Background and Purpose: Blocking the voltage-gated proton channel H V 1 is a promising strategy for the treatment of diseases like ischaemia stroke and cancer. However, few H V 1 channel antagonists have been reported. Here, we have identified a novel H V 1 channel antagonist from scorpion venom and have elucidated its action mechanism.Experimental Approach: H V 1 and NaV channels were heterologously expressed in mammalian cell lines and their currents recorded using whole-cell patch clamp. Sitedirected mutagenesis was used to generate mutants. Toxins were recombinantly produced in Escherichia coli. AGAP/W38F-H V 1 interaction was modelled by molecular dynamics simulations.Key Results: The scorpion toxin AGAP (anti-tumour analgesic peptide) potently inhibited H V 1 currents. One AGAP mutant has reduced Na V channel activity but intact H V 1 activity (AGAP/W38F). AGAP/W38F inhibited H V 1 channel activation by trapping its S4 voltage sensor in a deactivated state and inhibited H V 1 currents with less pH dependence than Zn 2+ . Mutation analysis showed that the binding pockets of AGAP/W38F and Zn 2+ in H V 1 channel partly overlapped (common sites are His140 and His193). The E153A mutation at the intracellular Coulombic network (ICN) in H V 1 channel markedly reduced AGAP/W38F inhibition, as observed for Zn 2+ . Experimental data and MD simulations suggested that AGAP/W38F inhibited H V 1 channel using a Zn 2+ -like long-range conformational coupling mechanism.Conclusion and Implications: Our results suggest that the Zn 2+ binding pocket in H V 1 channel might be a hotspot for modulators and valuable for designing H V 1 channel ligands. Moreover, AGAP/W38F is a useful molecular probe to study H V 1 channel and a lead compound for drug development.

Pain is a medical condition that interferes with normal human life and work and reduces human well-being worldwide. Human voltage-gated sodium channel NaV1.7 (hNaV1.7) 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 NaV-targeting spider toxin (NaSpTx) family 1, has shown negligible activity on mammalian voltage-gated sodium channels (VGSCs), including the hNaV1.7 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 hNaV1.7. 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-I on hNaV1.7 (IC50 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 hNaV1.7 channel, while variable residues may determine pharmacological specificity. Therefore, this study provides a profile for drug design targeting the hNaV1.7 channel.

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.

Edited by Mike ShipstonHuman 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 33residue 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.Voltage-gated sodium channels (VGSCs or Na v s) are essential for the initiation and propagation of action potentials in excitable tissues such as nerve, muscle, and other excitable cells (1, 2). Structurally, all VGSCs consist of an approximately 260-kDa α subunit and associated smaller β subunits. Nine distinct VGSC α subunit subtypes (Na v 1.1-Na v 1.9) have been

Human genetic and pharmacological studies have demonstrated that voltage-gated sodium channels (VGSCs) are promising therapeutic targets for the treatment of pain. Spider venom contains many toxins that modulate the activity of VGSCs. To date, only 0.01% of such spider toxins has been explored, and thus there is a great potential for discovery of novel VGSC modulators as useful pharmacological tools or potential therapeutics. In the current study, we identified a novel peptide, µ-TRTX-Ca1a (Ca1a), in the venom of the tarantula Cyriopagopus albostriatus. This peptide consisted of 38 residues, including 6 cysteines, i.e. IFECSISCEIEKEGNGKKCKPKKCKGGWKCKFNICVKV. In HEK293T or ND7/23 cells expressing mammalian VGSCs, this peptide exhibited the strongest inhibitory activity on Na v 1.7 (IC 50 378 nM), followed by Na v 1.6 (IC 50 547 nM), Na v 1.2 (IC 50 728 nM), Na v 1.3 (IC 50 2.2 µM) and Na v 1.4 (IC 50 3.2 µM), and produced negligible inhibitory effect on Na v 1.5, Na v 1.8, and Na v 1.9, even at high concentrations of up to 10 µM. Furthermore, this peptide did not significantly affect the activation and inactivation of Na v 1.7. Using site-directed mutagenesis of Na v 1.7 and Na v 1.4, we revealed that its binding site was localized to the DIIS3-S4 linker region involving the D816 and E818 residues. In three different mouse models of pain, pretreatment with Cala (100, 200, 500 µg/kg) dose-dependently suppressed the nociceptive responses induced by formalin, acetic acid or heat. These results suggest that Ca1a is a novel neurotoxin against VGSCs and has a potential to be developed as a novel analgesic.