Virtual screening of a random tripeptide library for easily prepared inhibitors of insect chitinolytic enzymes.

Insect molting requires chitinolytic enzymes to hydrolyze the chitin in the old cuticle, making chitinolytic enzyme inhibitors potential insecticides. Naturally occurring peptide chitinase inhibitors, such as argadin and argifin, possess complex non-natural structures, making large-scale production and application difficult. Given that chitinolytic enzymes recognize a core trisaccharide unit, this study integrates both in silico and in vitro approaches to explore the feasibility of using tripeptides as insect chitinolytic enzyme inhibitors. Virtual screening of 8000 random tripeptides identified 202 candidate peptides, which were found to be more hydrophobic and enriched in aromatic amino acids, complementing the properties of the chitinase substrate-binding pocket residues. Four selected peptides were synthesized and their inhibitory activities against chitinolytic enzymes from were quantitatively assessed, with QWW exhibiting an IC value of 0.2 mM against Chi-h. Molecular dynamics simulations indicated that strong hydrogen bonds and π-π interactions were key factors in the high activity of QWW. This study not only expands the chemical space for agricultural chemicals targeting chitinase but also establishes an extendable, peptide-based inhibitor discovery process, which may apply to drug development for other targets.