Enzymological and transcriptomic analyses reveal the insecticidal mechanism of 4-propargyloxybenzenesulfonamide as a V-ATPase inhibitor.

The vacuolar-type H+-ATPase (V-ATPase) has emerged as a promising molecular target for the development of new pesticides. In our previous work, we employed computer-aided drug design (CADD) strategies targeting the V-ATPase H subunit of the Mythimna separata. Through homology modeling, virtual screening, and lead compound synthesis, we successfully identified 4-propargyloxybenzenesulfonamide as a potent insecticidal compound. This study revealed that the compound exhibits significant toxicity, with LC50 values of 1.206 mg/mL against M. separata and 0.551 mg/mL against Plutella xylostella, demonstrating approximately 20-fold higher insecticidal potency compared to the natural insecticidal Celangulin V. Enzymatic assays confirmed that the compound inhibits midgut V-ATPase activity in a concentration-dependent suppression, reaching 69.3 % inhibition at 100 μM-significantly surpassing the 24.6 % inhibition observed with Celangulin V at the same concentration. Molecular docking simulations further revealed that 4-propargyloxybenzenesulfonamide forms a stable hydrogen bond with the Asn433 residue of the V-ATPase H subunit, suggesting a strong and specific interaction underlying its inhibitory efficacy. Furthermore, transcriptomic analysis of treated larvae showed significant enrichment of genes associated with the phagosome pathway, highlighting disruption of V-ATPase-mediated lysosomal acidification as a key mechanism of action. Collectively, these findings validate 4-propargyloxybenzenesulfonamide as a novel insecticidal lead compound targeting V-ATPase H subunit, providing a chemically optimizable scaffold for the development of environmentally sustainable insecticides.