Cell-Active Peptide Inhibitors of the FANCM-RMI Interaction

The FANCM-RMI protein-protein interaction plays an essential role in cancers that extend their telomeres through the Alternative Lengthening of Telomeres (ALT) pathway. Here, we report the first cell-active peptide inhibitors of FANCM-RMI. Screening of mRNA-displayed peptide libraries treated with <i>trans</i>-1,4-dibromo-2-butene led to the discovery of both linear and cyclic peptide hits that bind RMI at the FANCM interaction site. The most potent peptides engage RMI with nanomolar affinity (<i>K</i>_D = 4-31 nM) and outcompete the native peptide mimic of FANCM (IC₅₀ = 24-155 nM). A bound X-ray crystal structure of the top hit revealed novel interactions at the RMI binding site that were not found in the native interaction. Conjugation to a cell-penetrating peptide resulted in inhibitors that induced an antiproliferative effect in ALT-positive osteosarcoma cell lines. These inhibitors represent the first bioactive RMI binders that can be used as chemical tools for studying the involvement of FANCM-RMI in ALT-driven cancers.