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

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-1H-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.

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 μm; half-maximal inhibitory concentration (IC₅₀) = 37.55 μm]. 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.

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.