Identification of Hotspots in Synthetic Peptide Inhibitors of the FOXO4:p53 Interaction

Abstract

Abstract Background: Many stresses trigger cellular senescence, including telomere shortening, oncogene activation, DNA-replication stress, oxidative stress, and resistance to apoptosis. Forkhead box protein O4 (FOXO4) plays a pivotal role in cellular senescence, by binding to and inactivating p53, thereby leading to cellular senescence and inhibiting p53 mediated apoptosis. As a result, misregulation of FOXO4-p53 is linked to numerous diseases including cancers and aging-related diseases, pulmonary fibrosis and neurodegenerative diseases. Targeting of the FOXO4-p53 interface has been accomplished using a synthetic D retro inverse peptide (DRI), however recognition of the key molecular drivers of the interactions remains unknown. Understanding these interactions on a molecular level could lead to the development of small compounds, with enhanced pharmacological qualities, such as high stability, membrane permeability, and oral bioavailability, that mimics the activity of the DRI peptide. Methods and Results: Using an in silico model of the FOXO4-p53 interaction, we designed shorter peptides within the DRI to identify the key interactions. A biophysical assay demonstrates these shorter peptides retain strong binding affinity to p53, suggesting that the DRI's hot spot is located within these peptides. Conclusions: Taken together, this data provides information at the molecular level that is key to understanding the interplay of two proteins responsible for cellular senescence and provides the basis for the design of small molecules to inhibit this interaction.