Study On Cloning And Expression Of The Key Enzymes IspC/D/E In The MEP Pathway And Screening Methods Of Their Inhibitors

The development of modern pesticides face great challenges including weed resistance,biological tolerance and environmental pollution.Due to the long-term repeat use of herbicides and fungicides with only a few modes of action,weed resistance and biological tolerance develop rapidly,the research and development of pesticide based on new targets is an effective way to solve these problems.The MEP pathway widely exists in most bacteria,plants,as well as the parasitic apicomplexans while not in humans and other animals,the inhibitors targeting the key enzymes in the MEP pathway would possess the merits such as low toxicity to mammal,enhanced selectivity,and reduced undesired side effects of anti-infectives.Therefore,the design of inhibitors that target the MEP pathway will have an important role in the development of new herbicides and fungicides.The main work in this thesis focused on the use of gene recombination technology to clone and express the key enzymes IspC/E in the MEP pathway,the establishment of high-throughput screening methods for these enzyme inhibitors,expecting to discover new inhibitors with high activity based on the activity determination of these enzymes.The contents of the thesis are described as follows:1.The first chapter summarizes the research progress of pesticides discovery based on target structure,the key enzymes IspC/D/E in the MEP pathway,and the application of photoaffinity labeling technology in the identification of target protein.2.The recombinant expression plasmids of the IspC enzymes from Arabidopsis thaliana,Plasmodium falciparum and Escherichia coli were constructed by using the gene recombination technology.The recombinant proteins were purified by prokaryotic induction and Ni2+-NTA affinity chromatography.The recombinant enzyme activity and other enzymatic properties were determined by spectrophotometry and a high throughput screening method of inhibitors for these IspC enzymes was established.The screening method was used to screen the newly synthesized 37 lead structures in our research group and to determine the IC50 values of those inhibitors with high inhibitory activity.The structure-activity relationship of these compounds were prelimilarily summarized.3.Using the already established high throughput screening method of AtIspD enzyme inhibitors,300 lead structures of enzyme inhibitors synthesized in our research group were screened,and the IC50 values of the inhibitors with high inhibitory activity determined.Among them,the phenylpyrazole inhibitors were found to have high activity,of which,the compound Z17400 showed an inhitotary efficacy of IC50=3.98 μM,which is more active than the reported pseudilin(IC50=13 μM)and PIX PIX(IC50=9.3 μM).The structure-activity relationship for these compounds was prelimilarily discussed.4.Using a strategy based on the biological orthogonal "post-click" reporter group(fluorescent molecule BODIPY-N3),the interaction between inhibitor probe molecule and AtIspD protein was investigated.The specific interaction was verified by gel electrophoresis and fluorescence imaging technology.It was confirmed that three trizaolopyrimidine compounds were able to be used as the lead structure of AtIspD enzyme inhibitors for the discovery of new herbicides and fungicides.5.Using the gene recombination technology,the recombinant expression plasmid of IspE enzyme of Arabidopsis thaliana was constructed.The protein,namely AtIspE,was purified by prokaryotic induction and Ni-NTA affinity chromatography.The enzyme activity was qualitatively determined by high performance liquid chromatography(HPLC)and liquid chromatography-mass spectrometry(LC-MS).The results laid a foundation for the subsequent establishment of the inhibitor screening method on the basis of the coupled AtIspD and AtIspE enzymatic reaction.