Suppression of p53 response by targeting p53-Mediator binding with a stapled peptide

DNA-binding transcription factors (TFs) remain challenging to target with molecular probes. Many TFs function in part through interaction with Mediator, a 26-subunit complex that controls RNA polymerase II activity genome-wide. We sought to block p53 function by disrupting the p53-Mediator interaction. Through rational design and activity-based screening, we characterize a stapled peptide, with functional mimics of both p53 activation domains, that blocks p53-Mediator binding and selectively inhibits p53-dependent transcription in human cells; importantly, this “bivalent” peptide has negligible impact, genome-wide, on non-p53 target genes. Our proof-of-concept strategy circumvents the TF entirely and targets the TF-Mediator interface instead, with desired functional outcomes (i.e., selective inhibition of p53 activation). Furthermore, these results demonstrate that TF activation domains represent viable starting points for Mediator-targeting molecular probes, as an alternative to large compound libraries. Different TFs bind Mediator through different subunits, suggesting this strategy could be broadly applied to selectively alter gene expression programs. [Display omitted] •Proteomics identifies Mediator (MED) as “top hit” interactor with p53AD•Stapled peptide inhibits p53-MED binding and p53 transcription; other TFs not affected•TF activation domains viable starting points for design of Mediator-targeting probes•TF function can be controlled by avoiding TF entirely and targeting Mediator instead Allen et al. combine chemical synthesis and biochemistry to develop a Mediator-targeting “bivalent” peptide that blocks p53-Mediator binding and selectively inhibits p53 target gene expression in human cells. This proof-of-concept study shows that p53 activity can be blocked by molecules targeting p53-Mediator binding, circumventing p53 itself.