Atovaquone as a potent and safe anthelmintic against Gyrodactylus kobayashii in goldfish: Efficacy, mechanism, and structure-based optimization.

Gyrodactylosis, caused by Gyrodactylus spp., poses a significant challenge to sustainable aquaculture, highlighting the need for effective and safe anthelmintics. In this study, atovaquone, a specific inhibitor of the mitochondrial cytochrome bc1 complex, was repurposed as a therapeutic agent against Gyrodactylus kobayashii. In vivo bath treatments demonstrated high potency, with a 48-h EC50 of 0.005 mg/L, which is lower than that reported for most existing natural and synthetic compounds. Notably, atovaquone exhibited greater efficacy in vivo than in vitro, likely due to its lipophilic accumulation in fish mucus and its slow-acting mechanism of energy depletion. Biochemical assays and molecular docking analyses indicated that atovaquone binds to the Qo site of cytochrome b, thereby inhibiting electron transport and leading to intracellular ATP depletion. Scanning electron microscopy further revealed pronounced tegumental damage and morphological alterations, consistent with a combined effect of energy depletion and structural impairment, ultimately leading to neuromuscular dysfunction and parasite detachment from the host. In addition, atovaquone exhibited a wide therapeutic index (TI = 112.6) in goldfish, indicating a favorable safety profile. Structural modification of the naphthoquinone scaffold followed by biological evaluation led to the identification of optimized compounds, including Y114, with high anthelmintic activity. Collectively, these findings highlight atovaquone as a highly effective treatment for closed-system applications and validate the cytochrome b - targeting naphthoquinone scaffold as a promising basis for developing environmentally safe antiparasitic drugs in aquaculture.