MOLECULAR CHARACTERIZATION OF THE HDM2-P53 INTERACTION

A number of viral oncogenes target the tumour suppressor protein p53 a nd inactivate its function. This is an important step in tumourogenesi s. The cellular oncogene hdm2 acts through a similar mechanism. It bin ds the N terminus of p53, thereby interfering with the ability of p53 transcriptionally to activate genes responsible for growth arrest or a poptosis after genotoxic insults. The disruption of the interaction of the two proteins therefore comprises a promising therapeutic target f or treatment of the subset of human cancers in which this pathway is a ctive. In this paper we attempt to characterize the p53-hdm2 interacti on biochemically. We analyse the potential of a series of peptide inhi bitors, derived from previously described mdm2 binding peptide display phage, to disrupt this interaction in ELISA assays. We conclude that F19, W23 and L26 of p53 are critical contact points for p53 binding to hdm2. Furthermore, ave show the potential of the monoclonal antibody 3G5 to interfere with binding of p53 to hdm2 in ELISA assays. Conseque ntly, we define the binding site of 3G5 on hdm2 using overlapping pept ides derived from the N terminus of hdm2 and phage display libraries. The result indicates L66, Y67 and E69 on hdm2 as critical binding poin ts for 3G5. In electrophoretic mobility shift assay we demonstrate the formation of hdm2-p53 complexes that can be disrupted in the presence of 3G5 or inhibitory peptides. Finally, we describe the effects of NE M and DTT on the interaction between the two molecules in ELISA assays . All our results are discussed in the light of the recently published crystal structure of the mdm2-p53 complex. A striking correspondence between our findings and the crystal structure is revealed. (C) 1997 A cademic Press Limited.