Design Of Indole Compounds Based On P53-MDM2 Target And Its Anti-tumor Mechanism

Tumors are considered to be polygenic diseases,and once the genes controlling cell differentiation are defective,they lead to out-of-control growth regulation.The study found that p53 gene is the tumor suppressor gene with the highest correlation with human tumors,and has the function of maintaining genome stability,inhibiting or preventing cell transformation.MDM2 is a major regulator of p53.As its expression product,MDM2 protein can bind to p53 gene and inhibit the normal biological function of p53,thus leading to the pccurrence and development of tumors.In theory,blocking the effect of MDM2 on p53 can suppress the overexpression of MDM2 and release the normal function of p53.Therefore,inhibiting the interaction between MDM2 protein and p53 is considered to be one of the important targets for anti-tumor.So far,although a variety of small molecular p53-MDM2 binding inhibitors have been reported in the literature,small molecular inhibitors of heterocyclic spiral indoles based on natural product skeletons have been able to inhibit the binding of p53-MDM2,showing the highest activity in vitro,but not all types of compounds can be shown the obvious antitumor activity in the vivo.In order to get a more efficient p53-MDM2 binding inhibitor,in this study,p53-MDM2 was used as the target,we designed a series of novel oxindole skeleton with the active motif isoxazole by the principle of pharmacophore skeleton migration.The antitumor activity of novel oxindole compounds was studied by in vitro and in vivo animal experiments,and the antitumor mechanism of the p53 pathway was also studied.The work of this paper is summarized as follows:(1)?The three-component 1,3-dipolar cycloaddition reaction of(E)-nitro-isoxazole arylalkenyl with azomethine ylides(thermally generated in situ from isatins and proline,sarcosine or thioproline)under CH3 CN which was the bestchoice among all solvents.More than 40 novel polycyclic3,3-pyrrolidinyl-dispirooxindole were synthesized in high yield(up to 90%)with good diastereoselectivity(up to >20:1).All the target compounds were determined by nuclear magnetic resonance(NMR)spectroscopy and mass spectroscopy.(2)?With more than 40 new oxidant indoles synthesized as the research object,the cytotoxicity experiment of lung cancer cell A549 was conducted by MTT assay.The results showed that most of the compounds on A549 cells expressing wild-type p53 with moderate to strong inhibitory activity,especially compound 5hh(IC50=15.574 mM)and compound 6hh(IC50=10.842 mM)inhibitory activity than the positive control drug cisplatin on A549(IC50=18.794 m M),and the activity was similar to that of the control drug Nutlin-1(IC50=11.53 m M),and all had concentration gradient dependence and time gradient dependence.On this basis,MDM2 protein molecules were selected to carry out molecular docking experiments.The mode of action and binding ability of compound 5hh and compound 6hh and MDM2 protein were preliminarily explored,and further verified that the designed small molecular compound was related to the target of p53-MDM2.(3)?Through the experiment of cytotoxicity and the docking experiment of MDM2 protein molecules,two p53-mdm2 binding inhibitors with good anti-tumor activity were screened,5hh and 6hh.In order to explore the anti-tumor mechanism of compound 5hh and compound 6hh,this topic mainly studies the anti-tumor mechanism of compound 5hh and compound 6hh from the aspects of cell proliferation,cell apoptosis,cell cycle,cell migration and invasion.First of all,the experiment of soft agar colony formation and cell clones was used to investigate the proliferation ability of compound 5hh and compound 6hh on lung cancer cell A549.The results showed that both compound 5hh and compound 6hh could inhibit the proliferation of A549 cells and were concentration dependent(P <0.01).Afterwards,the apoptosis of A549 cells induced by compound 5hh and compound 6hh was explored by cell morphology observation,AO/EB fluorescent staining and Annexin V-FITC/PI double staining method.The results showed that the different concentrations of compounds 5hh and compound 6hh could cause the A549 cells toproduce different degrees of apoptosis.Then the cell cycle changes induced by compound 5hh and compound 6hh were detected by PI single dye combined with flow cytometry.The results showed that the cells induced by compound 5hh and compound 6hh were blocked in G0/G1 phase,which could inhibit the proliferation of tumor cells.On this basis,the expression of p53 and MDM2 protein and m RNA was detected by WB assay and RT-PCR experiment.The results showed that the protein and m RNA of p53 and MDM2 were expressed in A549 cells induced by compound 5hh and compound 6hh.Different expressions once again prove that the design of small molecule compounds is related to the p53-MDM2 target.At the same time,the expression of apoptosis-related factors Bax and Bcl-2 was detected by WB assay.The results showed that compound 5hh and compound 6hh induced the apoptosis of A549 cells and caused the up regulation of the proportion of protein Bax/Bcl-2,indicating that Bax protein and Bcl-2 protein play an important role in the regulation of apoptosis of A549 cells induced by compound 5hh and compound6 hh.In addition,the effects of compound 5hh and compound 6hh on migration and invasive ability of lung cancer cell A549 were initially investigated by cell scratch assay,Transwell chamber migration and invasion experiments.It was found that both compound 5hh and compound 6hh can inhibit the migration and invasion of A549 cells.(4)?In order to further explore the in vivo antitumor activity of compound5 hh and compound 6hh,this topic successfully established a mouse model of Lewis lung cancer xenografts.The results of in vivo animal experiments demonstrated that compound 5hh has good in vivo antitumor activity at high concentrations.The above results show that compounds 5hh and 6hh designed for p53-MDM2 targets can inhibit the proliferation,growth and metastasis of A549 in lung cancer cells,and compound 5hh has good antitumor activity in the vivo at high concentration.This study provides theoretical and experimental evidence for the further development and clinical application of novel yttrium oxide compounds,providing a valuable reference for the development of new generation of anti-tumor drugs and drug mechanism research.