Bovine pancreatic trypsin inhibitor and soybean Kunitz trypsin inhibitor: Differential effects on proteases and larval development of the soybean pest Anticarsia gemmatalis (Lepidoptera: Noctuidae).

Pest management is challenged with resistant herbivores and problems regarding human health and environmental issues. Indeed, the greatest challenge to modern agriculture is to protect crops from pests and still maintain environmental quality. This study aimed to analyze by in silico, in vitro, and in vivo approaches to the feasibility of using the inhibitory protein extracted from mammals - Bovine Pancreatic Trypsin Inhibitor (BPTI) as a potential inhibitor of digestive trypsins from the pest Anticarsia gemmatalis and comparing the results with the host-plant inhibitor - Soybean Kunitz Trypsin Inhibitor (SKTI). BPTI and SKTI interacts with A. gemmatalis trypsin-like enzyme competitively, through hydrogen and hydrophobic bonds. A. gemmatalis larvae exposed to BPTI did not show two common adaptative mechanisms i.e., proteolytic degradation and overproduction of proteases, presenting highly reduced trypsin-like activity. On the other hand, SKTI-fed larvae did not show reduced trypsin-like activity, presenting overproduction of proteases and SKTI digestion. In addition, the larval survival was reduced by BPTI similarly to SKTI, and additionally caused a decrease in pupal weight. The non-plant protease inhibitor BPTI presents intriguing element to compose biopesticide formulations to help decrease the use of conventional refractory pesticides into integrated pest management programs.

Arginine-containing dipeptides decrease affinity of gut trypsins and compromise soybean pest development.

The design and production of molecules capable of mimicking the binding or/and functional sites of proteins inhibitors represent a promising strategy for the exploration and modulation of gut trypsin function in insect pests, specifically Lepidoptera. Here, for the first time, we characterized the trypsin activity present in the gut, performance and development of Anticarsia gemmatalis (Lepidoptera: Noctuidae) larvae when exposed to arginine-containing dipeptides. In silico assessment showed that arginine-containing dipeptides have a greater affinity for the active site of A. gemmatalis trypsins than lysine-containing peptides due to the presence of the double-charged guanidinium group that enhances the interaction at the S1 subsite of trypsins. Furthermore, the inhibitory and anti-insect potential of the peptides was demonstrated through kinetic and larval life cycle parameters, respectively. These dipeptides showed structural stability, binding to the active site, corroborated in vitro (competitive inhibition), and significant reduction of trypsin enzyme activity in the gut, survival, and weight of the A. gemmatalis larvae. Our findings reinforce the idea that small peptides are promising candidates for lepidopteran pest management. The optimization of DI2 and DI1 peptides, enhancing uptake and affinity to trypsins, may turn the use of these molecules feasible in agriculture.

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.

Novel proteinase inhibitor from the hemolymph of soybean pest Anticarsia gemmatalis (lepidóptera: Noctuidae): Structural modeling and enzymatic kinetic.

New approaches are needed to reduce risks to the environment and natural enemies and to avoid or delay the onset of insecticide resistance. The use of insecticides based on proteinase inhibitors of hemolymph is an alternative for the control of Lepidoptera pests primarily by having low toxicity and short persistence in the environment. Thus, in this study, we describe the purification process and identification of protease inhibitors from hemolymph Anticarsia gemmatalis and their activities against trypsin enzymes. Furthermore, the three-dimensional (3D) structure of the inhibitor and binding mode to trypsin enzymes was determined, and the stability of the inhibitory activity in several pHs and temperature values was evaluated. The inhibitor was characterized as a serpin family inhibitor and named A. gemmatalis hemolymph serpin inhibitor (AHSI), with an approximate mass of 38 ± 2 kDa, highly stable to temperature and pH variations, and with inhibitory capacity on bovine trypsin and gut trypsin of A. gemmatalis demonstrated by calculated Ki values and affinity energy through molecular docking, being a reversible competitive inhibitor that binds to the active site of trypsin-like enzymes. We conclude that the AHSI inhibitor identified from the hemolymph of the soybean pest A. gemmatalis preserves the original structure of the serpin family with a good overall stereochemical quality confirmed from molecular modeling. The docking analysis showed that the reactive site of the inhibitor is in contact with the catalytic cavity of the trypsin with high-affinity energy.

Small peptides inhibit gut trypsin-like proteases and impair Anticarsia gemmatalis (Lepidoptera: Noctuidae) survival and development.

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.

Leaf metabolic profiles of two soybean genotypes differentially affect the survival and the digestibility of Anticarsia gemmatalis caterpillars.

