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Acid-sensing ion channels (ASICs) are expressed in the nervous system, activated by acidosis, and implicated in pain pathways. Mambalgins are peptide inhibitors of ASIC1 and analgesic in rodents via inhibition of centrally expressed ASIC1a and peripheral ASIC1b. This activity has generated interest in mambalgins as potential therapeutics. However, most mechanism and structure–activity relationship work on mambalgins has focused on ASIC1a, and neglected the peripheral analgesic target ASIC1b. Here, we compare mambalgin potency and mechanism of action at heterologously expressed rat and human ASIC1 variants. Unlike the nanomolar inhibition at ASIC1a and rodent ASIC1b, we find mambalgin-3 only weakly inhibits human ASIC1b and ASIC1b/3 under severe acidosis, but potentiates currents under mild/moderate acidosis. Our data highlight the importance of understanding the activity of potential ASIC-targeting pharmaceuticals at human channels.

Acid-sensing ion channels (ASICs) are neuronal voltage-independent Na+ channels that are activated by extracellular acidification. ASICs play essential roles in a wide range of physiological processes, including sodium homeostasis, synaptic plasticity, neurodegeneration, and sensory transduction. Mambalgins, a family of three-finger toxins isolated from black mamba venom, specifically inhibit ASICs to exert strong analgesic effects in vivo, thus are thought to have potential therapeutic values against pain. However, the interaction and inhibition mechanism of mambalgin on ASICs remains elusive. Here, we report a cryo-electron microscopy (cryo-EM) structure of chicken ASIC1a (cASIC1a) in complex with mambalgin-1 toxin at 5.4 Å resolution. Our structure provides the first experimental evidence that mambalgin-1 interacts directly with the extracellular thumb domain of cASIC1a, rather than inserting into the acid-sensing pocket, as previously reported. Binding of mambalgin-1 leads to relocation of the thumb domain that could disrupt the acidic pocket of cASIC1a, illustrating an unusual inhibition mechanism of toxins on ASIC channels through an allosteric effect. These findings establish a structural basis for the toxicity of the mambalgins, and provide crucial insights for the development of new optimized inhibitors of ASICs.

International audience; Mambalgins are 57-amino acid peptides isolated from mamba venom. They produce potent analgesic effects in mice against inflammatory pain upon central intrathecal (i.t.), and peripheral local (i.pl.) injections, through inhibition of different ASICs subtypes and involvement of opioid-independent pathways. They produce fewer side effects than morphine and no apparent toxicity. We now show that mambalgins also have an opioid-independent effect on both thermal and mechanical pain upon systemic intravenous (i.v.) administration and are effective against neuropathic pain by i.v., i.t. and i. pl. injections. By combining the use of knockdown and knockout animals, we show the critical involvement of peripheral ASIC1b-containing channels in the i.v. effects of mambalgins against inflammatory pain. The potent analgesic effect on neuropathic pain involves two different mechanisms depending on the route of administration, a naloxone-insensitive and ASIC1a-independent effect associated with i.v. injection, and an ASIC1a-dependent and partially naloxone-sensitive effect associated with i.t. injection. We have done in collaboration with the CEA iBiTecS Institute in Gif-sur-Yvette, the full stepwise solid-phase peptide synthesis of mambalgin-1, solved its 3D crystal structure, mapped the pharmacophore, and identified the binding site and the inhibitory mechanism on ASIC1a channels. These findings identify new roles for ASICs in pain pathways, and mambalgins as new potential analgesics against inflammatory and chronic neuropathic pain.

In lumbar radiculopathy, the dorsal root or dorsal root ganglia (DRG) are compressed or affected by herniated discs or degenerative spinal canal stenosis. The disease is multi-factorial and involves almost all types of pain, such as ischemic, inflammatory, mechanical, and neuropathic pain. Acid-sensing ion channels (ASICs) activated by extracellular acidosis play an important role in pain generation, and the effects of ASICs are widespread in lumbar radiculopathy. ASICs may be involved in the disc degeneration process, which results in disc herniation and, therefore, the compression of the dorsal roots or DRG. ASIC3 is involved in inflammatory pain and ischemic pain, and, likely, mechanical pain. ASIC1a and ASIC3 may have an important effect on control of the vascular tone of the radicular artery. In the central nervous system, ASIC1a modulates the central sensitization of the spinal dorsal horn. Thus, toxins targeting ASICs, because of their specificity, may help elucidate the roles of ASICs in lumbar radiculopathy and could be developed as novel analgesic agents.

