Search

Hi1a is a naturally occurring bivalent spider-venom peptide that is being investigated as a promising molecule for limiting ischemic damage in strokes, myocardial infarction, and organ transplantation. However, the challenges associated with the synthesis and production of the peptide in large quantities have slowed the progress in this area; hence, access to synthetic Hi1a is an essential milestone for the development of Hi1a as a pharmacological tool and potential therapeutic.

The King Baboon spider, Pelinobius muticus , is a burrowing African tarantula. Its impressive size and appealing coloration are tempered by reports describing severe localized pain, swelling, itchiness, and muscle cramping after accidental envenomation. Hyperalgesia is the most prominent symptom after bites from P. muticus , but the molecular basis by which the venom induces pain is unknown. Proteotranscriptomic analysis of P. muticus venom uncovered a cysteine-rich peptide, δ/κ-theraphotoxin-Pm1a (δ/κ-TRTX-Pm1a), that elicited nocifensive behavior when injected into mice. In small dorsal root ganglion neurons, synthetic δ/κ-TRTX-Pm1a (sPm1a) induced hyperexcitability by enhancing tetrodotoxin-resistant sodium currents, impairing repolarization and lowering the threshold of action potential firing, consistent with the severe pain associated with envenomation. The molecular mechanism of nociceptor sensitization by sPm1a involves multimodal actions over several ion channel targets, including Na V 1.8, K V 2.1, and tetrodotoxin-sensitive Na V channels. The promiscuous targeting of peptides like δ/κ-TRTX-Pm1a may be an evolutionary adaptation in pain-inducing defensive venoms., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Hi1a is a venom peptide from the Australian funnel-web spider Hadronyche infensa with a complex tertiary structure. Hi1a has neuroprotective and cardioprotective properties due to its potent inhibition of acid-sensing ion channel 1a (ASIC1a) and is currently being pursued as a novel therapy for acute ischemic events. Herein, we describe the total synthesis of Hi1a using native chemical ligation. The synthetic peptide was successfully folded and exhibited similar inhibitory activity on ASIC1a to recombinant Hi1a.

Acid-sensing ion channels (ASICs) are primary acid sensors in the mammalian nervous system that are activated by protons under conditions of local acidosis. They have been implicated in a range of pathologies including ischemic stroke (ASIC1a subtype) and peripheral pain (ASIC1b and ASIC3). Although the spider venom peptide PcTx1 is the best-studied ASIC modulator and is neuroprotective in rodent models of ischemic stroke, little experimental work has been done to examine its molecular interaction with human ASIC1a or the off-target ASIC1b. The complementary face of the acidic pocket binding site of PcTx1 is where these channels differ in sequence. We show here that although PcTx1 is 10-fold less potent at human ASIC1a than the rat channel, the apparent affinity for the two channels is comparable. We examined the pharmacophore of PcTx1 for human ASIC1a and rat ASIC1b, and show that inhibitory and stimulatory effects at each ASIC1 variant is driven mostly by a shared set of core peptide pharmacophore residues that bind to the thumb domain, while peptide residues that interact with the complementary face of the biding site underlie species and subtype-dependent differences in activity that may allow manipulation of ASIC1 variant selectivity. Finally, the stimulatory effect of PcTx1 on rat ASIC1a when applied under mildly alkaline pH correlates with low receptor occupancy. These new insights into the interactions between PcTx1 with ASIC1 subtypes demonstrates the complexity of its mechanism of action, and highlights important implications to consider when using PcTx1 as a pharmacological tool to study ASIC function., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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 IC 50 of 1-2 nM and potentiated ASIC1b with an EC 50 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.', (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Voltage-gated sodium (Na V ) channels are essential for the transmission of pain signals in humans making them prime targets for the development of new analgesics. Spider venoms are a rich source of peptide modulators useful to study ion channel structure and function. Here we describe β/δ-TRTX-Pre1a, a 35-residue tarantula peptide that selectively interacts with neuronal Na V channels inhibiting peak current of hNa V 1.1, rNa V 1.2, hNa V 1.6, and hNa V 1.7 while concurrently inhibiting fast inactivation of hNa V 1.1 and rNa V 1.3. The DII and DIV S3-S4 loops of Na V channel voltage sensors are important for the interaction of Pre1a with Na V channels but cannot account for its unique subtype selectivity. Through analysis of the binding regions we ascertained that the variability of the S1-S2 loops between Na V channels contributes substantially to the selectivity profile observed for Pre1a, particularly with regards to fast inactivation. A serine residue on the DIV S2 helix was found to be sufficient to explain Pre1a's potent and selective inhibitory effect on the fast inactivation process of Na V 1.1 and 1.3. This work highlights that interactions with both S1-S2 and S3-S4 of Na V channels may be necessary for functional modulation, and that targeting the diverse S1-S2 region within voltage-sensing domains provides an avenue to develop subtype selective tools.

