BAG-1 interacts with multiple partners, particularly with c-Raf, and promotes cancer cell survival. Hence, modulating the BAG-1-associated interactions with novel inhibitors could provide benefit for cancer therapy. Using HDX-MS, we first demonstrate the higher-order structure of BAG-1S and identify a potential "druggable" site on its BAG domain. An LC-MS/MS-coupled cell-free binding experiment is then used to map the BAG-1S:c-Raf interface, uncovering a 20-amino acid-length region of BAG-1S that is most likely to interact with c-Raf. Site-directed mutagenesis experiments reveal that K149 and L156 are hot spots for BAG-1S:c-Raf interaction, and their substitutions with alanine attenuate the survival of MCF-7 cells. We then show that a peptide derived from the BAG-1S-interacting c-Raf region hinders BAG domain-associated partners. The peptide, engineered with a cell-penetrating peptide motif, can penetrate cells, and it induces apoptosis in cancer cells. The anticancer activity of the peptide might lead to improved treatments for BAG-1-overexpressed and/or MAPK-driven tumors.
© 2025. The Author(s).