Structural dissection reveals a general mechanistic principle for group II chitinase (ChtII) inhibition

Abstract

Small-molecule inhibitors of insect chitinases have potential applications for controlling insect pests. Insect group II chitinase (ChtII) is the most important chitinase in insects and functions throughout all developmental stages. However, the possibility of inhibiting ChtII by small molecules has not been explored yet. Here, we report the structural characteristics of four molecules that exhibited similar levels of inhibitory activity against OfChtII, a group II chitinase from the agricultural pest Asian corn borer Ostrinia furnacalis These inhibitors were chitooctaose ((GlcN)₈), dipyrido-pyrimidine derivative (DP), piperidine-thienopyridine derivative (PT), and naphthalimide derivative (NI). The crystal structures of the OfChtII catalytic domain complexed with each of the four inhibitors at 1.4-2.0 Å resolutions suggested they all exhibit similar binding modes within the substrate-binding cleft; specifically, two hydrophobic groups of the inhibitor interact with +1/+2 tryptophan and a -1 hydrophobic pocket. The structure of the (GlcN)₈ complex surprisingly revealed that the oligosaccharide chain of the inhibitor is orientated in the opposite direction to that previously observed in complexes with other chitinases. Injection of the inhibitors into 4th instar O. furnacalis larvae led to defects in development and pupation. The results of this study provide insights into a general mechanistic principle that confers inhibitory activity against ChtII, which could facilitate rational design of agrochemicals that target ecdysis of insect pests.

Keywords: Asian corn borer (Ostrinia furnacalis); X-ray crystallography; ecdysis; glycoside hydrolase; group II chitinase; inhibition mechanism; inhibitor; inhibitor design; insect; protein crystallization.

Figure 1.

The chemical structures of inhibitors.

Figure 2.

Overall structure of OfChtII-C1 in complex with (GlcN)₈. A, cartoon representation of OfChtII-C1 complexed with (GlcN)₈. The ligands are shown as sticks with yellow carbon atoms. B, close view of the substrate-binding cleft with details of the interactions between (GlcN)₈ and OfChtII-C1. The composite omit map around the ligand is contoured at the 2.0 σ level. The catalytic residues and the amino acids that interact with the ligand are labeled and shown as sticks with cyan carbon atoms. The numbers indicate the subsite to which the sugar is bound. Hydrogen bonds are drawn as dashed lines.

Figure 3.

Comparison of OfChtII-C1 interactions with distinct inhibitors. A–C, the binding conformation of DP (A), PT (B), and NI (C) are shown in sticks with green, cyan, and orange color, respectively. The 2 F_o − F_c electron-density map around the ligand is contoured at the 1.0 σ level. D, merged view of the active site region of OfChtII-C1 shows that different inhibitors are bound in a similar conformation. The residues of OfChtII-C1 participating in the interactions with each inhibitor are shown in blue sticks and labeled with residue numbers. Hydrogen bonds are displayed as dashed lines.

Figure 4.

In vivo evaluation of OfChtII inhibitors. The inhibitors dissolved in 4% DMSO at a concentration of 0.5 mm were injected into 4th instar, day 1 O. furnacalis larvae. The numbers of larva, pupa, and dead larva were counted 5 days and 12 days after injection. The larvae injected 4% DMSO were used as control. The results are the average of three independent repeats.