Trypsinogen Pro‐Region as a Molecular Scaffold for the Design of Competitive Peptide Inhibitors of Trypsin‐Like Proteases

Soybean ( Glycine max L. Merrill) productivity is severely impacted by the velvetbean caterpillar Anticarsia gemmatalis Hübner, 1818 (Lepidoptera: Erebidae), a major defoliating pest in soybean‐producing regions. Current management strategies rely heavily on chemical insecticides, which are associated with environmental risks and the development of resistance. Protease inhibitors have emerged as a promising alternative, as protein digestion in lepidopteran larvae is primarily mediated by trypsin‐like serine proteases. In this context, the rational design of short inhibitory peptides represents a potential strategy for developing selective and environmentally safer pest control agents. This study introduces the regulatory pro‐region of trypsinogen as a previously underexplored scaffold for the rational design of competitive peptide inhibitors targeting digestive trypsin‐like enzymes in agricultural insect pests. Fifteen peptides derived from the trypsinogen pro‐region were initially screened in silico against A. gemmatalis trypsin‐like enzymes. Molecular docking analyses revealed favorable binding energies and interactions with residues located in the catalytic region. Among the candidates, the peptide Pep12 (designated GORE3) was selected for further optimization by substituting amino acids at the P1 position, yielding the analogs GORE4 and GORE5. Enzyme inhibition assays with bovine trypsin and partially purified A. gemmatalis trypsin‐like enzymes demonstrated that all peptides are competitive inhibitors. The inhibition constants (Ki) obtained for A. gemmatalis enzymes were 1.80 mM for GORE3, 1.63 mM for GORE4, and 0.78 mM for GORE5, with GORE5 exhibiting the lowest Ki among the evaluated peptides, although its inhibitory affinity remained within the millimolar range. Collectively, these findings underscore the potential of trypsinogen‐derived peptides as promising scaffolds for developing peptide‐based inhibitors targeting insect digestive proteases.