The subunit of Mediator, Med25, is part of transcriptional protein-protein interaction (PPI) networks that regulate stress-response and motility pathways. Furthermore, the dysregulation of these PPI networks contributes to oncogenesis and metastasis. It has been shown that homozygous deletion of Med25 is nonlethal and impacts approximately 900 genes, which presents the need to study the absence of Med25 in metastatic cancer cell types. Herein we show the development of a library of proteolysis-targeting chimeras (PROTACs) that target the Activator-Interacting Domain (AcID) domain of Med25 and the E3 ligases Von Hippel−Lindau (VHL) and Cereblon (CRBN). The previously mentioned library of PROTACs was built using native peptide Med25 AcID ligands as the necessary protein-of-interest ligand portion of the PROTAC and known small-molecule ligands of E3 ligases, such as pomalidomide, a CRBN ligand, coupled together by a variety of linkers. Several PROTACs containing cell-penetrating peptides were developed to address emerging cell-permeability issues due to the peptidic nature of the PROTACs. Whole-cell and cell lysate dose-response experiments were performed with the PROTACs using patient-derived triple-negative breast cancer VARI-068 cells, where degradation of Med25 was observed. Our results show that peptide-based PROTACs built from native ligands of Med25 AcID are a useful tool to study the absence of Med25 <i>in vitro</i>.

Degradation of Med25 with peptide based PROTACs

ACS Spring 2022

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