Background: The word “venom” has always been taken as the synonym for poison or a vile liquid with catastrophic effects. Out of several different venomous creatures, snakes have perked special interest of scientists and researchers in this matter across the world. Many scientists and researchers around the world are trying to explore and put forward the positive aspects of snake venom and in the past years they have come to the conclusion that snake venom has some potential effects against serious ailments like cancer, diabetes and hypertension. Objective: The aim of current research is to make people realize that the snake venom, despite being a deadly poison, may prove to be a treatment option in several rather incurable diseases. Methodology: A survey was conducted that was based on 200 (n = 200) people including common public, medical students and health practitioners to know their general opinion and to awake people regarding the beneficial aspects of snake venom. Result: The study shows that about 48.5% of them think that snake venom can be used for beneficial purposes, while 31% think of it only as a deadly poison, whereas 20.5% of the people have absolutely no idea about it. The researchers believe that proteins like “eristostatin” and “contortrostatin” found in the venoms of Asian sand viper and South American rattlesnake respectively have the ability to fight against cancers. “Mambalgins” (from the venom of black mamba) are believed to have analgesic and anti-inflammatory effects. Venoms are also being used in skin care products. Conclusion: We conclude that medical practitioners should come forward and explore more on this topic, so that maybe snake venom can prove to be another successful source of medication for several serious diseases.

Background: Despite a wide variety of current analgesia regimens, chronic pain is an incredibly difficult condition to treat. Its pathophysiology, initiation, development and course involve a range of different receptors and transmitters. The acid-sensing ion channels (ASICs) are being attributed to an increasingly larger significance in pain aetiology. Over the last few years, the mechanism of ASICs action, influence of their antagonists/agonists and clinical applications have been well described. However, the importance of this protein is significantly larger, not only from the perspective of pain management, but also in psychiatry of addiction or fear. Recently discovered peptides from three-finger toxin group, called mambalgins (isolated from Dendroaspis polylepis polylepsis) exhibit potent analgesic mechanisms of action on ASICs in animal model. Aims & Methods: The article reviews current knowledge in the field of mambalgins and assesses their potential analgesic application, based on the recent experimental evidence. Results: The mambalgins seem to decrease the intensity of the inflammatory, neuropathic and mechanic pain. This has been demonstrated in animal studies of different pain models, including carrageenan- induced inflammatory pain, chronic constrictive injury-induced neuropathic pain and thermal pain. The mechanism of mambalgin action is not clearly defined, but it is suspected that they bind directly to the pH-sensitive region of the ASIC. Conclusion: In this short review, we attempted to summarise the current knowledge about mambalgins and their potential applications as a new substance in searching for the ideal analgesia without common side effects of the other drug groups.

International audience; Mambalgins are 57-amino acid peptides isolated from snake venom that evoke naloxone-resistant analgesia after local (intraplantar) and central (intrathecal) injections through inhibition of particular subtypes of acid-sensing ion channels (ASICs). We now show that mambalgins also have an opioid-independent effect on both thermal and mechanical inflammatory pain after systemic intravenous (i.v.) administration and are effective against neuropathic pain. By combining the use of knockdown and knockout animals, we show the critical involvement of peripheral ASIC1b-containing channels, along with a contribution of ASIC1a-containing channels, in the i.v. effects of these peptides against inflammatory pain. The potent analgesic effect on neuropathic pain involves 2 different mechanisms depending on the route of administration, a naloxone-insensitive and ASIC1a-independent effect associated with i.v. injection and an ASIC1a-dependent and partially naloxone-sensitive effect associated with intrathecal injection. These data further support the role of peripheral and central ASIC1-containing channels in pain, demonstrate their participation in neuropathic pain, and highlight differences in the repertoire of channels involved in different pain conditions. They also strengthen the therapeutic potential of mambalgin peptides that are active in a broader range of experimental pain models and through i.v. systemic delivery.

Acid-sensing ion channels (ASICs) are neuronal proton-gated cation channels associated with nociception, fear, depression, seizure, and neuronal degeneration, suggesting roles in pain and neurological and psychiatric disorders. We have recently discovered black mamba venom peptides called mambalgin-1 and mambalgin-2, which are new three-finger toxins that specifically inhibit with the same pharmacological profile ASIC channels to exert strong analgesic effects in vivo. We now combined bioinformatics and functional approaches to uncover the molecular mechanism of channel inhibition by the mambalgin-2 pain-relieving peptide. Mambalgin-2 binds mainly in a region of ASIC1a involving the upper part of the thumb domain (residues Asp-349 and Phe-350), the palm domain of an adjacent subunit, and the β-ball domain (residues Arg-190, Asp-258, and Gln-259). This region overlaps with the acidic pocket (pH sensor) of the channel. The peptide exerts both stimulatory and inhibitory effects on ASIC1a, and we propose a model where mambalgin-2 traps the channel in a closed conformation by precluding the conformational change of the palm and β-ball domains that follows proton activation. These data help to understand inhibition by mambalgins and provide clues for the development of new optimized blockers of ASIC channels.

International audience; Acid-sensing ion channels (ASICs) are voltage-independent proton-gated cation channels that are largely expressed in the nervous system as well as in some non-neuronal tissues. In rodents, six different isoforms (ASIC1a, 1b, 2a, 2b, 3 and 4) can associate into homo-or hetero-trimers to form a functional channel. Specific polypeptide toxins targeting ASIC channels have been isolated from the venoms of spider (PcTx1), sea anemone (APETx2) and snakes (MitTx and mambalgins). They exhibit different and sometimes partially overlapping pharmacological profiles and are usually blockers of ASIC channels, except for MitTx, which is a potent activator. This review focuses on the use of these toxins to explore the structure-function relationships, the physiological and the pathophysiological roles of ASIC channels, illustrating at the same time the therapeutic potential of some of these natural compounds.

