Screening Of Novel MdmX/Mdm2 Inhibitors And Their Structure-activity

The p53 protein is one of the most important tumor suppressors that are closely related to the tumor occurance.In nearly half of the tumor cells,their p53 gene are mutated,resulting in inactivation of p53,in addition to nearly half of the tumor cells,the activity of the wild-type p53 protein was inhibited by overexpressed Mdm2 and/or MdmX,providing promising targets for designing small molecule drugs to disrupt these aberrant p53-MdmX/Mdm2 interactions to restore the p53 anticancer activity.Mdm2 and MdmX are highly homologous regaring their amino acid sequences and three-dimensional structures are highly similar.Although few potent Mdm2-specific inhibitors have already been under clinical investigations,the discovery of clinically-potential MdmX inhibitors has been left very behind.The inhibitory activity of the currently-discovered small molecule inhibitors of Mdm2 and/ or MdmX exhibited margin affinity for MdmX,let alone that some high-affinity MdmX inhibitors can cause serious side-effect as their binding goes through the formation of covalent bonding.Therefore,it is essential to develop more efficient strategy for evaluating potential scaffolds or drug leads of MdmX inhibitors.In this work,considering that Trp23 in the p53 transaction domain?p53p?plays an important role in determining the p53-MdmX/Mdm2 interactions,it can be used as an intrinsic fluorescence to monitor the interaction of p53 p with MdmX.A novel fusion protein?p53p-N-MdmX?was constructed by tethering p53 p to the p53 binding domain of MdmX?N-MdmX?with a flexible amino acid sequence.The fusion protein was evaluated with a small compounds library containing currently-available thirty-three Mdm2 inhibitors using fluorescence spectroscopy.Furthermore,to explore whether the fusion protein strategy can be used for searching effective fragments,the other two key amino acids?i.e.,Phe19 and Leu26?in the p53 p segment of the fusion protein were mutated to Ala,respectively,resulting in two novel fusion protein models(defined as p53p^F19A-N-MdmX and p53pL26A-N-MdmX,respectively).As a proof of concept,the p53p^F19A-N-MdmX fusion protein model was used to screen the aforementioned small molecules library.As a result,the 1,4-benzodiazepine-2,5-diones was found to interact with the F19 binding pocket via an allosteric effect.This fragment was further tested to modify the imidazo-indole scaffold of nutlin-3a for improve its biding activity.In summary,the results from this work reveals that it is feasible to ultilize the designed fusion proteins to screen small molecule library for new saffolds or fragments.More significally,an allosteric fragement?1,4-benzodiazepine-2,5-diones?was identified from the current library.The fragment has been sucessfuly used to modify nutlin-3a into a potent MdmX inhibitor which was defined as H201.We believe that the fusion protein strategy described in this work also can be applicable for other important proteins for drug discovery with or without further modifications.