Page 74 - GPD-2-3

Gene & Protein in Disease

ORIGINAL RESEARCH ARTICLE
Identification of hotspots in synthetic peptide

inhibitors of the FOXO4:p53 interaction

Ran Zhang , Kai Gao , Afsaneh Sadremomtaz 1,2,3 , Angel J. Ruiz-Moreno ,
1
1
1
Alessandra Monti , Zayana M. Al-Dahmani , Benjamin B. Gyau ,
4
1
1
Nunzianna Doti , and Matthew R. Groves *
4
1
1 XB20 Drug Design, Groningen Research Institute of Pharmacy, University of Groningen, AD
Groningen, The Netherlands
2 Department of Nanoengineering, North Carolina Agricultural and Technical State University,
Greensboro, North Carolina, USA
3 Department of Nanoengineering, Joint School of Nanoscience and Nanoengineering, North
Carolina Agricultural and Technical State University, Greensboro, North Carolina, USA
4 Institute of Biostructures and Bioimaging (IBB), National Council of Research, Napoli, Italy
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
*Corresponding author: of FOXO4 (FOXO4-FHD) and the transactivation domain of p53 (p53-TAD), emerging
Matthew R. Groves
(m.r.groves@rug.nl) evidence suggests that the interaction within the binary complex forms a highly
interconnected network, including a predicted interaction between FOXO4-FHD and
Citation: Zhang R, Gao K,
Sadremomtaz A, et al., 2023, the DNA-binding domain of p53 (p53-DBD). In this study, we investigated the DRI: p53-
Identification of hotspots in synthetic DBD interaction by measuring the binding affinities of DRI and the native peptide of
peptide inhibitors of the FOXO4:p53 FOXO4, from which it is derived, to p53-DBD using microscale thermophoresis and
interaction. Gene Protein Dis,
2(3): 1491. computational modeling. Our in vitro measurements reveal that DRI binds to p53-DBD
https://doi.org/10.36922/gpd.1491 with high affinity (K ~50 nM), while the native peptide exhibits significantly weaker
d
Received: August 10, 2023 binding affinity (K ~2.5 mM), implying distinct modes of interaction. Subsequently,
d
Accepted: September 11, 2023 we created an in silico model of the DRI: p53-DBD interaction, which we analyzed
Published Online: September 29, to identify potential interaction hotspots. The analysis of this model suggests that a
2023 truncated DRI peptide (FOXO4-FHD amino acids 101–109) retains the majority of the
Copyright: © 2023 Author(s). binding affinity, as subsequently demonstrated in vitro (K ~40 nM). Collectively, this
d
This is an Open-Access article data furnishes molecular-level insights that contribute to the understanding of the
distributed under the terms of the
Creative Commons Attribution interplay of the amino acids between DRI and p53, further supporting the notion of
License, permitting distribution, domain rearrangement or refolding during the formation of the FOXO4:p53 complex.
and reproduction in any medium, In addition, this data provides an additional basis for the design of small molecules
provided the original work is
properly cited. aimed at inhibiting this interaction.
Publisher’s Note: AccScience
Publishing remains neutral with Keywords: FOXO4-DRI: p53 interaction; Protein–protein interaction; Computational
regard to jurisdictional claims in
published maps and institutional docking; Microscale thermophoresis
affiliations.

Volume 2 Issue 3 (2023) 1 https://doi.org/10.36922/gpd.1491