| There are a variety of glucose decomposition pathways in cells,the more common ones are glycolysis,tricarboxylic acid cycle and pentose phosphate pathway(pentose phosphate pathway,PPP).The PPP pathway is carried out in the cytoplasm.Because the needs of cells are different in different periods,PPP will produce different products in different life activities and physiological conditions.It can be divided into oxidation branch and non-oxidation branch.The oxidative branch can produce NADPH and ribose to support cell activity,including three irreversible reactions catalyzed by glucose-6-phosphate dehydrogenase(G6PD),6-phosphate gluconolactone(6PGL)and 6-phosphate glucose dehydrogenase(6PGD).The most representative function of G6PD is to maintain the oxidative balance of cells.The loss of G6PD activity or maladjusted G6PD state will prevent normal cell proliferation and the development of embryos and organisms.As G6PD mediates and affects the occurrence,diagnosis,treatment and prognosis of many diseases,it is necessary to research and develop G6PD inhibitors.Moreover,the discovery of small molecular inhibitors targeting G6PD will further accelerate the research of tumor therapy targeting G6PD.In the second and third chapters of the graduation thesis,we first constructed the plasmid encoding G6PD,expressed the G6PD protein,and purified the target protein with a purity of more than 95%for follow-up experiments.After that,a high-throughput screening platform for targeted G6PD inhibitors based on enzyme kinetic cycle and NADPH absorbance detection was established and optimized.on this basis,this study carried out high-throughput screening of the self-built chemical library in the laboratory.Through the confirmation of the compounds screened,the compounds with better activity were determined and the false positive was excluded.The combination of MST and SPR was verified by experiments,and the combination of the two promoted further experimental research.Through the dilution inhibition kinetics experiment,it was found that the inhibitory effect of Wedelolactone on the enzyme activity of G6PD was reversible and recoverable.At the same time,through the enzyme dynamic substrate competition experiment,it is found that Wedelolactone is non-competitive to NADP+and G6P,the two substrates of G6PD.Combining the results of the two aspects,Wedelolactone is a substrate non-competitive reversible inhibitor targeting G6PD.However,according to these experiments,it is impossible to determine which specific site Wedelolactone acts on G6PD protein.Therefore,this study wants to study the more precise interaction mode between Wedelolactone and G6PD protein by co-crystallization.First,point crystals and crystal growth of G6PD are carried out under different conditions,and a series of conditions for crystal accumulation and growth are found,and then the empty crystals of G6PD are incubated with selected Wedelolactone compounds.The crystal diffraction is carried out,which is helpful for the further optimization of eutectic growth conditions and eutectic diffraction.In the fourth chapter,this study explores the effect of Wedelolactone on G6PD at the cellular level.The effects of budding compound Wedelolactone on the proliferation of liver cancer cells and ovarian cancer cells were characterized by cell viability test,cell clone formation test and cell glucose consumption test on Hepa1-6 and Ovcar3 cells.At the same time,the results showed that Wedelolactone affected the glucose consumption of Hepa1-6 and Ovcar3 cells,which affected the growth of tumor.In this graduation thesis,the main work of this study is to establish a high-throughput screening system for G6PD-targeted inhibitors,which provides a new strategy for the study of other oxidoreductases and has application value.At the same time,the non-covalent and non-competitive small molecule inhibitor Wedelolactone,of G6PD is found,which provides a new idea for the discovery of G6PD inhibitors and disease anti-tumor strategy. |
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