Computer-aided drug screening of anti-Neobenedenia melleni drugs based on annexin B1 in farmed pearl grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus ♂)
Monogenean capsalids of the genus Neobenedenia are widespread parasites of wild and farmed marine fish and represent a major threat to grouper mariculture in China, whose production reached approximately 294,000 tonnes in 2025. The development of effective drugs to control and prevent these infections is therefore urgently needed. Annexins, which have been identified in Neobenedenia and other parasites, differ markedly from their host counterparts, making them potentially attractive targets for antiparasitic therapeutics. Here, we performed a computer-aided drug discovery screen, employing Neobenedenia melleni annexin B1 as the molecular target against a library of 1,456,161 small molecules. The three-dimensional structure of annexin B1 was first predicted using AlphaFold 3, SWISS-MODEL, and I-TASSER, and the most accurate model (AlphaFold) was selected for structure-based virtual screening. In vivo validation of eleven compounds identified abamectin (Aba) as the most effective anti-Neobenedenia agent, achieving complete parasite elimination at 0.16 mg/L. Owing to its low toxicity to the host grouper (24-h LC50 = 0.254 mg/L), abamectin was selected for further investigation. Abamectin exhibited potent anthelmintic activity against N. melleni, with a 24-h bath exposure yielding an EC50 of 0.033 mg/L and complete parasite elimination at 0.16 mg/L, corresponding to a therapeutic index of approximately 7.7. To elucidate the antiparasitic mechanism, we performed long-timescale (1000 ns) molecular dynamics simulations of the annexin B1-abamectin complex, enabling atomic-level analysis of the essential protein motions involved in their interaction. The interaction profile between annexin B1 and abamectin was dominated by hydrophobic contacts and water bridges, involving residues TYR-210, GLU-214, GLU-244, and SER-247, which path a way for further drug optimization.