The economic loss in soybean crops caused by the Lepidoptera insects has encouraged the search for new strategies to control this pest, which are currently based on synthetic insecticides. This paper evaluated the ability of ApTI (Adenanthera pavonina trypsin inhibitor) to inhibit trypsin-like proteins from Anticarsia gemmatalis by docking, molecular dynamics, and enzymatic and survival assay. The docking and molecular dynamic simulation between trypsin and ApTI were performed using the program CLUSPRO and NAMD, respectively. The inhibitory constant K i and the inhibition type were determined through chromogenic assays. The survival assay of neonatal larvae under treatment with artificial diet supplemented with ApTI was also performed. The ApTI binding site was predicted to block substrate access to trypsin due to four interactions with the enzyme, producing a complex with a surface area of 1,183.7 Å 2 .The kinetic analysis revealed a noncompetitive tightbinding mechanism. The survival curves obtained using Kaplan-Meier estimators indicated that the highest larvae mortality was 60%, using 1.2 mg of ApTI per 100 ml of artificial diet. The in vitro, in vivo, and in silico studies demonstrated that ApTI is a strong noncompetitive inhibitor of trypsin with biotechnological potential for the control of A. gemmatalis insect. K E Y W O R D S binding, insects, Kunitz inhibitor, molecular docking, noncompetitive, trypsin enzyme

Proteases present in the salivary glands of phytosuccivorous insects are responsible for the initial breakdown of dietary protein into the amino acids necessary for insect growth and development. Here, we characterised the total proteases and trypsin‐like activities in the salivary glands of the Neotropical brown stink bug, Euschistus heros (F.) and used in silico and biochemical approaches to determine the interactions between trypsin‐like enzymes and the soybean Kunitz trypsin inhibitor (SKTI). Additionally, we evaluated whether these enzyme activities were affected in individuals resistant to the actions of the insecticide imidacloprid. We used an originally field‐collected strain to select individuals resistant to imidacloprid alone (ImiGoiasRes) or synergised with piperonyl butoxide (ImiPBORes). A laboratory‐susceptible (ImiSusc) strain was also used to select piperonyl butoxide synergised imidacloprid‐resistant (ImiPBOSel) individuals. Our results revealed that pH and temperature significantly affected the specific trypsin‐like activities in imidacloprid‐susceptible individuals. Significant alterations in the total protease activities were recorded for field‐ (ImiGoiasRes) and laboratory‐selected imidacloprid‐resistant individuals (ImiPBOSel and ImiPBORes). Our computational docking analysis demonstrated that the molecular interactions and the presence of the SKTI inhibitor (0.5 mg mL−1) could reduce up to 40% of the trypsin‐like activities of imidacloprid‐susceptible individuals. However, all imidacloprid‐resistant strains exhibited significantly less trypsin‐like activities and exhibited lower hydrolysing velocity (VMAX) and affinity (KM) rates, and the prior exposure to SKTI only synergised the imidacloprid toxicity for individuals of ImiFieldRes strain. Collectively, our findings demonstrate, for the first time, insecticide resistance detrimentally affected the activities of proteolytic enzymes in phytosuccivorous stink bug pests.

Although the importance of intestinal hydrolases is recognized, there is little information on the intestinal proteome of lepidopterans such as Anticarsia gemmatalis. Thus, we carried out the proteomic analysis of the A. gemmatalis intestine to characterize the proteases by LC/MS. We examined the interactions of proteins identified with protease inhibitors (PI) using molecular docking. We found 54 expressed antigens for intestinal protease, suggesting multiple important isoforms. The hydrolytic arsenal featured allows for a more comprehensive understanding of insect feeding. The docking analysis showed that the soybean PI (SKTI) could bind efficiently with the trypsin sequences and, therefore, insect resistance does not seem to involve changing the sequences of the PI binding site. In addition, a SERPIN was identified and the interaction analysis showed the inhibitor binding site is in contact with the catalytic site of trypsin, possibly acting as a regulator. In addition, this SERPIN and the identified PI sequences can be targets for the control of proteolytic activity in the caterpillar intestine and serve as a support for the rational design of a molecule with greater stability, less prone to cleavage by proteases and viable for the control of insect pests such as A. gemmatalis.

BACKGROUND Anticarsia gemmatalis larvae are key defoliating pests of soybean plants. Inorganic insecticides, harmful to the environment and human health, are the main molecules used in the control of this pest. To apply more sustainable management methods, organic molecules with high specificities, such as proteinaceous protease inhibitors, have been sought. Thus, molecular docking studies, kinetics assays, and biological tests were performed to evaluate the inhibitory activity of two peptides (GORE1 and GORE2) rationally designed to inhibit trypsin‐like enzymes, which are the main proteases of A. gemmatalis midgut. RESULTS The molecular docking simulations revealed critical hydrogen bonding patterns of the peptides with key active site residues of trypsin‐like proteases of A. gemmatalis and other Lepidopteran insects. The negative values of binding energy indicate that hydrogen bonds potentiate the tight binding of the peptides with trypsin‐like proteases, predicting an effective inhibition. The inhibition's rate constants (Ki) were 0.49 and 0.10 mM for GORE1 and GORE2, resulting in effective inhibition of the activity trypsin on the L‐BApNA substrate in the in vitro tests, indicating that the peptide GORE2 has higher inhibitory capacity on the A. gemmatalis trypsins. In addition, the two peptides were determined to be reversible competitive inhibitors. The in vivo test demonstrated that the peptides harm the survival and development of A. gemmatalis larvae. CONCLUSION These results suggest that these peptides are potential candidates in the management of A. gemmatalis larvae and provide baseline information for the design of new trypsin‐like inhibitors based on peptidomimetic tools. © 2020 Society of Chemical Industry