The Inhibitory Mechanism Of Chitinase OfChtⅡ From Ostrinia Furnacalis

Chitinase plays an important role in insect growth and development.Small-molecule inhibitors of insect chitinases have potential applications for pest control.Three insect chitinases(ChtⅠ,ChtⅡ and Chi-h)are reported to be involved in the cuticle degradation during ecdysis.Among them,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.In the previous study,we have obtained the crystal structure of OfChtⅡ,a group Ⅱ chitinase from the agricultural pest Asian corn borer Ostrinia furnacalis.Based on the crystal structure,we obtained four small-molecule inhibitors targeting OfChtⅡ by screening.These inhibitors were chitooctaose[(GlcN)8],dipyrido-pyrimidine derivative(DP),piperidine-thienopyridine derivative(PT),and naphthalimide derivative(NI).(GlcN)8 only showed moderate inhibitory activity,while DP,PT and NI exhibited excellent inhibitory activity,with Ki values of 1.99 μM,1.72 μM and 2.18 μM,respectively.To elaborate the inhibitory mechanism,we further resolved the crystal structures of four enzyme-inhibitor complexes with high-resolution(OfChtⅡ-C1/(GlcN)8,1.60 A;OfChtⅡ-C1/DP,1.50 A;OfChtⅡ-C1/PT,1.44 A;OfChtⅡ-C1/NI,2.00 A).Although DP,PT and NI possess distinctively different structures,the crystal structures showed that they all exhibited similar binding modes within the substrate-binding cleft of OfChtⅡ.Specifically,one hydrophobic group of the inhibitor interacted with conserved Trp1691/Trp 1809 at the+1/+2 subsites of substrate-binding cleft,while another hydrophobic group of the inhibitor bound at the-1 subsite of substrate-binding cleft and interacted with a hydrophobic pocket formed by Trp1621,Tyr1803,Tyr1856,Phe1899 and Trp1961.In vivo bioactivity tests showed that 30%of the larvae in three experimental groups were arrested at the 5th instar and failed to molt into pupa.In summary,this study reported multiple small molecule inhibitors targeting OfChtⅡ and revealed a general mechanistic principle that confers inhibitory activity against ChtⅡ.These results will facilitate rational design of green pesticides that target ecdysis of insect pests.