p53 is a tumor suppressor protein that is inactivated in as many as 50% of human cancers, which, despite advances in understanding and treatment, remains the 2nd leading cause of death in the US. The vast majority of p53 mutations that lead to cancer are missense, and result in the accumulation of high levels of misfolded p53 in the tumor cells. Because of the prevalence of p53 mutations in human disease and their high level of accumulation in cancer cells, p53 reactivation has been a long sought-after approach to cancer chemotherapy. p53 is a Zn2+-dependent DNA-binding protein. Thus, its core domain requires both a single, appropriately coordinated Zn2+ and an intact DNA binding surface to function. Our collaborators in the Carpizo lab at Rutgers recently identified compound NSC319726 (726) as an allele-specific reactivator for cells expressing p53 hotspot mutants with impaired Zn2+ binding. Our lab has since determined that 726 is a Zn2+ metallochaperone. It delivers Zn2+ to the appropriate binding sites in the p53 DNA-binding domain (DBD), restoring stability and function to the mutants. Interestingly, it seems to function solely through its interaction with Zn2+, without binding to the protein at all. This finding underscores both the nee to understand how p53 and its mutants interact with Zn2+, and what physical parameters of 726 allow it to deliver Zn2+ to the protein. The goal of this project is to quantitatively define the parameters that allow 726 to reactivate Zn2+-deficient p53 mutants. Our approach is to measure the kinetic and thermodynamic parameters governing the p53-Zn2+ interaction in vitro. We will use the results to predict the optimal Zn2+-binding characteristics of synthetic metallochaperones using a quantitative model for the p53-Zn2+ binding interaction in silico. We also propose to determine the physical characteristics that allow 726 to deliver Zn2+ to p53 in live cells. This wil provide clear direction to efforts to translate 726 into a viable pharmaceutical.
p53 inactivation is seen in as many as 50% of human cancers, often through disruption of a critical Zn2+- binding interaction. Compound NSC319726 is a thiosemicarbazone that restores Zn2+ to Zn2+-deficient p53 mutants, restoring their function. Determining the physical parameters of p53 and NSC319726 that allow for this reactivation could be critical to developing drugs that work by the same mechanism for clinical use.