French researchers have discovered potent painkilling peptides from an unlikely source—the venom of black mamba snakes (Nature, DOI: 10.1038/nature11494). The peptides, which relieve pain in mice as powerfully as morphine does, act by a mechanism different from that of available painkillers. The discovery could help researchers expand the legions of painkillers, which currently rely heavily on opioids that carry side effects and potential for abuse. Sylvie Diochot, Anne Baron, and colleagues at the Institute of Molecular & Cellular Pharmacology near Nice isolated the peptides, which they named mambalgins. The mambalgins appeal as leads for pain drugs because, unlike morphine, they don’t cause respiratory complications, Baron says. Through studies using knockout mice, her team found that the peptides relieve pain by blocking acid-sensing ion channels in the skin and central nervous system. Although scientists have known that this family of channels plays a role in pain pathways, Baron says, none of ...

Acid-sensing ion channels (ASICs) are a family of proton-activated cation channels expressed in a variety of neuronal and non-neuronal tissues. As proton-gated channels, they have been implicated in many pathophysiological conditions where pH is perturbed. Venom derived compounds represent the most potent and selective modulators of ASICs described to date, and thus have been invaluable pharmacological tools to study ASIC structure, function, and biological roles. There are now eleven ASIC modulators described from animal venoms, with those from snakes and spiders favouring ASIC1, while the sea anemone and cone snail modulators preferentially target ASIC3. Chapter 1 reviews the current state of knowledge on venom derived ASIC modulators, with a particular focus on their molecular interaction with ASICs, what they have taught us about channel structure, and what they may still reveal about ASIC function and pathophysiological roles.Venom peptides are often disulfide bonded making their recombinant expression challenging. Chapter 2 describes a periplasmic Escherichia coli protocol for production of correctly folded peptides that was used throughout this thesis. Using the two-electrode voltage-clamp technique, Chapter 3 shows that a wide variety of voltage- and ligand-gated ion channels have the same channel properties and pharmacological profiles when expressed in either Xenopus laevis or X. borealis oocytes. This voltage-clamp technique was heavily used throughout this thesis to perform functional studies of venom peptides.The spider venom peptide PcTx1 is the best studied ASIC modulator; it has an IC50 of ~1 nM at rat ASIC1a and is neuroprotective in rodent models of ischemic stroke. Chapter 4 examines the molecular interaction between PcTx1 and both human ASIC1a and the off- target ASIC1b subtype, where little experimental work has been done. We show that although PcTx1 is 10-fold less potent at human ASIC1a than the rat channel, the apparent affinity for the two channels is comparable. The pharmacophore of PcTx1 for human ASIC1a and rat ASIC1b was examined via alanine scanning mutagenesis and uncovered residues that show subtle ASIC1 species and subtype-dependent differences in activity that may allow for further manipulation to develop more selective PcTx1 analogues.The ASIC3 inhibitor APETx2 is analgesic in rodent models of chemically-induced, inflammatory and postoperative pain, providing strong evidence for the ASIC3 subtype as a potential pain target. Despite the comprehensive structure-activity studies of APETx2, the mechanism of action and channel binding site have remained elusive. Chapter 5 fills this gap in knowledge of an important ASIC research tool and also reveals subtle differences in the pharmacophore of APETx2 for inhibition of ASIC3 and potentiation of ASIC1b. We propose that APETx2 binds to a novel region for peptide modulators of ASICs and acts by preventing the closed-to-open gating transition of rat ASIC3.Mambalgins are a group of three-finger toxins isolated from black and green mamba snake venoms that target ASIC1a and ASIC1b containing channels. Unique among ASIC modulators, the potent inhibitory activity of mambalgins at rodent ASIC1b was crucial in demonstrating that ASIC1b, and not ASIC1a, is important for peripheral pain sensing in rodents. Chapter 6 shows that the efficacy of mambalgins varies between the ASIC1 splice variants ASIC1a and ASIC1b, in both human and rat channels. Strikingly, mambalgin is a potentiator of human ASIC1b under certain conditions. Furthermore, these pharmacological differences are due to both the molecular interactions at the binding sites and the different ways mambalgin can modify the gating characteristics of specific ASIC variants.Chapters 7 describes the isolation, pharmacological characterisation and chemical stability of the novel spider venom peptide Hm3a from the venom of the tarantula Heteroscodra maculata. Hm3a is a close homolog of PcTx1 with five amino acid substitutions and a three residue C-terminal truncation. Despite its high sequence similarity with PcTx1 and similar pharmacology, Hm3a showed higher levels of stability over 48 h 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.Chapter 8 describes the characterisation of conorfamides As1a and As2a from the venom of the cone snail Conus austini. Amidated peptide variants altered desensitization of ASIC1a and 3, and a lysine to arginine mutation introduced ASIC1a peak current potentiation. These conorfamides also inhibited a7 and muscle-type nicotinic acetylcholine receptors (nAChR) at nanomolar concentrations. These are the first conorfamides with the dual pharmacology described to date.