NanoBRET Target Engagement Enables Validation of p53 Y220C Ligands in a Cellular Context

The p53 Y220C mutation is a clinically relevant conformational mutant that creates a surface cavity exposed to potential small-molecule-based stabilization and reactivation. Although p53 Y220C has emerged as an attractive therapeutic target, approaches to directly quantify intracellular target engagement remain limited, hindering compound characterization in a cellular context. Here, we report the synthesis of BODIPY-based fluorophore tracers for development and validation of a NanoBRET-based target engagement assay for p53 Y220C in living cells. The assay showed robust performance with excellent signal windows and Z' values and was further applied to the p53 Y220S mutant, demonstrating its utility beyond Y220C. The results establish the first NanoBRET assay for direct measurement of p53 Y220C target engagement in living cells and provide a valuable platform for the discovery, characterization, and optimization of next-generation p53 Y220C targeting therapeutics.