Anin vitroassay to investigate venom neurotoxin activity on muscle-type nicotinic acetylcholine receptor activation and for the discovery of toxin-inhibitory molecules (original) (raw)
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PLOS Neglected Tropical Diseases, 2020
The aim of this study was to develop an in vitro assay for use in place of in vivo assays of snake venom lethality and antivenom neutralizing potency. A novel in vitro assay has been developed based on the binding of post-synaptically acting α-neurotoxins to nicotinic acetylcholine receptor (nAChR), and the ability of antivenoms to prevent this binding. The assay gave high correlation in previous studies with the in vivo murine lethality tests (Median Lethal Dose, LD 50), and the neutralization of lethality assays (Median Effective Dose, ED 50) by antisera against Naja kaouthia, Naja naja and Bungarus candidus venoms. Here we show that, for the neurotoxic venoms of 20 elapid snake species from eight genera and four continents, the in vitro median inhibitory concentrations (IC 50s) for α-neurotoxin binding to purified nAChR correlated well with the in vivo LD 50s of the venoms (R 2 = 0.8526, p < 0.001). Furthermore, using this assay, the in vitro ED 50s of a horse pan-specific antiserum against these venoms correlated significantly with the corresponding in vivo murine ED 50 s, with R 2 = 0.6896 (p < 0.01). In the case of four elapid venoms devoid or having a very low concentration of α-neurotoxins, no inhibition of nAChR binding was observed. Within the philosophy of 3Rs (Replacement, Reduction and Refinement) in animal testing, the in vitro αneurotoxin-nAChR binding assay can effectively substitute the mouse lethality test for toxicity and antivenom potency evaluation for neurotoxic venoms in which α-neurotoxins predominate. This will greatly reduce the number of mice used in toxicological research and antivenom production laboratories. The simpler, faster, cheaper and less variable in vitro assay should also expedite the development of pan-specific antivenoms against various medically important snakes in many parts of the world.
Snake Venoms in Drug Discovery: Valuable Therapeutic Tools for Life Saving
Toxins
Animal venoms are used as defense mechanisms or to immobilize and digest prey. In fact, venoms are complex mixtures of enzymatic and non-enzymatic components with specific pathophysiological functions. Peptide toxins isolated from animal venoms target mainly ion channels, membrane receptors and components of the hemostatic system with high selectivity and affinity. The present review shows an up-to-date survey on the pharmacology of snake-venom bioactive components and evaluates their therapeutic perspectives against a wide range of pathophysiological conditions. Snake venoms have also been used as medical tools for thousands of years especially in tradition Chinese medicine. Consequently, snake venoms can be considered as mini-drug libraries in which each drug is pharmacologically active. However, less than 0.01% of these toxins have been identified and characterized. For instance, Captopril® (Enalapril), Integrilin® (Eptifibatide) and Aggrastat® (Tirofiban) are drugs based on snak...
Toxicology Letters, 2012
Pseudonaja textilis (Eastern Brown snake) and Oxyuranus scutellatus scutellatus (Coastal taipan) are clinically important Australian elapid snakes, whose potent venoms contain the presynaptic () neurotoxins, textilotoxin and taipoxin, respectively, and a number of postsynaptic neurotoxins. However, while taipan envenoming frequently results in neurotoxicity, Brown snake envenoming causes an isolated coagulopathy and neurotoxicity is rare. This phenomenon is called the 'Brown snake paradox'. This study compared the pharmacology of both venoms and their respective presynaptic neurotoxins to investigate this phenomenon. From size-exclusion high performance liquid chromatography (HPLC) analysis textilotoxin represents a significantly smaller proportion (5.7%) of P. textilis venom compared to taipoxin in O. s. scutellatus venom (20.4%). In the chick biventer cervicis nerve-muscle (CBCNM) preparation both venoms caused concentration-dependent neurotoxicity, with P. textilis venom being significantly more potent than O. s. scutellatus venom. Conversely, taipoxin was significantly more potent than textilotoxin when compared at the same concentration. Textilotoxin only partially contributed to the overall neurotoxicity of P. textilis venom, while taipoxin accounted for the majority of the neurotoxicity of O. s. scutellatus venom in the CBCNM preparation. Compared with taipoxin, textilotoxin is less potent and constitutes a smaller proportion of the venom. This is likely to be the reason for the absence of neurotoxicity in envenomed humans thus explaining the 'Brown snake paradox'.
