obtained H4 [K0G1-P18K-A21L-V] based on molecular docking of HNTX-III and hNa v 1.7 with a 30-fold improved potency (IC 50 0.007 ± 0.001 μM) and >1000-fold selectivity against Na v 1.4 and Na v 1.5. H4 also showed robust analgesia in the acute and chronic inflammatory pain model and neuropathic pain model. Thus, our results provide further insight into peptide toxins that may prove useful in guiding the development of inhibitors with improved potency and selectivity for Na v subtypes with robust analgesia., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021. Published by Elsevier Inc.)

Spider venoms contain a vast array of bioactive peptides targeting ion channels. A large number of peptides have high potency and selectivity toward sodium channels. Na v 1.7 contributes to action potential generation and propagation and participates in pain signaling pathway. In this study, we describe the identification of μ-TRTX-Ca2a (Ca2a), a novel 35-residue peptide from the venom of Vietnam spider Cyriopagopus albostriatus ( C. albostriatus ) that potently inhibits Na v 1.7 (IC 50 = 98.1 ± 3.3 nM) with high selectivity against skeletal muscle isoform Na v 1.4 (IC 50 > 10 μM) and cardiac muscle isoform Na v 1.5 (IC 50 > 10 μM). Ca2a did not significantly alter the voltage-dependent activation or fast inactivation of Na v 1.7, but it hyperpolarized the slow inactivation. Site-directed mutagenesis analysis indicated that Ca2a bound with Na v 1.7 at the extracellular S3-S4 linker of domain II. Meanwhile, Ca2a dose-dependently attenuated pain behaviors in rodent models of formalin-induced paw licking, hot plate test, and acetic acid-induced writhing. This study indicates that Ca2a is a potential lead molecule for drug development of novel analgesics.

Pain is a medical condition that interferes with normal human life and work and reduces human well-being worldwide. The voltage-gated sodium channel (VGSC) human Na V 1.7 (hNa V 1.7) is a compelling target that plays a key role in human pain signaling. The 33-residue peptide µ-TRTX-Hhn2b (HNTX-I), a member of Na V -targeting spider toxin (NaSpTx) family 1, has shown negligible activity on mammalian VGSCs, including the hNa V 1.7 channel. We engineered analogues of HNTX-I based on sequence conservation in NaSpTx family 1. Substitution of Asn for Ser at position 23 or Asp for His at position 26 conferred potent activity against hNa V 1.7. Moreover, multiple site mutations combined together afforded improvements in potency. Ultimately, we generated an analogue E1G⁻N23S⁻D26H⁻L32W with >300-fold improved potency compared with wild-type HNTX-1 on hNa V 1.7 (IC 50 0.036 ± 0.007 µM). Structural simulation suggested that the charged surface and the hydrophobic surface of the modified peptide are responsible for binding affinity to the hNa V 1.7 channel, while variable residues may determine pharmacological specificity. Therefore, this study provides a profile for drug design targeting the hNa V 1.7 channel.