Multifunctional metallochaperone modifications for targeting subsite cavities in mutant p53-Y220C

The p53 protein, known as the 'guardian of the genome', plays an important role in cancer prevention. Unfortunately, p53 mutations result in compromised activity with over 50% of cancers resulting from point mutations to p53. There is considerable interest in mutant p53 reactivation, with the development of small-molecule reactivators showing promise. We have focused our efforts on the common p53 mutation Y220C, which causes protein unfolding, aggregation, and can result in the loss of a structural Zn from the DNA-binding domain. In addition, the Y220C mutant creates a surface pocket that can be stabilized using small molecules. We previously reported the bifunctional ligand L5 as a Zn metallochaperone and reactivator of the p53-Y220C mutant. Herein we report two new ligands L5-P and L5-O that are designed to act as Zn metallochaperones and non-covalent binders in the Y220C mutant pocket. For L5-P the distance between the Zn-binding di-(2-picolyl)amine function and the pocket-binding diiodophenol was extended in comparison to L5, while for L5-O we extended the pocket-binding moiety via attachment of an alkyne function. While both new ligands displayed similar Zn-binding affinity to L5, neither acted as efficient Zn-metallochaperones. However, the new ligands exhibited significant cytotoxicity in the NCI-60 cell line screen as well as in the NUGC3 Y220C mutant cell line. We identified that the primary mode of cytotoxicity is likely reactive oxygen species (ROS) generation for L5-P and L5-O, in comparison to mutant p53 reactivation for L5, demonstrating that subtle changes to the ligand scaffold can change the toxicity pathway.