Venom peptides are natural ligands of ion channels and have been used extensively in pharmacological characterization of various ion channels and receptors. In this chapter, we survey all known venom peptide ion-channel modulators. Our survey reveals that the majority of venom peptides characterized to date target voltage-gated sodium or potassium channels. We further find that the majority of these peptides are found in scorpion and spider venoms. We discuss the influence of the pharmacological tools available in biasing discovery and the classical "toxin-to-sequence" approach to venom peptide biodiscovery. The impact of high-throughput sequencing on the existing discovery framework is likely to be significant and we propose here an alternative "sequence-to-toxin" approach to peptide screening, relying more on recently developed high-throughput methods. Methods for production and characterization of disulfide rich toxins in a high-throughput setting are then described, focusing on bacterial protein expression and solution state structural characterization by NMR spectroscopy. Finally, the role of X-ray crystallography and cryo-EM are highlighted by discussing the currently known channel-peptide complexes., (© 2017 Elsevier Inc. All rights reserved.)

Acid-sensing ion channel 1a (ASIC1a) is the primary acid sensor in mammalian brain and plays a major role in neuronal injury following cerebral ischemia. Evidence that inhibition of ASIC1a might be neuroprotective following stroke was previously obtained using "PcTx1 venom" from the tarantula Psalmopeous cambridgei. We show here that the ASIC1a-selective blocker PcTx1 is present at only 0.4% abundance in this venom, leading to uncertainty as to whether the observed neuroprotective effects were due to PcTx1 blockade of ASIC1a or inhibition of other ion channels and receptors by the hundreds of peptides and small molecules present in the venom. We therefore examined whether pure PcTx1 is neuroprotective in a conscious model of stroke via direct inhibition of ASIC1a. A focal reperfusion model of stroke was induced in conscious spontaneously hypertensive rats (SHR) by administering endothelin-1 to the middle cerebral artery via a surgically implanted cannula. Two hours later, SHR were treated with a single intracerebroventricular (i.c.v.) dose of PcTx1 (1 ng/kg), an ASIC1a-inactive mutant of PcTx1 (1 ng/kg), or saline, and ledged beam and neurological tests were used to assess the severity of symptomatic changes. PcTx1 markedly reduced cortical and striatal infarct volumes measured 72 h post-stroke, which correlated with improvements in neurological score, motor function and preservation of neuronal architecture. In contrast, the inactive PcTx1 analogue had no effect on stroke outcome. This is the first demonstration that selective pharmacological inhibition of ASIC1a is neuroprotective in conscious SHRs, thus validating inhibition of ASIC1a as a potential treatment for stroke., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background and Purpose: The spider-venom peptide PcTx1 is the most potent and selective inhibitor of acid-sensing ion channel (ASIC) 1a. It has centrally acting analgesic activity and is neuroprotective in rodent models of ischaemic stroke. Understanding the molecular details of the PcTx1 : ASIC1a interaction should facilitate development of therapeutically useful ASIC1a modulators. Previously, we showed that several key pharmacophore residues of PcTx1 reside in a dynamic β-hairpin loop; conclusions confirmed by recent crystal structures of the complex formed between PcTx1 and chicken ASIC1 (cASIC1). Numerous peptide : channel contacts were observed in these crystal structures, but it remains unclear which of these are functionally important., Experimental Approach: We combined molecular dynamics (MD) simulations of the PcTx1 : cASIC1 complex with mutagenesis of PcTx1 and rat ASIC1a., Key Results: Crystal structures of the PcTx1 : cASIC1 complex indicated that 15 PcTx1 residues form a total of 57 pairwise intermolecular contacts (<5 Å) with 32 channel residues. MD simulations, however, suggested that about half of these interactions do not persist in solution. Mutation to alanine of only eight of 15 PcTx1 contact residues substantially altered ASIC1a inhibition by PcTx1. Our data reveal that many of the peptide-channel interactions observed in the PcTx1 : cASIC1 crystal structures are not important for PcTx1 inhibition of rat ASIC1a., Conclusions and Implications: We identified the atomic interactions that are critical for PcTx1 inhibition of ASIC1a. Our data highlight the value of combining structural information, MD and functional experiments to obtain detailed insight into the molecular basis of protein : protein interactions., (© 2015 The British Pharmacological Society.)

