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

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.

Intestinal proteolytic profile changes during larval development of Anticarsia gemmatalis caterpillars.

Soybean is one of most consumed and produced grains in the world, and Anticarsia gemmatalis is a pest that causes great damage to this crop due to severe defoliation during its larval phase. Plants have mechanisms that lead to the inhibition of proteases in the intestine of these herbivores, hampering their development. Understanding this complex protease inhibitor is important for pest control. The objective of this study was to evaluate the enzymatic profiles of the intestinal proteases of the soybean caterpillar at different instars. For this, the proteolytic profile of the gut in the third, fourth, and fifth instars were analyzed. Irreversible inhibitors of proteases were separately incubated with A. gemmatalis enzyme extracts at the third, fourth, and fifth instar to assess the contribution of these proteases to total proteolytic activity. The enzymatic extracts were also evaluated with specific substrates to confirm changes in the specific activities of trypsin-like, chymotrypsin-like, and cysteine proteases at different instars. The results showed that the protease profile of A. gemmatalis gut changes throughout its larval development. The activity of cysteine proteases was more intense in the first instar. On the contrary, the serine proteases showed major activities in the late stages of the larval phase. Zymogram analysis and protein identification by liquid chromatography-mass spectrometry indicated serine protease as the main protease class expressed in the fifth instar. These results may shift the focus from the rational development of the protease inhibitor to A. gemmatalis and other Lepidoptera, as the expression of major proteases is not constant.