Noncompetitive tight‐binding inhibition ofAnticarsia gemmatalistrypsins byAdenanthera pavoninaprotease inhibitor affects larvae survival

Abstract

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

Extensive reprogramming of protein isoforms and histopathological alterations in the midgut of Anticarsia gemmatalis fed with protease inhibitors

“…At the kinetic level, SKTI show lower inhibition constant (Ki) compared with those from synthetic inhibitors, which allow them to occupy all the binding sites available in the active centre of the enzyme and thus an optimised adjustment occurs in the enzyme‐inhibitor (EI) complex formation. The synthetic inhibitors, being smaller, do not occupy all the available sites of the enzyme, forming less stable EI complex (Meriño‐Cabrera, de Oliveira Mendes, et al, 2020; Meriño‐Cabrera, Severiche Castro, et al, 2020; Patarroyo‐Vargas et al, 2020; Volpicella et al, 2003). In this study, we found that protein synthesis is accompanied by changes in the insect's enzymes profile.…”

Trypsins: Structural Characterization and Inhibition Focus in Insects

“…Trypsins (EC 3.4.21.4) are serine proteases and are considered the most important digestive proteases of most insects. Trypsins are involved in the initial phase of protein digestion and are characterized by containing a catalytic triad consisting of the amino acid residues Hys, Asp, and Ser; in A. gemmatalis trypsin are Ser 229, Hys 85, and Asp 132 [16,29,30].…”

“…Several works show that chronic exposure of insects, mainly those of the order Lepidoptera, to plant-derived protease inhibitors such as SKTI (Soybean Kunitz Trypsin Inhibitor), SBBI (Soybean Bowman-Birk Inhibitor), APTI (Adenanthera pavonina Trypsin Inhibitor), ILTI (Inga Laurina Trypsin Inhibitor) showed negative effects on the larval cycle of these herbivores (Figure 3). These adverse effects include reduced weight, survival, and delayed larval cycle [17,29,[71][72][73][74][75]. However, the use of these molecules so far in agriculture has not proven effective, mainly due to the long exposure period required to cause effective control rates.…”

“…The reference [11] evaluated the effect of this inhibitor throughout the insect cycle and concluded that although it caused an increase in the larval cycle and a higher percentage of mortality, most larvae were able to adapt to the inhibitor by remodeling the amount and type of enzyme present during digestion of the artificial diet. In addition, negative effects of other protease inhibitors on A. gemmatalis, mainly the organic ones [29,30], were shown. Peptide protease inhibitors have also been tested, which were developed from molecular minimization using docking-molecular techniques in developing insects of the order Lepidoptera [81].…”

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

“…Thus, given the abovementioned adaptive mechanisms exhibited by Lepidopteran insects when exposed to PIs type proteins, minor and specific molecules have been proposed as inhibitors of their main proteases in the gut (Chen et al, 2013; Meriño‐Cabrera, Castro, et al, 2020; Meriño‐Cabrera, de Oliveira Mendes, et al, 2020; Saikhedkar et al, 2019). In this investigation, we characterized of stability in silico by molecular dynamic (MD) and in vitro of the previously proposed tripeptides GORE1 (Val–Leu–Lys) and GORE2 (Val–Leu–Arg) (Barros et al, 2021).…”