Isolation and pharmacological characterisation of novel neurotoxins from the venoms of Australian copperheads and the efficacy of tiger snake antivenom to prevent or reverse neurotoxicity

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NO FULL TEXT AVAILABLE. This thesis contains 3rd party copyright material. ----- The venom of the Australian lowlands copperhead, Austrelaps superbus, is capable of producing significant and potentially lethal neurotoxic paralysis in cases of clinical envenomation. However, little is known about the neurotoxic components within this venom. Furthermore, there are no pharmacological data available regarding the suspected neurotoxic components in the venoms of related alpine (A. ramsayi) or pygmy (A. labialis) copperheads (Austrelaps labialis). In the isolated chick biventer cervicis nerve-muscle (CBCNM) preparation, all Austrelaps venoms caused a potent and rapid inhibition of nerve-evoked twitch contractions that was accompanied by a block of contractures to nicotinic agonists. These actions are consistent with the presence of postsynaptic neurotoxicity. Subsequent separation of Austrelaps spp. venoms under non-reducing conditions using size-exclusion liquid chromatography revealed that all three venoms contained a high molecular mass fraction with only weak phospholipase A₂ activity. In all three venoms, this fraction caused a slow inhibition of nerve-evoked twitch contractions in the CBCNM preparation, without inhibiting contractures to nicotinic agonists or KCl. These actions are consistent with the presence of presynaptic neurotoxicity, in the absence of overt myotoxicity, attributed to by the presence of snake presynaptic PLA₂ neurotoxin (SPAN) complexes. This is the first report of presynaptic neurotoxicity in Austrelaps spp. venoms. Subsequently, the first isolated SPAN complex from A. superbus venom, P-elapitoxin-As1a (P-EPTX-As1a), was pharmacologically characterised in the CBCNM preparation. P-EPTX-As1a causes ‘tetanic fade’ in muscle tension under high frequency nerve stimulation and produces a triphasic alteration to neurotransmitter release. These actions have been previously noted with other multimeric SPAN complexes, such as taipoxin. Moreover, the neurotoxic α-subunit of P-EPTX-As1a shows high homology to taipoxin α-chain. Furthermore, the presence of a class PIII snake venom metalloproteinase, C-type lectin, L-amino acid oxidase, acetylcholinesterase and phospholipase B, with potential coagulopathic and myotoxic effects, were also identified in the same high molecular mass fraction as P-EPTX-As1a. This thesis also describes the pharmacological characterisation of α-elaiptoxin-Al2a (α-EPTX-Al2a), the first snake postsynaptic α-neurotoxin from A. labialis venom. α-Elapitoxin-Al2a was isolated from A. labialis venom using a series of liquid chromatography techniques. In the CBCNM preparation, α-EPTX-Al2a (8072.77 Da) caused a concentration-dependent block of nerve-evoked twitch contractions and a complete block of contractures to nicotinic agonists. α-Elapitoxin-Al2a produces a pseudo-irreversible antagonism of chick muscle nicotinic acetylcholine receptors (nAChRs), with an estimated pA₂ value of 7.902 (KB = 12.5 nM) but failed to inhibit ganglionic α3β2/ α3β4 nAChRs in a fluorescence-based FLIPR assay using SH-SY5Y cells. The toxin contains 75 amino acid residues with five disulphide bonds and shows significant homology to classical long-chain α-neurotoxins, however it is unique among long-chain α-neurotoxins as it only produces a modest block of neuronal α7 nAChR (IC₅₀ = 1.2 μM), despite containing the critical residue, Arg³³. Monovalent tiger snake antivenom (TSAV) is the recommended treatment following Austrelaps spp. envenomation. The efficacy of TSAV in preventing or reversing neurotoxicity induced by either whole venom or isolated neurotoxins was determined in the CBCNM preparation by adding TSAV 10 minutes prior to the addition of venom or toxin or at 90% neuromuscular blockade, respectively. This thesis confirms the effectiveness of TSAV in neutralising the neurotoxicity of A. superbus whole venom. In addition, this thesis provides the first in vitro study to confirm the effectiveness of TSAV to neutralise the neurotoxicity of A. labialis and A. ramsayi whole venoms as well as isolated P-EPTX-As1a and α-EPTX-Al2a. Thus, this thesis supports the current clinical recommendation for the use of TSAV in the treatment of envenomation by all Austrelaps spp. This thesis also found that TSAV was unable to effectively reverse the neurotoxicity of Austrelaps spp. venoms, prejunctional SPAN neurotoxicity caused by the SPAN complex in all three venoms, or postsynaptic neurotoxicity caused by α-EPTX-Al2a once established. In conclusion, this thesis is the first study that confirms the suspected presence of both presynaptic and postsynaptic neurotoxic components in all Austrelaps spp. venoms. Furthermore, this thesis represents the first in vitro study to assess the efficacy of TSAV in preventing and reversing neurotoxicity caused by Austrelaps spp. venoms and their isolated neurotoxic components. Given that the neurotoxic effects caused by these venoms are only partially reversible once neurotoxicity is well established, clinicians should be aware that early antivenom intervention may be necessary to prevent the onset of irreversible neurotoxicity caused by irreversible multimeric SPAN complexes and snake postsynaptic α-neurotoxins in Austrelaps spp. venoms.
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