Discovery and molecular interaction studies of a highly stable, tarantula peptide modulator of acid-sensing ion channel 1.

Acute pharmacological inhibition of acid-sensing ion channel 1a (ASIC1a) is efficacious in rodent models in alleviating symptoms of neurological diseases such as stroke and multiple sclerosis. Thus, ASIC1a is a promising therapeutic target and selective ligands that modulate it are invaluable research tools and potential therapeutic leads. Spider venoms have provided an abundance of voltage-gated ion channel modulators, however, only one ASIC modulator (PcTx1) has so far been isolated from this source. Here we report the discovery, characterization, and chemical stability of a second spider venom peptide that potently modulates ASIC1a and ASIC1b, and investigate the molecular basis for its subtype selectivity. π-TRTX-Hm3a (Hm3a) is a 37-amino acid peptide isolated from Togo starburst tarantula (Heteroscodra maculata) venom with five amino acid substitutions compared to PcTx1, and is also three residues shorter at the C-terminus. Hm3a pH-dependently inhibited ASIC1a with an IC50 of 1-2 nM and potentiated ASIC1b with an EC50 of 46.5 nM, similar to PcTx1. Using ASIC1a to ASIC1b point mutants in rat ASIC1a revealed that Glu177 and Arg175 in the palm region opposite α-helix 5 play an important role in the Hm3a-ASIC1 interaction and contribute to the subtype-dependent effects of the peptide. Despite its high sequence similarity with PcTx1, Hm3a showed higher levels of stability over 48 h. Overall, Hm3a represents a potent, highly stable tool for the study of ASICs and will be particularly useful when stability in biological fluids is required, for example in long term in vitro cell-based assays and in vivo experiments. This article is part of the Special Issue entitled 'Venom-derived Peptides as Pharmacological Tools.'

Isolation of two insecticidal toxins from venom of the Australian theraphosid spider Coremiocnemis tropix.

Sheep flystrike is caused by parasitic flies laying eggs on soiled wool or open wounds, after which the hatched maggots feed on the sheep flesh and often cause large lesions. It is a significant economic problem for the livestock industry as infestations are difficult to control due to ongoing cycles of larval development into flies followed by further egg laying. We therefore screened venom fractions from the Australian theraphosid spider Coremiocnemis tropix to identify toxins active against the sheep blowfly Lucilia cuprina, which is the primary cause of flystrike in Australia. This screen led to isolation of two insecticidal peptides, Ct1a and Ct1b, that are lethal to blowflies within 24 h of injection. The primary structure of these peptides was determined using a combination of Edman degradation and sequencing of a C. tropix venom-gland transcriptome. Ct1a and Ct1b contain 39 and 38 amino acid residues, respectively, including six cysteine residues that form three disulfide bonds. Recombinant production in bacteria (Escherichia coli) resulted in low yields of Ct1a whereas solid-phase peptide synthesis using native chemical ligation produced sufficient quantities of Ct1a for functional analyses. Synthetic Ct1a had no effect on voltage-gated sodium channels from the American cockroach Periplanata americana or the German cockroach Blattella germanica, but it was lethal to sheep blowflies with an LD50 of 1687 pmol/g.

Selective inhibition of ASIC1a confers functional and morphological neuroprotection following traumatic spinal cord injury.

Tissue loss after spinal trauma is biphasic, with initial mechanical/haemorrhagic damage at the time of impact being followed by gradual secondary expansion into adjacent, previously unaffected tissue. Limiting the extent of this secondary expansion of tissue damage has the potential to preserve greater residual spinal cord function in patients. The acute tissue hypoxia resulting from spinal cord injury (SCI) activates acid-sensing ion channel 1a (ASIC1a). We surmised that antagonism of this channel should provide neuroprotection and functional preservation after SCI. We show that systemic administration of the spider-venom peptide PcTx1, a selective inhibitor of ASIC1a, improves locomotor function in adult Sprague Dawley rats after thoracic SCI. The degree of functional improvement correlated with the degree of tissue preservation in descending white matter tracts involved in hind limb locomotor function. Transcriptomic analysis suggests that PcTx1-induced preservation of spinal cord tissue does not result from a reduction in apoptosis, with no evidence of down-regulation of key genes involved in either the intrinsic or extrinsic apoptotic pathways. We also demonstrate that trauma-induced disruption of blood-spinal cord barrier function persists for at least 4 days post-injury for compounds up to 10 kDa in size, whereas barrier function is restored for larger molecules within a few hours. This temporary loss of barrier function provides a " treatment window" through which systemically administered drugs have unrestricted access to spinal tissue in and around the sites of trauma. Taken together, our data provide evidence to support the use of ASIC1a inhibitors as a therapeutic treatment for SCI. This study also emphasizes the importance of objectively grading the functional severity of initial injuries (even when using standardized impacts) and we describe a simple scoring system based on hind limb function that could be adopted in future studies.

