Discovery of conformation constrained tetracyclic compounds as potent chitinase Of Chi-h inhibitors with a novel binding mode

Abstract

Chitinase h (Chi-h) has been identified as a promising pesticide target due to its exclusive distribution in lepidopteran insects and its essential role in the moulting processes. In this study, we leverage OfChi-h from destructive agricultural pest Ostrinia furnacalis (Asian corn borer) as a model target to identify novel chitinase inhibitors. A conformational restriction approach was employed to design a series of novel OfChi-h inhibitors. Among these, compound 6a showed the highest inhibitory activity against OfChi-h, with a K_i value of 58 nM. Molecular docking analysis suggested that 6a tightly bound to three subsites (-3 to -1) of OfChi-h. The binding mode is further confirmed by the co-crystallization data of 6a with the SmChiA, a bacterial homologue of OfChi-h, at a resolution of 1.8 Å. This research presents a novel approach for the development of highly potent insect chitinase inhibitors, offering potential tools for effective pest control.

Keywords: Chitinase; conformation restriction; inhibitor; inhibitory mechanism.

Graphical abstract

Figure 1.

Binding clef of OfChi-h and its inhibitors. (a) OfChi-h in complex with (GlcN)₇ (PDB: 5GQB) and (b) representative chitinase OfChi-h inhibitors.

Figure 2.

Design strategy of novel tetracyclic chitinase inhibitors. (a) Predicted binding mode of compound 5a with OfChi-h; (b) superimposition of compound 5a and compound 6a; (c) novel conformation restriction strategy by ring closure.

Scheme 1.

Synthesis of target compounds 6a-l. Reagents and conditions: (a). neat, 110 °C; (b). POCl₃, DMF, 0 °C-80 °C; (c). aqueous ammonia, EtOH, 70 °C; (d). methyl cyanoacetate, r.t. (e). NaOH/EtOH, 70 °C; (f). DMFDMA, neat, 110 °C.

Figure 3.

Representative Dixon plots for inhibition chitinase SmChi-A by 5a (a), and chitinase SmChi-A (b), OfChi-h (c) and HsChit1 (d) by 6a. The trend lines represent three substrate concentrations.

Figure 4.

Binding mode of 6a with SmChiA and OfChi-h. A. Binding mode of 6a with SmChiA revealed by co-crystal complex structure; B. Binding mode of 6a with OfChi-h revealed by molecular docking. 6a is shown in violet sticks, the key residues involved in inhibitor binding are shown in gray sticks, hydrogen bonds are shown as black dashed lines and water molecules are shown as red spheres.

Figure 5.

Superposition of 6a (a) and 6e (b) with 2–8-s2 in the binding site of OfChi-h. 6a is shown in violet sticks, 6e was shown in red sticks, 2–8-s2 was shown in light green sticks and the key residues involved in inhibitor binding are shown in gray sticks, hydrogen bonds are shown as black dashed lines.