Design,synthesis And Biological Evaluation Of Dual Target Inhibitors Of P53-MDM2/MDMX Based On Pyrrolidone Scaffold

As a serious threat to human health,malignant tumor accounts for 1/4 of the new cancer cases in China every year.The pivotal tumor suppressor p53 protein plays a key role in the process of tumor cell cycle regulation,apoptosis and DNA repair.Currently,the deletion or mutation of p53 protein is present in more than 50%of tumors,while wild-type p53 is present in another 50%of tumor cells,and its activity is inhibited by MDM2 through a negative feedback regulatory loop.Thus,activation of the p53 pathway by blocking the MDM2-p53 interaction could be a promising therapeutic strategy in a variety of p53 wild-type tumors.According to relevant studies,the longterm use of single target p53-MDM2 inhibitors will lead to drug resistance and efficacy decline.The research progress of small molecule p53-MDM2/MDMX inhibitor is relatively slow.The polypeptide p53-MDM2/MDMX inhibitors have poor bioavailability and unstable conformation.Therefore,it is urgent to develop a new type of small molecule p53-MDM2/MDMX inhibitor.The cocrystal structure of p53 and MDM2,which p53 binds to the hydrophobic surface of the MDM2 N-terminal domain via its α-helical conformational peptide,reveals that three amino acid residues of p5 3(Trp23,Leu26,and Phe 19)are responsible for key hydrophobic contacts with the MDM2 protein.On the basis of the proteinprotein interaction between p53 and MDM2,we designed and synthesized p53MDM2/MDMX inhibitors with the help of molecular docking in this study.The designed compounds not only occupy the three key cavities of Trp23,Leu26 and Phe19,but also form hydrogen bonds and stopping with the residues in MDM2/MDMX cavities by other functional groups in the molecule to further enhance the activity.Chalcone,showing a variety of antitumor activities as a natural product,originally discovered as MDM2 inhibitors,can disrupt the p53-MDM2 protein interaction to activate p53.Molecular docking showed that chalcone only occupies two key active cavities of p53 binding to MDM2 protein(Phe19 and Leu26),containing great modification potential.Hence,we propose to introduce a third group into the chalcone skeleton to occupy the Trp23 cavity.In addition,in order to adapt to the narrow and deep cleft of MDM2,the α,β-unsaturated carbonyl part of chalcone is planned to be transformed into a ring.This work,a series of unsaturated pyrrolidone compounds were designed and synthesized by computer-aided drug design,which exhibited improved docking score with MDM2 and MDMX and occupied Phe19,Trp23 and Leu26 cavities of p53-MDM2 well by three benzenes in the scaffold.In this study,a total of 19 compounds were synthesized and their protein affinity was measured by fluorescence polarization method.After the concentration of MDM2 probe(PMDMF-6)and the concentration of MDM2 protein were investigated,the binding affinity of compounds to MDM2 at a specific concentration of 10 μM was determined.The results showed that compounds S5,S16 and S18 showed better protein binding affinity than the positive compound Nutlin-3,and the binding afinities of most compounds was greater than 50%,which laid a foundation for subsequent activity screening.Then,the in vitro anti-tumor proliferation inhibitory activity test and the preliminary structure-activity relationship study were carried out.All tumor cells with wild-type p53 were selected,including HCT116 cells with normal MDM2 expression,U87MG cells with high expression of MDM2 and SH-SY5Y cells with high expression of MDMX,for activity evaluation.In addition,we conducted selective investigation by artificially knocking out p53 in HCT116 cells.The results showed that the compounds with electron-withdrawing group showed better activity,while the benzene ring with electron-donating group showed no activity at all.Compounds S5,S9,S16,S17 and S18 with electron-withdrawing group had better activities and p53 selectivities.The activity of S5 in HCT116 cells was nearly 20 folds of Nutlin-3,and its selectivity was also nearly 10 times higher.Considering that compound S5 has the best binding affinity with MDM2 and anti-tumor activity in vitro,S5 was selected in this study for subsequent mechanism studies.Cell migration assay,Western Blot assay and cell cycle and apoptosis assay showed that compound S5 could successfully inhibit the migration of HCT116 cells.The expression level of p53 and its downstream protein p21 was increased by blocking the p53-MDM2/MDMX interaction.HCT116 cells were successfully induced to block G1 phase and cell apoptosis.In addition,molecular docking and molecular dynamics simulation were also used to predict the binding ability of compound S5 with different configurations to protein,and it was believed that its "S"configuration would exhibit better biological activity.With the aid of computer aided drug design and medicinal chemistry research method,we designed and synthesized a series of pyrrolidone compounds.By conducting the test of the binding affinity,proliferation inhibition in vitro,cell cycle and apoptosis,cell migration test,we finally screened an obvious antitumor activity compound.This study provides a research basis for the further development of small molecule p53-MDM2/MDMX dual target inhibitors.