Mutations in p53 disrupt its ability to induce apoptosis in response to DNA damage and are present in over 50% of all human cancers. Most mutations in p53 result in loss of the wild-type function, gain of various toxic functions, and a dominant negative effect on remaining wild-type p53. Recent evidence indicates that the dominant negative effect on wild-type p53 is caused by amyloid aggregation of mutant p53. As such, inhibiting aggregation of p53 has emerged as a novel therapeutic strategy. Previous work in the Meade group designing and characterizing targeted cobalt(III) Schiff base (Co(III)-sb) complexes has demonstrated their utility as protein inhibitors in a wide variety of applications. Addition of a targeting moiety, allows Co(III)-sb to specifically bind to histidine residues of a protein of interest. This binding has been shown to decrease aggregation of amyloidogenic proteins. The objective of this project is to design and synthesize Co(III)-sb complexes that specifically bind to mutant p53 and decrease its aggregation propensity. Synthesis of Co(III)-sb will be adjusted to include a non-B DNA targeting moiety that will confer specific binding to mutant p53 and not wild-type p53 (Specific Aim 1). After purification and molecular characterization of the compound, binding will be tested by mass spectrometry and EMSA (Specific Aim 1). Wild-type p53 and the linear p53 binding sequence will be used as controls to ensure selective binding of Co(III)-sb to mutant p53. Following successful synthesis of the target compound, aggregation inhibition by Co(III)-sb will be measured by a variety of techniques. Recombinant mutant p53 aggregation will be measured in buffer in the presence and absence of Co(III)-sb using ThT fluorescence, CD spectroscopy, and DLS. Aggregates will be morphologically characterized using EM. Aggregation inhibition by Co(III)-sb in heterozygous colorectal cancer cell lines will be measured using immunofluorescence and Western blots (Specific Aim 2).
|Effective start/end date||9/15/16 → 3/14/18|
- U.S. Army Medical Research and Materiel Command (W81XWH-16-1-0379)