Discovery And Activity Evaluation Of Insect Chitin Hydrolase Multi-target Inhibitors

The use of pesticides is still an effective way to effectively reduce pests and increase food production.The discovery and development of new targets to control the evolution of pest resistance to pesticides and reduce the toxicity to non-target organisms has become a hot research direction in recent years.Insect chitin hydrolase has been confirmed as a green pesticide target.In summary,the existing studies on chitin hydrolase inhibitors have found that natural products have better insect growth regulating activity than synthetic compounds.Because multi-target drugs can increase the possibility of their effects in insects,this paper uses high-throughput screening of natural product libraries to screen out compounds that have high-efficiency inhibitory effects on multiple chitin hydrolases and study their inhibitory mechanism and biological activity,the main experimental contents and results are as follows:High-throughput screening of natural product libraries.With the four enzymes of OfChtⅠ,OfChtⅡ,OfChi-h and OfHex1 from the Ostrinia furnacalis as targets,Through high-throughput screening,baicalein and deoxyshikonin were found to have inhibitory activity on all four enzymes.According to existing research,the final selection naphthoquinone series of deoxyshikonin,shikonin and alkannin,and flavonoid series of baicalein,3-hydroxyflavone,keampferol,galangin,wogonin,chrysin,quercetin and myricetin were used for the determination of inhibition constants,binding mode analysis and insect growth regulation activity analysis.Determination of the inhibition constant of naphthoquinone series and flavonoid series.The inhibition constants of alkannin,shikonin and deoxyshikonin on the four enzymes of OfChtⅠ,OfChtⅡ,OfChi-h and OfHex1 from the O.furnacalis were determined.Deoxyshikonin has the smallest inhibitory constant for these four enzymes,with K_i values of 27.88μM,0.68μM,5.5μM,and 12μM,respectively.The inhibitory constants of alkannin,shikonin and deoxyshikonin on OfHex1 are the smallest among the four enzymes,and the K_i value is about1μM.The overall inhibitory constant of flavonoids on OfChtⅠ is relatively large.Myricetin has the smallest inhibitory constant for OfChtⅡ,OfChi-h,and OfHex1,with K_i values of 5.3μM,8.2μM and 4.1μM,respectively.Analysis of the binding mode of inhibitors.The binding modes of deoxyshikonin and OfChtⅠ,OfChtⅡ,OfChi-h are similar.Both the naphthoquinone part and the conserved tryptophan residue in the-1 subsite of the substrate binding cleft form aπ-πstacking interaction.The side chain of deoxyshikonin extends to the non-reducing end of the cleft where it binds to the substrate.Compared with the combination of deoxyshikonin and enzymes,the side chain hydroxyl groups of alkannin and shikonin form steric hindrance,reducing the ability to bind to enzymes.The binding mode of the compound deoxyshikonin and OfHex1 is that the naphthoquinone ring of the compound forms aπ-πstacked sandwich structure with Trp⁴⁹⁰and Val²³⁷at the+1 position,and the isohexenyl tail of deoxyshikonin is inserted into the hydrophobic-1 of the substrate binding pocket.The flavonoid series of compounds have similar binding modes with OfChtⅠ,OfChtⅡ,OfChi-h.The conjugated largeπring of the benzopyran ring is formed by the conserved tryptophan residue in the-1 subsite of the substrate binding cleft the interaction ofπ-πstacking,the benzopyran ring forms hydrogen bonds with the key catalytic amino acid.The phenyl group of the side chain extends to the non-reducing end of the cleavage binding to the substrate,forming a hydrophobic interaction or hydrogen bonding interaction to stabilize the binding.The combination of the number of hydroxyl groups on the side chain phenyl of flavonoids and OfHex1 plays a very important role.Analysis of insect growth regulation activity.The three compounds of the naphthoquinone series have certain insect growth regulating activities against the O.furnacalis,and Shikonin has the most obvious effect.Also has a significant inhibitory effect on the growth of Mythimna seperata and Spodoptera frugiperda.After 6 days,the weight of the larvae of S.frugiperda fed with Shikonin is 99%lower than the control group,and the weight of the M.seperata is 98%lower than the control group.Among the five compounds of the flavonoid series,wogonin has obvious growth regulation activity on the O.furnacalis.After 6 days,the larval body weight is88%lower than the control group,and the larval mortality rate is 50%.Wogonin has an inhibitory effect on the growth of M.seperata and S.frugiperda.After 6 days,the weight of the M.seperata was 62%lower than that of the control group,and the weight of the M.seperata in the experimental group was 84%lower than that of the control group.In addition,wogonin has obvious insecticidal activity against M.separata,and the mortality rate of the experimental group of M.separata larvae after 5 days of rearing is as high as 88%.In summary,this paper uses chitin hydrolase OfChtⅠ,OfChtⅡ,OfChi-h and OfHex1 as targets to screen out natural compounds that simultaneously inhibit these four enzymes through high-throughput screening of natural product libraries,with a view to insect-mediated The disease control and agricultural pest control are of practical significance.