Monitoring Binding of p53 Reactivation Compound by CETSA

Saturday, February 13, 2016
Kyle P. Kisor, University of California, Irvine, Irvine, CA
According to the CDC, roughly fourteen million people each year are diagnosed with cancer, 8 million of which subsequently die. Previous studies have shown that nearly 50% of human cancers have a mutation in the tumor suppressor p53. Most cancers have full-length p53 proteins that lack tumor suppression capability only due to a single missense mutation. Interestingly, these mutations arise at six hotspot positions in the core domain of p53, which include R175H, G245S, R248W, R249S, R273H, and R282W implying there is a selective advantage for tumor growth conferred by these mutations. These single amino acid changes alter p53 conformation and therefore disable its ability to regulate cellular functions important for repression of tumors. However, presence of full-length but inactive p53 holds the promise of unconventional but exciting cancer therapeutics: small molecules that bind to mutated p53, restore its wild type conformation leading to p53 reactivation, and subsequently kill cancer cells. Approaches to validate this concept and monitor a compounds ability to restore the wild type p53 conformation are scarce, but necessary to develop effective p53 reactivation compounds. The aim of this study is to employ the biophysical properties of ligand induced thermal stabilization for a cellular thermal shift assay (CETSA) to examine the efficacy of novel p53 reactivation compounds in vivo. CETSA can directly measure if a drug can bind to its target in vivo and is built on the concept that the thermal stability of target proteins increases upon compound binding. To monitor the effect of compounds on thermal stability of p53 mutants, candidate reactivation molecules were added to cells carrying various p53 cancer mutants. Cells were harvested after 3 hours and heated to different temperatures. When the temperature reaches a point that disrupts the p53 structure, the protein aggregates and becomes insoluble.  The soluble fraction is separated from the precipitated fraction by centrifugation and the amount of soluble p53 was determined by immunoblotting. A melting temperature for p53 can be determined by analyzing a range of temperatures.  Our results show that when NCI-26 is added to cells carrying the G245S mutant, the melting temperature of p53 shifts to 45.0°C as opposed to 43.0°C without NCI-26. This suggests that NCI-26 binds to the p53 G245S mutant in vivo and increases the thermal stability of this cancer mutant. Overall, NCI-26 binding reactivates the p53-G245S cancer mutant and induces death of these cancer cells. Current results suggest that compound NCI-26 holds promise as a potential therapeutic for cancer patients with mutations in the p53 tumor suppressor.