An in vitro assay to investigate venom neurotoxin activity on muscle-type nicotinic acetylcholine receptor activation and for the discovery of toxin-inhibitory molecules

Abstract

Snakebite envenoming is a neglected tropical disease that causes over 100,000 deaths annually. Envenomings result in variable pathologies, but systemic neurotoxicity is among the most serious and is currently only treated with difficult to access and variably efficacious commercial antivenoms. Venom-induced neurotoxicity is often caused by α-neurotoxins antagonising the muscle-type nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel. Discovery of therapeutics targeting α-neurotoxins is hampered by relying on binding assays that do not reveal restoration of receptor activity or more costly and/or lower throughput electrophysiology-based approaches. Here, we report the validation of a screening assay for nAChR activation using immortalised TE671 cells expressing the γ-subunit containing muscle-type nAChR and a fluorescent dye that reports changes in cell membrane potential. Assay validation using traditional nAChR agonists and antagonists, which either activate or block ion fluxes, was consistent with previous studies. We then characterised antagonism of the nAChR by a variety of elapid snake venoms that cause muscle paralysis in snakebite victims, before defining the toxin-inhibiting activities of commercial antivenoms, and new types of snakebite therapeutic candidates, namely monoclonal antibodies, decoy receptors, and small molecules. Our findings show robust evidence of assay uniformity across 96-well plates and highlight the amenability of this approach for the future discovery of new snakebite therapeutics via screening campaigns. The described assay therefore represents a useful first-step approach for identifying α-neurotoxins and their inhibitors in the context of snakebite envenoming, and it should provide wider value for studying modulators of nAChR activity from other sources.

Keywords: Antibody discovery; Antivenom; Drug discovery; Snake venom neurotoxin; Three-finger toxin; nicotinic acetylcholine receptor (nAChR); α-neurotoxins.