Abstract

Spodoptera frugiperda is a major agricultural pest responsible for significant yield losses in a wide range of crops. Control strategies have traditionally relied on chemical insecticides; however, the recurrent evolution of resistance has compromised their long-term effectiveness. In this context, sustainable alternatives such as digestive enzyme inhibitors have emerged as promising tools for pest management. Molecular docking analyses predicted a conserved binding mode for the peptide GORE 3 across multiple S. frugiperda trypsin isoforms. GORE 3 occupies the S1/S1′ subsites and adjacent regions of the catalytic pocket, establishing a broader network of aromatic and hydrophobic interactions compared with the reference inhibitor benzamidine, which is primarily restricted to the S1 pocket. This binding topology is consistent with enzymatic assays, in which GORE 3 acted as a competitive inhibitor (Ki = 4.00 mM), while benzamidine exhibited higher potency (Ki = 1.64 mM). IC₅₀ determinations further confirmed GORE 3’s inhibitory effect on trypsin-like activity. In vivo bioassays demonstrated that dietary exposure to GORE 3 significantly reduced larval body mass, prolonged the larval developmental period, and increased mortality to up to 46.66%. Nutritional and kinetic parameters were also affected, including increased KM values, indicating reduced substrate affinity, and lower approximate digestibility at higher peptide concentrations. Notably, neither leaf area consumption nor feeding preference index differed from control treatments, suggesting that physiological impairment occurred independently of feeding behavior. Together, docking predictions and biological assays demonstrate that GORE 3 interacts with trypsin-like proteases through a robust multisite binding mechanism, leading to competitive inhibition and measurable developmental effects in S. frugiperda.