Discovery of novel covalent agonists for p53 Y220C through synergistic strategy combining covalent binding and scaffold hopping.

The Y220C mutation represents one of the most frequent mutational variants in p53. This mutant impairs p53-mediated tumor suppression and heightens oncogenic risk. Consequently, activating the antitumor function of p53 mutants emerges as a promising therapeutic strategy for diverse malignancies. Herein, we report a strategic concept targeting the shallow neomorphic pocket created by the p53 Y220C mutant with covalent small molecules. Utilizing scaffold hopping, we identified the privileged pyrrolopyrimidine scaffold and subsequently designed high-potency compounds through covalent warhead replacement and systematic structural optimization. The protein thermal shift assay confirmed enhanced thermal stabilization of p53 Y220C. Several compounds restored its DNA-binding capacity. Among them, LLQ-45 showed selective activity against p53 Y220C-mutant tumor cells, inhibiting their proliferation and markedly upregulating CDKN1A expression in a dose-dependent manner. These results demonstrate that LLQ-45 activates the antitumor function of p53 Y220C via covalent modification, thereby suppressing tumor cell growth. This study provides a framework for targeting neomorphic pockets and advances targeted cancer therapies.