Study On The Action Mechanism Of Small Molecular Compounds Of Indole Alkaloids Targeting P53 For Anti-tumor

Tumors are formed in some cases due to abnormalities in cell proliferation and apoptosis.They are mainly regulated by tumor suppressor genes and proto-oncogenes in cells.They are related to each other and restrict each other.Any mistake in the link may cause the cells to become cancerous.Among them,P53 gene is the most relevant inhibitor of tumor,and P53 gene mutation occurs in about 50%of tumors.P53 gene is the main pathway regulating the malignant metastasis of protective cells,and has the function of inhibiting or preventing cell proliferation,transformation and protecting genome stability.However,the most studied oncogene is MDM2.The MDM2 protein can bind to the acidic domain and partial transcriptional activation domain of P53 protein to form p53-MDM2 complex,which inhibits the normal function of P53 and induces tumorigenesis.In theory,the p53 signaling pathway in tumor cells can be activated by preventing the interaction between MDM2 and P53.Therefore,inhibition of the interaction between p53-MDM2 is considered to be one of the important anti-tumor drug targets,and as a target for drug discovery and screening.In addition,according to the literature,Indole alkaloids have good anti-tumor activity.This study mainly explores whether it has inhibitory effect on p53-MDM2target,and thus finds a new anti-tumor Indole alkaloid inhibitor for P53.In this study,we designed p53-MDM2 as the target,and used bioluminescence resonance transfer（BRET）technology to construct the p53-MDM2 interaction model,and then synthesized the activity of small molecule compounds of indole alkaloids with better activity in the previous laboratory.The compounds act on the interaction model to determine whether the small molecule compounds of indole alkaloid have an inhibitory effect on the p53-MDM2 target.thereby a lead compound that targets the P53 anti-tumor the small molecule compound of indole alkaloid was screened.This study is divided into the following three parts:The first part,the research team used the wt P53 A549 cells to screen the anti-tumor activity of more than 40 steroidal alkaloids in the in vitro.The half-inhibitory concentration(IC₅₀)and the positive compound Nutlin-3 were tested.Comparing,the results showed that the inhibitory activities of the compounds 2yy(IC₅₀=15.57μM)and 3zz(IC₅₀=10.84μM)on A549 cells were superior to the positive control compound Nutlin-3.Therefore,it can be preliminarily speculated that the compounds 2yy and 3zz may have good effects on cell viability and targeting p53anti-tumor selectivity,and the effects on p53-MDM2 interaction can be further studied.In the second part,the construction of p53-MDM2 interaction BRET model.The first is to clone the P53 and MDM2 genes,and then fuse the two genes with the fluorescent protein gene EGFP and the luciferase gene Rluc to form a fusion plasmid,and then use the transfection reagent to recombine HEK293 cells in a certain ratio for fusion protein.Expression,after adding luciferase substrate,the BRET ratiowas measured,so that the interaction of p53-MDM2 and the ability of small molecule drugs to disrupt their interaction can be judged.Finally,the clinical phase II positive compound Nutlin-3 was used to verify the BRET mechanism of p53-MDM2 interaction.The results show that the p53-MDM2 interaction BRET model is successfully constructed.It can be used to study the mechanism of action of small molecule compounds of proline alkaloids in the early stage.In the third part,the small molecule compounds of indole alkaloids with activity act on the BRET model of p53-MDM2 interaction.The BRET mechanism of p53-MDM2 interaction was studied by the small molecule compounds of indole alkaloids with better activity screened by MTT.The effects of these small molecule compounds on the BRET model of p53-MDM2 interaction were studied.The results showed that 3zz,a small molecule compound of indole alkaloids,had a good inhibitory effect on the interaction between P53 and MDM2,and could be further optimized as a target P53 anti-tumor lead compound.