Mambalgins are a novel class of snake venom components that exert potent analgesic effects mediated through the inhibition of acid-sensing ion channels (ASICs). The 57-residue polypeptide mambalgin-2 (Ma-2) was synthesized by using a combination of solid-phase peptide synthesis and native chemical ligation. The structure of the synthetic toxin, determined using homonuclear NMR, revealed an unusual three-finger toxin fold reminiscent of functionally unrelated snake toxins. Electrophysiological analysis of Ma-2 on wild-type and mutant ASIC1a receptors allowed us to identify α-helix 5, which borders on the functionally critical acidic pocket of the channel, as a major part of the Ma-2 binding site. This region is also crucial for the interaction of ASIC1a with the spider toxin PcTx1, thus suggesting that the binding sites for these toxins substantially overlap. This work lays the foundation for structure-activity relationship (SAR) studies and further development of this promising analgesic peptide., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Disulfide-rich peptides are the dominant component of most animal venoms. These peptides have received much attention as leads for the development of novel therapeutic agents and bioinsecticides because they target a wide range of neuronal receptors and ion channels with a high degree of potency and selectivity. In addition, their rigid disulfide framework makes them particularly well suited for addressing the crucial issue of in vivo stability. Structural and functional characterization of these peptides necessitates the development of a robust, reliable expression system that maintains their native disulfide framework. The bacterium Escherichia coli has long been used for economical production of recombinant proteins. However, the expression of functional disulfide-rich proteins in the reducing environment of the E. coli cytoplasm presents a significant challenge. Thus, we present here an optimised protocol for the expression of disulfide-rich venom peptides in the periplasm of E. coli, which is where the endogenous machinery for production of disulfide-bonds is located. The parameters that have been investigated include choice of media, induction conditions, lysis methods, methods of fusion protein and peptide purification, and sample preparation for NMR studies. After each section a recommendation is made for conditions to use. We demonstrate the use of this method for the production of venom peptides ranging in size from 2 to 8 kDa and containing 2-6 disulfide bonds.

Amphibian skin secretions are well known as a rich source of bioactive peptides. However, little is known about the presence or role of peptides in the highly toxic, parotid secretion of the cane toad or giant toad, Bufo marinus (Rhinella marina), though small molecule bufadienolides, which act as potent cardiotoxins, have been described. In the current study we used RP-HPLC, MALDI-TOF mass spectrometry and tandem mass spectrometry to analyze and determine the first sequences of peptides from the parotid secretion of B. marinus. We show that peptides in the range of 900-2500 Da are indeed present, however in extremely low abundance. Despite the low abundance, the sequences of 14 peptides were determined, several of which match fragments of larger cellular proteins, yet none share substantial homology with defensive or anti-microbial peptides reported from frog skin secretions. We conclude that peptides are present in the parotid glands of B. marinus only in very low quantities and that they are likely to be breakdown products of proteins involved in cell maintenance. Given these results, we conclude that peptides are unlikely to contribute directly to the high toxicity of the cane toad., (Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.)

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.

Stroke is the second-leading cause of death worldwide, yet there are no drugs available to protect the brain from stroke-induced neuronal injury. Acid-sensing ion channel 1a (ASIC1a) is the primary acid sensor in mammalian brain and a key mediator of acidosis-induced neuronal damage following cerebral ischemia. Genetic ablation and selective pharmacologic inhibition of ASIC1a reduces neuronal death following ischemic stroke in rodents. Here, we demonstrate that Hi1a, a disulfide-rich spider venom peptide, is highly neuroprotective in a focal model of ischemic stroke. Nuclear magnetic resonance structural studies reveal that Hi1a comprises two homologous inhibitor cystine knot domains separated by a short, structurally well-defined linker. In contrast with known ASIC1a inhibitors, Hi1a incompletely inhibits ASIC1a activation in a pH-independent and slowly reversible manner. Whole-cell, macropatch, and single-channel electrophysiological recordings indicate that Hi1a binds to and stabilizes the closed state of the channel, thereby impeding the transition into a conducting state. Intracerebroventricular administration to rats of a single small dose of Hi1a (2 ng/kg) up to 8 h after stroke induction by occlusion of the middle cerebral artery markedly reduced infarct size, and this correlated with improved neurological and motor function, as well as with preservation of neuronal architecture. Thus, Hi1a is a powerful pharmacological tool for probing the role of ASIC1a in acid-mediated neuronal injury and various neurological disorders, and a promising lead for the development of therapeutics to protect the brain from ischemic injury. [ABSTRACT FROM AUTHOR]