Insect pests such as Anticarsia gemmatalis cause defoliation and yield losses. Soybean breeding has obtained resistant genotypes, however the mechanism remains unknown. Studies indicated the presence of deterrents compounds in the resistant genotype IAC17, and their leaf metabolite profiles were compared to the susceptible genotype UFV105, which was elicited or not by caterpillar infestation. Cluster analysis indicated a significative distinction between these profiles as well as differences in plant defense pathways. Methylquercetins were constitutively present in the largest concentrations, specifically in the IAC17. Relationship between the resistance and the levels of phytohormones jasmonic acid, abscisic acid and salicylic acid was not observed. However, 1-aminocyclopropane -1carboxylic acid levels indicated that the ethylene may be involved in the constitutive biosynthesis of bioactive compounds. Extracts were added to the diets at three different concentrations to evaluate the effect on caterpillar survival. Lowest survival rates were observed when extracts from the resistant IAC 17 were used, at the lowest concentrations. Survival rates were not higher when IAC 17 infested by caterpillars were used. On the other hand, when extracts from the susceptible were used, the survival reductions were only observed in the highest extract concentrations. These supplementations of the diet reduced the digestive capacity, agreeing with the proteolytic activities, whereas malformations of the intestinal cells were dose dependent. The inhibitory effects persisted in higher dilutions only for the IAC17. Constitutive resistance was also explained by higher levels of protease inhibition. These results can be useful to elucidate the genes and cascades controlling the resistance.

Rational design of mimetic peptides based on the interaction between Inga laurina inhibitor and trypsins for Spodoptera cosmioides pest control.

The interaction of Inga laurina Kunitz inhibitor with insect trypsins is an example of protein-protein interaction with potential application for the pest control. However, the crop field application of proteins as inhibitors is limited due to high production cost, the large molecular size and low environmental stability. The use of mimetic peptides that have molecular features associated with the protein inhibitor can result in a product with lower cost and higher efficiency for the agricultural application. Here, we designed mimetic peptides deriving from globular domains of ILTI that are predicted to interact with trypsin enzymes of Lepidoptera pest. Two linear peptides were identified and synthetized from the interface of interaction between trypsin-ILTI complexes. These peptides were derived due to its high-energy contribution for the biding affinity between the enzyme-protein inhibitor. The peptides showed structural stability, propensity to adopt the bound conformation also without the context of the protein, inhibitory activity of digestive trypsins and toxic effects on the S. cosmioides, indicating that they can be used as potential inhibitor for pest control.

Noncompetitive tight-binding inhibition of Anticarsia gemmatalis trypsins by Adenanthera pavonina protease inhibitor affects larvae survival.

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 Ki 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 Å2 . The kinetic analysis revealed a noncompetitive tight-binding 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.

Kinetic Characterization of Anticarsia gemmatalis Digestive Serine- Proteases and the Inhibitory Effect of Synthetic Peptides.

BACKGROUND: Enzyme kinetics contributes to understanding the structure and function of insect digestive serine proteases. Kinetic parameters allow to understanding active sites and mechanisms of enzymes efficacy, identifying the inhibition of the insects digestive protease system by inhibitors produced by plants, or via the application of synthetic inhibitors Objectives: The aim of this study was to purify digestive serine proteases of A. gemmatalis, determining their kinetic properties using the chromogenic substrates tripeptidyl and characterizing the effects of synthetic inhibitors on their activity. In order to provide new opportunities for sustainable pest management through the development of protease inhibitors. METHODS: The enzymes were purified on p-aminobenzamidine agarose affinity column in an FPLC system using electrophoresis with 12.5% polyacrylamide gel. Michaelis-Menten constants and the inhibition model were determined according to the Dixon methodology and Lineweaver-Burk's double reciprocal. RESULTS: The KM values and catalytic constants of peptide substrates show that A. gemmatalis trypsin- like has a higher affinity for substrates with arginine in the P1 position. Inhibition by Gor 3, Gor 4, and Gor 5, in the presence of L-BApNA, was linear competitive. The inhibition constant for the Gor 5 peptide was higher due to its strong interaction with hydrophobic residues in the secondary site region of A. gemmatalis trypsin-like. CONCLUSION: It is observed that among the three peptides analyzed, the Gor 5 presented lower inhibition constant and therefore, the most potent among the tested ones. The predominance of hydrophobic residues in the region of the secondary site of the enzymes favored the interaction of the peptide. After characterization by three different types of graphs profiles, it is possible to verify that the inhibition model of the trypsin-like enzymes for the tested peptides is of the linear competitive type, in the concentration range of inhibitors and substrates analyzed. However, by the graphing profiles it is observed that the inhibition occurred due to the interaction of the peptides at the secondary site S2' in the hydrophobic cavity of the enzymes analyzed.