Seven novel modulators of the analgesic target NaV 1.7 uncovered using a high-throughput venom-based discovery approach.

BACKGROUND AND PURPOSE: Chronic pain is a serious worldwide health issue, with current analgesics having limited efficacy and dose-limiting side effects. Humans with loss-of-function mutations in the voltage-gated sodium channel NaV 1.7 (hNaV 1.7) are indifferent to pain, making hNaV 1.7 a promising target for analgesic development. Since spider venoms are replete with NaV channel modulators, we examined their potential as a source of hNaV 1.7 inhibitors. EXPERIMENTAL APPROACH: We developed a high-throughput fluorescent-based assay to screen spider venoms against hNaV 1.7 and isolate 'hit' peptides. To examine the binding site of these peptides, we constructed a panel of chimeric channels in which the S3b-S4 paddle motif from each voltage sensor domain of hNaV 1.7 was transplanted into the homotetrameric KV 2.1 channel. KEY RESULTS: We screened 205 spider venoms and found that 40% contain at least one inhibitor of hNaV 1.7. By deconvoluting 'hit' venoms, we discovered seven novel members of the NaSpTx family 1. One of these peptides, Hd1a (peptide µ-TRTX-Hd1a from venom of the spider Haplopelma doriae), inhibited hNaV 1.7 with a high level of selectivity over all other subtypes, except hNaV 1.1. We showed that Hd1a is a gating modifier that inhibits hNaV 1.7 by interacting with the S3b-S4 paddle motif in channel domain II. The structure of Hd1a, determined using heteronuclear NMR, contains an inhibitor cystine knot motif that is likely to confer high levels of chemical, thermal and biological stability. CONCLUSION AND IMPLICATIONS: Our data indicate that spider venoms are a rich natural source of hNaV 1.7 inhibitors that might be useful leads for the development of novel analgesics.

Spider-venom peptides that target voltage-gated sodium channels: pharmacological tools and potential therapeutic leads.

Voltage-gated sodium (Na(V)) channels play a central role in the propagation of action potentials in excitable cells in both humans and insects. Many venomous animals have therefore evolved toxins that modulate the activity of Na(V) channels in order to subdue their prey and deter predators. Spider venoms in particular are rich in Na(V) channel modulators, with one-third of all known ion channel toxins from spider venoms acting on Na(V) channels. Here we review the landscape of spider-venom peptides that have so far been described to target vertebrate or invertebrate Na(V) channels. These peptides fall into 12 distinct families based on their primary structure and cysteine scaffold. Some of these peptides have become useful pharmacological tools, while others have potential as therapeutic leads because they target specific Na(V) channel subtypes that are considered to be important analgesic targets. Spider venoms are conservatively predicted to contain more than 10 million bioactive peptides and so far only 0.01% of this diversity been characterised. Thus, it is likely that future research will reveal additional structural classes of spider-venom peptides that target Na(V) channels.

Spider-venom peptides as therapeutics.

Spiders are the most successful venomous animals and the most abundant terrestrial predators. Their remarkable success is due in large part to their ingenious exploitation of silk and the evolution of pharmacologically complex venoms that ensure rapid subjugation of prey. Most spider venoms are dominated by disulfide-rich peptides that typically have high affinity and specificity for particular subtypes of ion channels and receptors. Spider venoms are conservatively predicted to contain more than 10 million bioactive peptides, making them a valuable resource for drug discovery. Here we review the structure and pharmacology of spider-venom peptides that are being used as leads for the development of therapeutics against a wide range of pathophysiological conditions including cardiovascular disorders, chronic pain, inflammation, and erectile dysfunction.