Cellular activity upregulation of the thermolabile p53 cancer mutant Y220C by small molecule indazole derivatives

The TP53 gene is one of the most frequently mutated genes in human cancers. Mutations often result in loss of tumor-suppressive functions and acquisition of oncogenic properties by p53, contributing to tumor progression and resistance to therapy. Among structural p53 mutations, Y220C is particularly notable for creating a surface-exposed hydrophobic pocket that destabilizes the protein while preserving partial function, making it a promising target for pharmacological reactivation. In this study, we performed a structure-guided phenotypic screen of an in-house heterocyclic compound library to identify novel small-molecule modulators of p53-Y220C. This led to the identification of a series of (1H-pyrrol-1-yl)indazole derivatives (JC16, JC36, JC65), structurally inspired by known Y220C binders. JC16 and JC36 exhibited selective cytotoxicity and pro-apoptotic activity in p53-Y220C mutant cancer cell lines, with minimal effects in wild-type or p53-null cells. These compounds induced a mutant-to-wild-type conformational shift in cellular p53-Y220C, accompanied by transcriptional activation of canonical p53 target genes, including BBC3 (PUMA) and MDM2. Western blot analysis revealed that in HUH7 cells, these effects occurred without a corresponding increase in total p53 protein levels, suggesting a mechanism based on conformational reactivation. Our findings position JC16 and JC36 as early-stage chemical leads with potential to restore mutant p53 function in a context-dependent manner. While their exact mechanism of action remains to be fully elucidated, these results provide a foundation for further development of indazole-based scaffolds as reactivators of the p53-Y220C mutant in cancer therapy.