De novo design and evaluation of dual inhibitory peptide binders targeting MDMX-p53 and MDM2-p53 interactions

Abstract

The MDMX-p53 and MDM2-p53 interactions are critical regulators of the tumor-suppressive functions of p53, often disrupted in cancers through overexpression of murine double minute X (MDMX) and murine double minute 2 (MDM2). In this study, we designed and computationally evaluated dual inhibitory peptides using RFdiffusion, ProteinMPNN, AlphaFold Multimer, and AlphaFold 3. Electrostatic complementarity, thermal stability, and binding affinity were assessed, followed by 300 ns molecular dynamics (MD) simulations. Mb2 and Mb4 (MDMX/2 binder 2 and 4) exhibited improved predicted binding affinity, enhanced electrostatic complementarity, and higher thermal stability relative to p53. Structural modeling and comparative validation confirmed reliable peptide–protein interactions. MD simulations further demonstrated stable trajectories, reduced conformational fluctuations, and persistent binding of Mb2 and Mb4 to both MDMX and MDM2. These findings identify Mb2 and Mb4 as promising dual inhibitory peptides with potential for restoring p53 activity. This study provides a computational foundation for future experimental validation and therapeutic development.