Identification of hotspots in synthetic peptide inhibitors of the FOXO4:p53 interaction

Abstract

Forkhead box protein O4 (FOXO4) plays a pivotal role in cellular senescence by binding to and inactivating p53. Consequently, misregulation of the FOXO4:p53 complex is associated with numerous diseases. Targeting the FOXO4-p53 interface has been achieved using a synthetic D-retro-inverso (DRI) peptide derived from the forkhead-homology domain of FOXO4 (FOXO4-FDH), also known as DRI (FOXO4-FHD residues 91–124). However, a comprehensive understanding of the key amino acids driving the interaction between DRI and p53 remains incomplete. While previous publications have demonstrated a robust interaction between the forkhead homology domain of FOXO4 (FOXO4-FHD) and the transactivation domain of p53 (p53-TAD), emerging evidence suggests that the interaction within the binary complex forms a highly interconnected network, including a predicted interaction between FOXO4-FHD and the DNA-binding domain of p53 (p53-DBD). In this study, we investigated the DRI: p53-DBD interaction by measuring the binding affinities of DRI and the native peptide of FOXO4, from which it is derived, to p53-DBD using microscale thermophoresis and computational modeling. Our in vitro measurements reveal that DRI binds to p53-DBD with high affinity (Kd ~50 nM), while the native peptide exhibits significantly weaker binding affinity (Kd ~2.5 mM), implying distinct modes of interaction. Subsequently, we created an in silico model of the DRI: p53-DBD interaction, which we analyzed to identify potential interaction hotspots. The analysis of this model suggests that a truncated DRI peptide (FOXO4-FHD amino acids 101–109) retains the majority of the binding affinity, as subsequently demonstrated in vitro (Kd ~40 nM). Collectively, this data furnishes molecular-level insights that contribute to the understanding of the interplay of the amino acids between DRI and p53, further supporting the notion of domain rearrangement or refolding during the formation of the FOXO4:p53 complex. In addition, this data provides an additional basis for the design of small molecules aimed at inhibiting this interaction.