Inhibitors Of Chitin Synthase And Chitinolytic Enzymes And Their Inhibitory Mechanism

Plant pests and diseases threaten agricultural production and food security.Eco-friendly and efficient agro-chemicals are the inevitable choice for managing these agriculturally-threatening organisms.Chitin is a critical polysaccharide for the growth of parasitic fungi,nematodes,and insects,but is absent in plants and mammals.Thus,inhibition of chitin synthesis and chitinolytic enzymes using small molecules can control pests and pathogens,without affecting plants and mammals,therebyfore protect crops.Chitin synthesis and degradation requires a series of chitin metabolic enzymes including ?-N-acetyl-hexosaminidase,chitinase,and chitin synthase.In this work,inhibitors were screened against insect ?-N-acetyl-hexosaminidase,nematode chitinase,and fungal chitin synthase and their mechanisms of inhibition were studied.1)Inhibition of fungal chitin synthase and its mechanismFungi is an important plant pathogenic microorganism.In this study,an inhibitor screening method for fungal chitin synthase,using the model fungi Saccharomyces cerevisiae,was established,and a series of novel chitin synthase inhibitors were found.The most potent inhibitor,S-16,was 2.5-fold more effective than the classical chitin synthase inhibitor,Nikkomycin Z.Moreover,all of these inhibitors exhibited antifungal activities against the growth of the agriculturally-destructive fungi,Fusarium graminearum,Botrytis cinerea,and Colletotrichum lagenarium at 100 ?g/mL.In addition,based on the complex structure of cellulose synthase BcsA/B with UDP,a model of the S.cerevisiae chitin synthase(ScCHS1)complexed with the UDP moiety of its substrate,UDP-GlcNAc,was established.Then according to the structural similarity of the inhibitors and UDP-GlcNAc,an inhibitor binding model to the chitin synthase active center was proposed.In this model the Uridine-N-acylhydrazone moiety binds to the UDP binding site,the GlcNAc mimicry moiety binds to the GlcN Ac binding site,and the hydrophobic alky chain moiety binds to the hydrophobic entrance of the acceptor binding channel.2)Inhibition of insect ?-N-acetyl-hexosaminidase and its mechanismOstrinia furnacalis is a major crop pest.A series of bis-aromatic naphthalimide derivatives with high ?-N-acetyl-hexosaminidase inhibitory activity was screened against the?-N-acetyl-hexosaminidase from O.furnacali(OfHex2).Compound 1-09 was the most effective inhibitor of OfHex2,with a Ki value of 0.37 ?M,and was 7-fold lower than the most efficient known ?-N-acetyl-hexosaminidase inhibitor,M-31850.Then,a structure model of OfHex2 was built using the homologous structure model of human ?-N-acetyl-hexosaminidase(HsHex)as the template.According to this structure model,a binding model of naphthalimide derivatives with OfHex2 was assembled from molecular docking and tryptophan fluorescent assay data.This predicted that the inhibition was achieved by the naphthalimide moiety stacking with the tryptophan residues in the active pocket;the other aromatic moiety interacting with a hydrophobic site outside of the active pocket;and hydrogen bonding between the secondary amine of the linker connecting the two aromatic moieties and the side chain of residue E345.3)Inhibition of nematode chitinase and its mechanismPlant parasitic nematodes have a great impact on agriculture.Inhibitor screening and inhibition mechanism study were performed using chitinase CeChtl,a key enzyme in nematode egg hatching,from the model nematode Caenorhabditis elegans.Firstly,recombinant CeChtl was expressed and purified.Then,a series of novel naphthalimide CeChtl inhibitors were screened.Compound I-26 was the most efficient inhibitor with a Ki value of 4.68 ?M.In addition,a structure model of CeChtl was built using the homologous structure of chitinase O.furnacali(OfChtI)as the template.Using this model,molecular docking was performed and the inhibitory mechanism for the bis-aromatic naphthalimide derivatives was attributed to the naphthalimide moiety binding to a hydrophobic area near the +2 glycosyl binding site;the other aromatic moiety stacking with residue W394;and hydrogen bonding between the linker secondary amine moiety and residue E179.For the mono-aromatic naphthalimide derivatives,the inhibition was attributed to the stacking interaction between the naphthalimide moiety and residue W394,and the hydrogen bonding between the linker secondary amine moiety and both residues E179 and D177.In summary,this work found novel effective inhibitors against the key chitin metabolic enzymes ?-N-acetyl-hexosaminidase,chitinase,and chitin synthase,and proposed their inhibitory mechanisms.This lays the foundation for the development of novel agro-chemicals against key chitin metabolic enzymes...