Identification of threonyl‐ tRNA synthetase as a potential fungicidal target of 4‐fluoro‐2‐aryl‐ 1H ‐indole

Abstract BACKGROUND Discovering novel pesticide targets based on newly synthesized compounds is of great importance for the development of new pesticides and sustainable agricultural. 4‐Fluoro‐2‐aryl‐1 H ‐indole (W1), a 2‐arylindole derivative obtained via one‐pot synthesis in our previous studies, exhibited potent antifungal activity against Botrytis cinerea B05.10, with a half‐maximal effective concentration of 6.78 mg L −1 . RESULTS To discover the novel fungicide target, in this study, western blot–drug affinity responsive target stability, surface plasmon resonance and enzymatic assays were used to explore the potential target of W1, and B. cinerea threonyl‐tRNA synthetase (BcThrRS) was identified as a direct target of W1 [equilibrium dissociation constant ( K D ) = 5.28 μ m ; half‐maximal inhibitory concentration (IC 50 ) = 37.55 μ m ]. Molecular docking and mutagenesis analyses identified Pro256 as a key residue mediating ligand binding, with mutation of this residue substantially reducing target affinity and enzymatic inhibition. Metabolomic profiling suggested that inhibition of BcThrRS by W1 disrupts threonyl‐tRNA formation, resulting in defective protein translation, amino acid metabolic imbalance, and downstream perturbation of cellular metabolism. CONCLUSION These findings indicate that BcThrRS is a promising antifungal target and highlight W1 as a privileged scaffold for the development of fungicides with a novel mode of action. They open up new avenues for the rational design of next‐generation antifungals targeting aminoacyl‐tRNA synthetases. © 2026 Society of Chemical Industry.