Zebrafish Serotonin Transporter Inhibition Enables Identification of Neuroactive Chemicals: Structural Insights into Interspecies Differences.

Antidepressants are widely detected in aquatic environments and can affect fish behavior through inhibition of the serotonin transporter (SERT). Notably, zebrafish SERT (zSERT) exhibits greater sensitivity to antidepressants than human SERT (hSERT), suggesting species-specific differences in transporter structures. However, the molecular mechanisms underlying this differential inhibition remain elusive, and prioritized antidepressants alone cannot fully explain the zSERT-mediated activity observed in the environmental samples. Herein, molecular simulation, computational screening, and experimental validation were adopted to identify environmental chemicals with potential activity toward zSERT. The binding affinity of 27 antidepressants for hSERT and zSERT was predicted by Boltz-2. The antidepressant with the largest interspecies difference was selected as a representative case, and the key amino acid residues involved in binding were characterized by using molecular dynamics simulations performed for at least 100 ns. The molecular simulation-optimized zSERT structure was then applied in structure-based virtual screening to identify potentially toxic compounds from a chemical library comprising high-risk chemicals from Europe and China. Fourteen of the 15 top-ranked candidates were found to inhibit zSERT in an in vitro assay. This integrative approach provides a mechanistically informed framework for identifying previously unrecognized zSERT-acting chemicals and improving hazard identification and cross-species risk assessments in aquatic environments.