| Cancer is the second largest disease after cardiovascular disease in terms of morbidity and mortality.Among these cancers,the incidence rate and mortality are higher for lung cancer,stomach cancer,skin cancer,liver cancer,colorectal cancer,skin cancer and female breast cancer.According to the data from world health organization in 2015 survey that Cancer is the first or second leading cause of death in most countries.Human fructose 1,6-bisphosphate aldolase(Aldolase),including three isoenzymes:ALDOA,ALDOB and ALDOC,is a key regulatory enzyme in human glycolysis and gluconeogenesis.Studies have shown that normal cells maintain the normal life activities of the human body through the energy produced by glucose metabolism.Compared with normal cells,in a variety of tumor cells such as oral squamous cell carcinoma,liver cancer and lung cancer,the glycolysis level was significantly increased,and the expression level of Aldolase in tumor cells is over-expressed.A large number of studies have shown that cancer can be treated by inhibiting the activity of Aldolase,and Aldolase is considered as a new target for treating cancer.Based on this,we first purified by affinity chromatography to obtain high-purity,high-yield,high-activity ALDOA,ALDOB,ALDOC and 17 ALDOB mutant proteins.On this basis,we tested the inhibitory effect of maleimide compounds on Aldolase,the structure-activity relationship was analyzed,and the binding mode was explored.Specific research work includes the following three aspects:1.Establishment of ALDOA,ALDOB,ALDOC expression and purification systems and study of enzymatic kineticsWe constructed the ALDO A,ALDOB,and ALDOC gene into the pET-28a(+)vector,and we used Escherichia coli BL21(DE3)as an expression strain to express ALDO A,ALDOB,and ALDOC heterologously.The expression condition was optimized.It including induction temperature,induction time and inducer concentration,we use the method of hydrophobic chromatography to get the purified high-activity enzyme ALDOA,ALDOB and ALDOC with the purity of more than 95% and high protein yield.We used the enzyme coupling reaction method to establish an enzyme activity-testing system for Aldolase,and we optimized the reaction systems of ALDOA,ALDOB,and ALDOC.The effects of reaction time,temperature,pH,TPI content and GPDH content on the enzyme activities of ALDOA,ALDOB and ALDOC were determined.Then the kinetic constants of the three enzymes were determined.2.Inhibitory effect of maleimide compounds on ALDOA,ALDOB and ALDOCWe determined the inhibitory effect of 33 maleimide compounds synthesized earlier on three isoenzymes.The results showed that the IC50 of Compound 127 to ALDOA was 60.04 ±3.50 μM,the IC50 of compound 127 to ALDOB was 1.20 ± 0.16 μM,and the IC50 of compound 127 to ALDOC was 80.92 ± 2.08 μM.Compared with ALDOB,the inhibitory effects of compound 127 on ALDOA and ALDOC were reduced by 50 and 67 times,The results indicate that compound 127 has good selectivity,with selectivity reaching 50-fold and 67-fold,respectively.We used Compound 127 as a probe molecule and use a combination of theoretical prediction and experimental verificationSite-directed mutagenesis experiments to explore the mechanism between the compound 127 and ALDOB.Covalent binding of cystine inhibits the activity of ALDOB.It provided theoretical guidance and ideas for the subsequent design and modification of inhibitors targeting Aldolase.3.Screening and optimization of ALDOA and ALDOB crystallization conditionsIn order to study the mechanism of action of Aldolase and maleimide compounds,we tried to culture protein crystals of ALDOA and ALDOB.The first is the screening of crystallization conditions,and then according to the crystallization conditions and crystallization conditions,we optimized the precipitating agent,salt concentration,buffer solution and pH.Finally,we obtained the ALDOA crystals with good diffraction quality.It lays the foundation for the subsequent structural biology research. |
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