Novel Inhibitors Of Chitinases And Structure-based Inhibitory Mechanisms

Glycoside hydrolase family 18(GH18)chitinases(EC 3.2.1.14)catalyze the degeradation of β-1,4 linkages in chitin and chitooligosaccharides.GH18 Chitinases are widely distributed in organisms,such as bacteria,fungi,nematodes,plants,arthropods and mammals,playing key roles in bacterial nutrition supplying,fungal cell wall remodeling,nematode developing,arthropod molting,and plant and mammalian immune defenses.Chitin is the main structural component in the exoskeleton,trachea and peritrophic membrane of insects.The RNA interference of molting-related GH18 chitinases results in abnormal molting and death,suggesting these chitinases are essential for insects’ development and molting.Thus,GH18 chitinases are potential targets for developing drugs and agrochemicals.Human express two GH18 chitinases,Chitl and AMCase,which are involved in the resistance to the invasion of chitinous pathogens or parasites.Although Chit1 was found to participate in pulmonary fibrosis and pathogenic type 2 inflammation,the exact role of Chitl has not yet been fully defined.Development of novel,highly potent,highly selective and in vivo active Chit1 inhibitors can play a significant part in understanding the biological role and implications of inhibiting this protein.In this dissertation,two insect GH18 chitinases OfChtI,OfChi-h from the insect pest Ostrinia furnacalis,and one human chitinase HsChitl,were selected as targets for exploring novel chitinase inhibitors.The binding mode,selective mechanism and biological activity of the obtained inhibitors were investigated.The main results are listed below:(1)A novel scaffold for developing OfChtI specific inhibitorBased on the crystal structure of OfCht1,17 compounds from a commercial chemical database were identified by two rounds virtual screening.Among these,three compounds from FQ series(furan-quinoline scaffold)inhibited the activity of OfChtI with single-digit-micromolar IC50 values.One compound from TP series(tetrahydrothieno-naphthyridin scaffold)exhibited a broad inhibitory activity not only toward OfChtI but also toward bacterial,fungal,and human chitinases.Molecular docking results indicated that FQ series were bound in the non-reducing end of the substrate binding cleft of OfChtI,forming hydrogen bonds and stacking interactions with Glu140 and Trp34,respectively.(2)OfChi-h inhibitors and inhibitory mechanismMost of agricultural pests belong to Lepidoptera.Developing inhibitors of Chi-h,which is the Lepidoptera-exclusive chitinase,is helpful to solve the toxicity of insecticides to other beneficial insects.By virtual screening,two series compounds with a dipyrido-pyrimidine scaffold,namely the 2-8 and 2-8-s series,were obtained as inhibitors of OfChi-h.The most potent inhibitor of the OfChi-h,compound 2-8-s2,exhibited a Ki value of 9 nM.Crystal structures of OfChi-h complexed with 2-8-s2 revealed that the dipyrido-pyrimidine scaffold formed stacking interactions with Trp268 and Trp389 in+1 and+2 subsites.Nitrogen atoms of 3-pyridinylmethyl carboxamide formed hydrogen bonds with the catalytic residues Glu308 and Asp384,respectively.Additionally,the oxygen atom of 1-furan formed a hydrogen bond with Arg439.Isothermal titration calorimetry(ITC)experiments were performed to determine the thermodynamic changes during the inhibitor binding.The enthalpy change of 2-8-s2 when binding to OjChi-h was larger than that of 2-8-s2 binding to HsChit1 or SmChiB,confirming the stronger interaction between 2-8-s2 and OfChi-h.(3)HsChitl inhibitors and inhibitory mechanismInhibitory activity assay for 2-8 and 2-8-s series compounds against HsChitl suggested 2-8-14 was the most potent compound,with a Ki value of 49 nm.The crystal structures of 2-8-14 complexed with HsChitl showed that 2-8-14 formed stacking interactions with Trp99 and Trp218 in+1 and+2 subsites,and also formed several water-mediated hydrogen bonds with Glu140,Glu297,Asp213 and Arg269.In a murine model of bleomycin-induced pulmonary fibrosis,2-8-14 was found to suppress the chitinase activity by 60%,leading to a significant increase in inflammatory cells and suggested that Chit1 played a protective role in immune homeostasis during early pulmonary firbosis.In summary,by utilizing a combination of in silico,in vitro and in vivo experiments,we identified chitinase inhibitors with high inhibitory activity and selectivity.Combining X-ray crystallography and computational modeling,we further revealed the molecular basis of the inhibition and selectivity.The identified novel scaffold of the chitinase inhibitor provides a new starting point for the design of green pesticides,molecular tools and inflammation regulating drugs.