Abstract
Parasitic gastrointestinal worms (i.e., helminths) remain a significant global health and economic burden. The increasing inefficacy of current anthelmintic drugs against parasitic diseases necessitates the discovery of novel therapeutic options. This study investigated the anthelmintic properties and therapeutic potential of stonefish ichthyocrinotoxins (i.e., secreted skin toxins). xWORM (xCELLigence Worm Real-Time Motility Assay) was used to evaluate the anthelmintic activity of ichthyocrinotoxins from two stonefish species, Synanceia horrida (Estuarine Stonefish) and Synanceia verrucosa (Reef Stonefish), against the infective third-stage larvae of Nippostrongylus brasiliensis (Rodent Hookworm). Both toxins demonstrated potent anthelmintic effects, with S. horrida ichthyocrinotoxin exhibiting greater potency (IC(50) = 196.0 µg/mL) compared to ichthyocrinotoxin from S. verrucosa (IC(50) = 329.7 µg/mL). Fractionation revealed that the anthelmintic activity of S. verrucosa is likely driven by synergistic interactions between the large (>3 kDa) and small (<3 kDa) components. In contrast, the small components isolated from S. horrida ichthyocrinotoxin were responsible for the majority of the observed activity, making them a more attractive therapeutic candidate. Furthermore, despite the cytotoxicity of crude S. horrida ichthyocrinotoxin against human skin and bile duct cell lines, the isolated small components exhibited potent anthelmintic effects (IC(50) = 70.5 µg/mL) with negligible cytotoxicity (<10% decrease in survival at 100 µg/mL). While further research is necessary to fully characterise these compounds and assess their clinical suitability, this study highlights the potential of stonefish ichthyocrinotoxins as a novel source of anthelmintic therapeutics.