# Interrogating stonefish venom: small molecules present in envenomation caused by Synanceia spp

## TL;DR

This study identifies new small molecules in stonefish venom and explores their effects on human receptors and cells.

## Contribution

The discovery of GABA, choline, and 0-acetylcholine in stonefish venom, including the first report of GABA in fish venom.

## Key findings

- Stonefish venom contains GABA, choline, and 0-acetylcholine, which are new to venom research.
- S. horrida venom activates specific nicotinic and GABA receptors in humans.
- Venoms show cell-specific cytotoxicity but no activity on pain-related sodium channels.

## Abstract

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 GABAA receptor (GABAAR) 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 GABAAR (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.

Molecular analysis of Synanceia horrida (ShV) and S. verrucosa (SvV) venoms identified three small molecules new to stonefish venom: Gamma‐aminobutyric acid (GABA), choline, and 0‐acetylcholine. Analysis on human nicotinic acetylcholine (nAChRs) and GABAA receptors showed activation of neuronal (α7) and adult muscle‐type (α1β1δε) nAChRs by ShV, while both ShV and SvV activated GABAAR (α1β2γ2).