Venom-derived peptides have attracted much attention as potential lead molecules for pharmaceutical development. A well-known example is Huwentoxin-IV (HwTx-IV), a peptide toxin isolated from the venom of the Chinese bird-eating spider Haplopelma schmitdi. HwTx-IV was identified as a potent blocker of a human voltage-gated sodium channel (hNaV1.7), which is a genetically validated analgesic target. The peptide was promising as it showed high potency at NaV1.7 (IC50 ~26 nM) and selectivity over the cardiac NaV subtype (NaV1.5). Mutagenesis studies aimed at optimising the potency of the peptide resulted in the development of a triple-mutant of HwTx-IV (E1G, E4G, Y33W, m3-HwTx-IV) with significantly increased potency against hNaV1.7 (IC50 = 0.4 ± 0.1 nM) without increased potency against hNaV1.5. The activity of m3-HwTx-IV against other NaV subtypes was, however, not investigated. Similarly, the structure of the mutant peptide was not characterised, limiting the interpretation of the observed increase in potency. In this study we produced isotope-labelled recombinant m3-HwTx-IV in E. coli, which enabled us to characterise the atomic-resolution structure and dynamics of the peptide by NMR spectroscopy. The results show that the structure of the peptide is not perturbed by the mutations, whilst the relaxation studies reveal that residues in the active site of the peptide undergo conformational exchange. Additionally, the NaV subtype selectivity of the recombinant peptide was characterised, revealing potent inhibition of neuronal NaV subtypes 1.1, 1.2, 1.3, 1.6 and 1.7. In parallel to the in vitro studies, we investigated NaV1.7 target engagement of the peptide in vivo using a rodent pain model, where m3-HwTx-IV dose-dependently suppressed spontaneous pain induced by the NaV1.7 activator OD1. Thus, our results provide further insight into the structure and dynamics of this class of peptides that may prove useful in guiding the development of inhibitors with improved selectivity for analgesic NaV subtypes. [ABSTRACT FROM AUTHOR]

Mambalgins are a novel class of snake venom components that exert potent analgesic effects mediated through the inhibition of acid-sensing ion channels (ASICs). The 57-residue polypeptide mambalgin-2 (Ma-2) was synthesized by using a combination of solid-phase peptide synthesis and native chemical ligation. The structure of the synthetic toxin, determined using homonuclear NMR, revealed an unusual three-finger toxin fold reminiscent of functionally unrelated snake toxins. Electrophysiological analysis of Ma-2 on wild-type and mutant ASIC1a receptors allowed us to identify α-helix 5, which borders on the functionally critical acidic pocket of the channel, as a major part of the Ma-2 binding site. This region is also crucial for the interaction of ASIC1a with the spider toxin PcTx1, thus suggesting that the binding sites for these toxins substantially overlap. This work lays the foundation for structure-activity relationship (SAR) studies and further development of this promising analgesic peptide. [ABSTRACT FROM AUTHOR]

From a systematic screening of animal venoms, we isolated a new toxin (APETx2) from the sea anemone Anthopleura elegantissima, which inhibits ASIC3 homomeric channels and ASIC3-containing heteromeric channels both in heterologous expression systems and in primary cultures of rat sensory neurons. APETx2 is a 42 amino-acid peptide crosslinked by three disulfide bridges, with a structural organization similar to that of other sea anemone toxins that inhibit voltage-sensitive Na+ and K+ channels. APETx2 reversibly inhibits rat ASIC3 (IC50=63 nM), without any effect on ASIC1a, ASIC1b, and ASIC2a. APETx2 directly inhibits the ASIC3 channel by acting at its external side, and it does not modify the channel unitary conductance. APETx2 also inhibits heteromeric ASIC2b+3 current (IC50=117 nM), while it has less affinity for ASIC1b+3 (IC50=0.9 µM), ASIC1a+3 (IC50=2 µM), and no effect on the ASIC2a+3 current. The ASIC3-like current in primary cultured sensory neurons is partly and reversibly inhibited by APETx2 with an IC50 of 216 nM, probably due to the mixed inhibitions of various co-expressed ASIC3-containing channels. [ABSTRACT FROM AUTHOR]

[Display omitted] Acid-sensing ion channel 1a (ASIC1a) is a proton-gated channel involved in synaptic transmission, pain signalling, and several ischemia-associated pathological conditions. The spider venom-derived peptides PcTx1 and Hi1a are two of the most potent ASIC1a inhibitors known and have been instrumental in furthering our understanding of the structure, function, and biological roles of ASICs. To date, homologous spider peptides with different pharmacological profiles at ASIC1a have yet to be discovered. Here we report the characterisation of Hc3a, a single inhibitor cystine knot peptide from the Australian funnel-web spider Hadronyche cerberea with sequence similarity to PcTx1. We show that Hc3a has complex pharmacology and binds different ASIC1a conformational states (closed, open, and desensitised) with different affinities, with the most prominent effect on desensitisation. Hc3a slows the desensitisation kinetics of proton-activated ASIC1a currents across multiple application pHs, and when bound directly to ASIC1a in the desensitised conformation promotes current inhibition. The solution structure of Hc3a was solved, and the peptide-channel interaction examined via mutagenesis studies to highlight how small differences in sequence between Hc3a and PcTx1 can lead to peptides with distinct pharmacology. The discovery of Hc3a expands the pharmacological diversity of spider venom peptides targeting ASIC1a and adds to the toolbox of compounds to study the intricacies of ASIC1 gating. [ABSTRACT FROM AUTHOR]