Selective Targeting of the p53-hDM2 Interaction Using Hydrocarbon-Stapled p53 Peptides.

p53 is a transcription factor that induces cell cycle arrest and apoptosis in response to DNA damage and cellular stress, and thereby plays a critical role in protecting cells from malignant transformation. The E3 ubiquitin ligase hDM2 controls p53 levels through a direct binding interaction that neutralizes p53 transactivation activity, exports nuclear p53, and targets it for degradation via the ubiquitination-proteasomal pathway. Loss of p53 activity, either by deletion, mutation, or hDM2 overexpression, is the most common defect in human cancer. Tumors with preserved expression of wild type p53 are rendered vulnerable to pharmacologic approaches that stabilize and upregulate p53. In this context, hDM2 inhibition has emerged as a validated approach to restore p53 activity and resensitize cancer cells to apoptosis in vitro and in vivo. The inhibition of the p53-hDM2 interaction has been studied intensively with the goal of developing novel therapeutics for cancer. Here we describe the synthesis and evaluation of hydrocarbon-stapled α-helical peptides based on the transactivation domain of the p53 tumor suppressor protein. Select p53 stapled peptides exhibit subnanomolar binding to hDM2, displaying the highest affinity hDM2 binders reported to date. We find that these structurally-stabilized peptides are resistant to proteolysis and exhibit cell permeability as documented by flow cytometry and confocal microscopy analyses. In vivo binding of p53 stapled peptides to nuclear hDM2 is highly specific as demonstrated by co-immunoprecipitation experiments. In response to stapled peptide treatment, we observe upregulation of p53 and its transcriptional targets, including p21 and Bax. As a consequence, the peptides are capable of activating apoptosis in hDM2-overexpressing tumor cells through the p53 tumor suppressor signaling pathway. Thus, we find that hydrocarbon-stapled p53 peptides can trigger apoptosis in cancer cells by effectively modulating the p53-hDM2 protein interaction.