Study On The Potent Target And Mechanism Of XWJ-1601 Based On Chemical Proteomics

The discovery of unknown targets of active small molecules is an important part of their further development.At present,the "target hooking" technology based on quantitative isotope labeling and high-resolution mass spectrometry has been widely used in the discovery of small molecular compound targets.In this thesis,the 2-substituted-6-(6-(4aminopiperidin-l-yl)-pyridin-3-yl)-1H-imidazo[4,5-b]pyridine derivatives(XWJ-16 series compounds)synthesized in our laboratory were tested for tumor cytotoxicity,and the structure-activity relationship of these compounds was preliminarily discussed.Then the representative compound XWJ-1601 was preliminarily studied in vivo and in vitro antitumor activity and anti-tumor mechanism.Finally,based on the "Biotin-Avidin System" technology(BAS)and "Proteolysis target chimeras" technology(PROTAC),we preliminary studied the possible target binding spectrum of XWJ-1601,and some important targets were verified.The project mainly achieved the following results:1)The tumor cytotoxicity result of the XWJ-16 series of compounds indicated that the compounds showed obvious anti-proliferative activity on the tumor cells,and these compounds had a greater impact of the proliferation inhibition rates of HepG2 and A498 cell lines than that of MCF-7 cell lines.Besides,the position of phenyl-substitution displayed a great influence on the activity of the compound.2)The in vitro and in vivo antitumor activity experiments of the representative compound XWJ-1601:The IC50 values of XWJ-1601 against tumor cells HepG2 was 363.4±7.4 nM,respectively.In the mice hepatoma HepG2 xenograft model,XWJ-1601 significantly inhibited the growth of tumors at the concentration of 25 mg/kg,but it had no obvious toxicity to the liver and spleen of mice.3)The research on the anti-cancer mechanism of the representative compound XWJ1601 showed that XWJ-1601 might down-regulate the potential therapeutic target of liver cancer-RAR y in a time-dependent and concentration-dependent manner.XWJ-1601 induced HepG2 cells to arrest in G2-M phase,and down-regulated anti-withering death protein BCl2,and induced PARP cleavage.The intervention effect of XWJ-1601 on the whole proteome of HepG2 cells showed that XWJ-1601 might block the cellular process by up-regulating the function of MAPK kinase or down-regulating the expressing level of genes such as POLR2A,thereby blocking the cell process and thereby accelerating cell apoptosis.4)Representative compound XWJ-1601 potential important target verification:①XWJ-1601-Biotin enriched CDK9 and EGFR which could be partially competed by XWJ1601;②The mass spectrum at 55 Kd determined that CDK9 was a target of XWJ-1601;③XWJ-1601-PO-4 down-regulated the protein expression levels of CDK9 and EGFR;④The results of kinase assay showed that XWJ-1601 inhibited EGFR kinase activity in a concentration-dependent manner with an IC50 value of 6.8 μM,but XWJ-1601 had no effect on the kinase activity of CDK9.It was speculated that XWJ-1601 did not directly affect the kinase activity of CDK9,but regulated CDK9 through protein-protein interaction.In summary,XWJ-1601 is a kinase inhibitor of EGFR and a small molecule ligand of CDK9.Therefore,XWJ-1601 can be used as a seedling compound for the treatment of liver cancer.Based on the existing biological data,the target binding spectrum and the mechanism of action of XWJ-1601 will be further determined,which will provide insights into the possible toxic and side effects and experimental basis for the further development of 1Himidazo[4,5-b]pyridine derivatives.