Isolation And Purification Of Native Spinach D1 Protease And Its Interaction With Novel Small Inhibitors

Drug discovery is the preliminary stage in the research and development of drug, including drug molecular design,synthesis,target discovery and confirmation,lead compounds screening and optimization,and so on.In nowadays,rational design and synthesis of lead compounds are the most important methods for target directed drug development.The process of pesticide discovery is similar to that of drug discovery. New target discovery and the new pesticide discovery based on it play an important role in the modern development of pesticide.D1 protein is the necessary component for the assembly of phtotosystemⅡin plants, and it comes from the D1 protein precursor(pD1).The precursor has an extension of polypeptide and which is cleaved by a carboxyl terminal processing protease of D1 protein(CtpA)to form mature D1 protein(mD1).D1 protein is the target of most herbicide used at present.However,the content of CtpA in plant is only 1%of that of D1 protein.Therefore,CtpA is considered as a high-efficient herbicidal target in novel herbicide development.It is very important for the methodology establishment of isolation and purification of CtpA as well as the herbicidal activity screening of novel potential inhibitors based on this novel target.In this thesis,the study mainly covers three aspects:1)isolation and purification of CtpA from spinach;2)determination of CtpA specific activity and Michaelis constant K_m; 3)investigation of the biological activity of novel potential herbicides targeting CtpA.1).Isolation and purification of spinach CtpATooking fresh spinach leaves as the raw material,the chloroplast was obtained by grinding,homogenizing,extraction with buffer solution.With the untrasonic fragmentation of chloroplast and protein salting,the total thylakoid membrane proteins were obtained.Through the fractional salting procedure with ammonium sulfate,the crude CtpA was obtained.The crude CtpA was further purified by anion exchange chromatography and gel filtration chromatography,reaching a purity of 90%.2).Determination of CtpA specific activity and Michaelis constant K_mThe synthetical 24-mer peptide was taken as the D1 protein mimic substrate for CtpA enzymatic hydrolysis,the determination of the specific activities of CtpA was obtained in each step of the purification process,through the raction monitoring by HPLC.The highest specific activity reached 0.172 nmol·mg^-1·min^-1;Analysis showed that the Michaelis constant K_m for 24-mer peptide was 19.6μM.3).Biological activities of the lead compounds based on CtpA target. Based on the inhibitive effect to the CptA enzymatic hydrolysis of 24-mer peptide and the HPLC analysis,the biological activities of four novel lead compounds were investigated.A new methodology for the lead compounds screening of CtpA inhibitors was established.The molecular structures of these lead compounds involved in this study were shown in the following:The four lead compounds of CtpA inhibitors were synthesized in our lab,which were designed and synthesized according to hits from the homology modeling of spinach CtpA tridimensional structure and the virtual screening of molecules generated with the fragment-based de novo design.The results show these compounds possess effective inhibition to CtpA with a competitive inhibition.The inhibition constants of a1,a2,b1 and b2 were 3.8,4.9,5.2 and 8.8μM,respectively.Compounds with relatively lower inhibiting constants,such as al and a2,can inhibit the growth of dicotyledonous rape in 100ppm,with an inhibition rate of 100%.Thus,we have established a new method to validate CtpA target and to screen the CtpA inhibitors by CtpA enzymatic reaction and HPLC analysis.