Inhibition constant and stability of tripeptide inhibitors of gut trypsin‐like enzyme of the soybean pest Anticarsia gemmatalis

Abstract

Insects overcome the action of natural protease inhibitors (PIs) due to evolutionary adaptations through endogenous proteolysis and reprogramming proteases. Insect adaptations complicate the formulation of IP‐based crop protection products. However, small peptides designed based on the active site of enzymes have shown promising results that could change this scenario. GORE1 and GORE2 are designed tripeptides that reduce the survival of Anticarsia gemmatalis when ingested orally. In this article, the stability and ability of the peptides to bind trypsin‐like enzymes of A. gemmatalis were evaluated by molecular dynamics (MD) simulations. The ability of the peptides to inhibit trypsin‐like enzymes in vivo was compared with the SKTI protein by feeding A. gemmatalis larvae at different concentrations, followed by an inhibition persistence assay. During the MD simulation of enzyme–ligand complexes, both peptides showed a small variation of root‐mean‐square deviation and root‐mean‐square fluctuation, suggesting that these molecules reach equilibrium when forming a complex with the trypsin‐like enzyme. Furthermore, both peptides form hydrogen bonds with substrate recognition sites of A. gemmatalis trypsin‐like enzyme, with GORE2 having more interactions than GORE1. Larvae of A. gemmatalis exposed to the peptides and SKTI showed a similar reduction in proteolytic activity, but the persistence of inhibition of trypsin‐like enzyme was longer in peptide‐fed insects. Despite their size, the peptides exhibit important active and substrate binding site interactions, stability during complex formation, and steadiness effects in vivo. The results provide fundamental information for the development of mimetic molecules and help in decision‐making for the selection of delivery methods for larger‐scale experiments regarding similar molecules.

Structural analysis and inhibition capacity of dioscorin protein yam: Theoretical study by molecular docking and dynamic simulations

“…On the other hand, in silico tools have recently become an unavoidable approach for the optimization of protein–protein complexes and the design of new molecules from the study of the interaction between these proteins related to pest management (de Almeida Barros, Meriño‐Cabrera, Severiche Castro, da Silva Junior, de Oliveira, et al, 2022; de Almeida Barros, Meriño‐Cabrera, Severiche Castro, da Silva Júnior, Schultz, et al, 2022; de Almeida Barros et al, 2021; Halim et al, 2022; Machado et al, 2020; Meriño‐Cabrera, de Oliveira Mendes, et al, 2020; Meriño‐Cabrera, Severiche Castro, et al, 2020; Meriño‐Cabrera, et al, 2022). Using tools such as molecular modeling of proteins, which allows us to determine the three‐dimensional (3D) structure of the sequences of receptors and ligands such as yam disocorins; protein–protein molecular docking, to determine the affinity site and energy of the inhibitor to the digestive enzymes of interest; and study of the interface using software such as Pymol and Discovery, which allow exploring inhibitory residues that bind to the active site of the enzyme and/or potential to reduce enzyme activity (hot‐spot residues), which would be expensive and slowly by experimental methods (Childers & Daggett, 2017); In addition, molecular dynamics (MD) simulations are performed to analyze the stability of the protein–protein complex and the free binding energy in aqueous solution estimation, allowing to understand several underlying biological phenomena, such as enzyme inhibition (Patel & Ytreberg, 2018).…”