Development of a peptide inhibitor of the FOXO4-p53 interaction based on the sequence of p53 transactivation domain

Abstract

Cellular senescence is the permanent cell-cycle arrest state triggered by various stressors, such as telomere erosion, oxidative stress, and oncogene activation while maintaining growth. It is known that the interaction between FOXO4 and p53 induces cellular senescence, disturbing the apoptosis of senescent cells and leading to the development of aging-related diseases. As one therapeutic strategy, a few peptide inhibitors targeting this interaction have been developed, such as FOXO4-DRI, the most well-known peptide inhibitor for the FOXO4-p53 interaction. However, it consists of 46 residues and the introduction of D-retro amino acids costs high. Therefore, there is a need to develop a shorter, effective peptide inhibitor. In this study, we selected the p53 TAD2 (42-57) sequence as the wild type for a novel peptide inhibitor targeting FOXO4 FHD, based on a previous study that elucidated the binding domain between FOXO4 and p53. To identify crucial residues for binding and determine the binding affinities of peptides, we conducted competitive fluorescence anisotropy (FPA) assays with the introduction of site-directed mutagenesis. Based on the results of the FPA assay, we designed several peptide candidates to enhance binding affinity. We observed that our inhibitory peptides could partially disrupt the FOXO4 FHD-p53 TAD interaction, through analysis of the chemical shift perturbations (CSP) of the complex between p53 TAD (13-61) and FOXO4 FHD (95-195) with titrations of increasing peptide concentrations. In addition, we used circular dichroism (CD) spectroscopy to investigate the secondary structures of peptides when bound to FOXO4 FHD. To validate the senolytic effect, which selectively eliminates senescent cells, of our peptides in cells, we first optimized the conditions of an MTS assay and performed the assay using our peptides. In this study, we suggested that our peptide inhibitors based on the p53 TAD2 sequence can disrupt the interaction in vitro, however, it needs further development for targeting in cells.