Interrogating stonefish venom: small molecules present in envenomation caused by <i>Synanceia</i> spp.

The stonefish Synanceia verrucosa and Synanceia horrida are arguably the most venomous fish species on earth and the culprits of severe stings in humans globally. Investigation into the venoms of these two species has mainly focused on protein composition, in an attempt to identify the most medically relevant proteins, such as the lethal verrucotoxin and stonustoxin components. This study, however, focused on medically relevant small molecules, and through nuclear magnetic resonance, mass spectroscopy, and fractionation techniques, we discovered and identified the presence of three molecules new to stonefish venom, namely γ-aminobutyric acid (GABA), choline and 0-acetylcholine, and provide the first report of GABA identified in a fish venom. Analysis of the crude venoms on human nicotinic acetylcholine receptors (nAChRs) and a GABA_A receptor (GABA_AR) showed S. horrida venom could activate neuronal (α7) and adult muscle-type (α1β1δε) nAChRs, while both crude S. horrida and S. verrucosa venoms activated the GABA_AR (α1β2γ2). Cytotoxicity studies in immunologically relevant cells (human PBMCs) indicated the venoms possess cell-specific cytotoxicity and analysis of the venom fractions on Na⁺ channel subtypes involved in pain showed no activity. This work highlights the need to further investigate the small molecules found in venoms to help understand the mechanistic pathways of clinical symptoms for improved treatment of sting victims, in addition to the discovery of potential drug leads.