Snake venom toxins targeting the central nervous system
Toxin Reviews, 2023
Snake venom is a blend of bioactive proteins, polypeptides, and various other substances with toxic and lethal properties that are known to modulate varied physiological and biological systems. During envenomation, venom toxins primarily target the hemostatic and nervous system for effective immobilization or death of the prey. The central (CNS) and the peripheral nervous system (PNS) are targeted through neuroreceptors, synaptic membranes, and critical ion channels, and some of these toxins also penetrate the blood-brain barrier. Despite its vital role and influence on the central nervous system, there exist limited information on the role of venom proteins and peptides associated with the manifestations of neurotoxicity. This review attempts to update the reader on the mechanism of direct and indirect interactions of snake venom protein (s) in the central nervous system as well as its effects on the physiology and behavior of the envenomated prey. Further, the role of these snake venom peptides in the field of neuropathic pain and neurodegenerative diseases has been reviewed for their therapeutic potential. Future investigations may provide valuable information to study the detailed mechanisms of such interactions to identify novel targets for the development of therapeutic interventions.
Journal of Biological Chemistry, 2015
Background: Different snake venom three-finger toxins interact with various receptors, channels, and membranes. Results: Here, we demonstrate that GABA A receptors are inhibited by ␣-cobratoxin, other long chain ␣-neurotoxins, nonconventional toxin from Naja kaouthia, and ␣-conotoxin ImI. Conclusion: Some toxin blockers of nicotinic acetylcholine receptors also inhibit GABA A receptors. Significance: Three-finger toxins offer new scaffolds for the design of GABA A receptor effectors. Ionotropic receptors of ␥-aminobutyric acid (GABA A R) regulate neuronal inhibition and are targeted by benzodiazepines and general anesthetics. We show that a fluorescent derivative of ␣-cobratoxin (␣-Ctx), belonging to the family of three-finger toxins from snake venoms, specifically stained the ␣13␥2 receptor; and at 10 M ␣-Ctx completely blocked GABA-induced currents in this receptor expressed in Xenopus oocytes (IC 50 ؍ 236 nM) and less potently inhibited ␣12␥2 ≈ ␣22␥2 > ␣52␥2 > ␣23␥2 and ␣13␦ GABA A Rs. The ␣13␥2 receptor was also inhibited by some other three-finger toxins, long ␣-neurotoxin Ls III and nonconventional toxin WTX. ␣-Conotoxin ImI displayed inhibitory activity as well. Electrophysiology experiments showed mixed competitive and noncompetitive ␣-Ctx action. Fluorescent ␣-Ctx, however, could be displaced by muscimol indicating that most of the ␣-Ctx-binding sites overlap with the orthosteric sites at the /␣ subunit interface. Modeling and molecular dynamic studies indicated that ␣-Ctx or ␣-bungarotoxin seem to interact with GABA A R in a way similar to their interaction with the acetylcholine-binding protein or the ligand-binding domain of nicotinic receptors. This was supported by mutagenesis studies and experiments with ␣-conotoxin ImI and a chimeric Naja oxiana ␣-neurotoxin indicating that the major role in ␣-Ctx binding to GABA A R is played by the tip of its central loop II accommodating under loop C of the receptors.
Venom-Derived Neurotoxins Targeting Nicotinic Acetylcholine Receptors
2021
Acetylcholine was the first neurotransmitter described. The receptors targeted by acetylcholine are found within organisms spanning different phyla and position themselves as very attractive targets for predation, as well as for defense. Venoms of snakes within the Elapidae family, as well as those of marine snails within the Conus genus, are particularly rich in proteins and peptides that target nicotinic acetylcholine receptors (nAChRs). Such compounds are invaluable tools for research seeking to understand the structure and function of the cholinergic system. Proteins and peptides of venomous origin targeting nAChR demonstrate high affinity and good selectivity. This review aims at providing an overview of the toxins targeting nAChRs found within venoms of different animals, as well as their activities and the structural determinants important for receptor binding.
Peptide Inhibitors of the α-Cobratoxin–Nicotinic Acetylcholine Receptor Interaction
Journal of Medicinal Chemistry, 2020
Venomous snakebites cause >100 000 deaths every year, in many cases via potent depression of human neuromuscular signaling by snake α-neurotoxins. Emergency therapy still relies on antibody-based antivenom, hampered by poor access, frequent adverse reactions, and cumbersome production/purification. Combining high-throughput discovery and subsequent structure−function characterization, we present simple peptides that bind α-cobratoxin (α-Cbtx) and prevent its inhibition of nicotinic acetylcholine receptors (nAChRs) as a lead for the development of alternative antivenoms. Candidate peptides were identified by phage display and deep sequencing, and hits were characterized by electrophysiological recordings, leading to an 8-mer peptide that prevented α-Cbtx inhibition of nAChRs. We also solved the peptide:α-Cbtx cocrystal structure, revealing that the peptide, although of unique primary sequence, binds to α-Cbtx by mimicking structural features of the nAChR binding pocket. This demonstrates the potential of small peptides to neutralize lethal snake toxins in vitro, establishing a potential route to simple, synthetic, low-cost antivenoms.