Construction of Small‐Molecule Deubiquitinase‐Targeting Chimeras to Reactivate and Stabilize Mutant p53 Y220C in Vitro and in Vivo

Abstract Wild‐type p53 is a tumor suppressor protein that is crucial in various pathological processes. The predominant strategy for p53 Y220C mutant involves binding reactivators to mutation‐induced pocket to stabilize the protein thermally and restore wild‐type structure. However, the corrected p53 Y220C remains vulnerable to ubiquitination, reducing protein levels and impairing function. Herein, we report the development of p53 Y220C‐specific deubiquitinase‐targeting chimeras (DUBTACs)—bifunctional small molecules concurrently recruiting both p53 Y220C and the deubiquitinase OTUB1. The most potent compound, A1 , effectively restored wild‐type conformation and potently removed ubiquitin. Furthermore, A1 demonstrated superior efficacy in inducing apoptosis in p53 Y220C‐mutant Huh7 cells and achieved a 61.06% reduction in tumor weight in Huh7 xenograft model. Our results demonstrated the viability of the DUBTAC strategy for p53 Y220C, successfully re‐establishing conformation and promoting deubiquitination. More importantly, this study provides the first proof‐of‐concept for DUBTAC application in vivo, establishing targeted deubiquitination as a therapeutic approach.