title: A fluorescence polarization assay for the identification of inhibitors of the p53-DM2 protein-protein interaction.
abstract: Improper function of the tumor suppressor protein p53 is a contributing factor in many human cancers. In normal cells, p53 acts to arrest the cell cycle in response to DNA damage or nucleotide depletion. One mechanism of regulating the amount of p53 in the cell is through the action of the Double Minute 2 protein, DM2 (also known as MDM2), which ubiquitinates p53 and targets it for proteosomal degradation. In a number of human cancers, the DM2 gene is amplified or overexpressed, leading to inadequate levels of p53 for cell cycle arrest or apoptosis. With the goal of restoring p53 function in cancers that overexpress DM2, we are developing inhibitors of the p53-DM2 protein-protein interaction that structurally mimic the N-terminal segment of p53 that binds to DM2. To assist this effort, we have devised a fluorescence polarization assay that quantifies the interaction between the N-terminal regions of both proteins in 384-well microtiter plates. Using this assay, we have demonstrated that a peptide with a nonhydrolyzable beta-amino acid substitution binds DM2 with an affinity comparable to a p53 peptide that is composed of only alpha-amino acids.
authors.affiliation: Department of Pharmacology, University of Wisconsin-Madison, Madison, Wisconsin 53706-1532, USA.
authors.forename: Stephen M G
authors.initials: SM
authors.lastname: Knight
authors.forename: Naoki
authors.initials: N
authors.lastname: Umezawa
authors.forename: Hee-Seung
authors.initials: HS
authors.lastname: Lee
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Gellman
authors.forename: Brian K
authors.initials: BK
authors.lastname: Kay
mesh_terms: D000595:Amino Acid Sequence; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D018716:Molecular Mimicry; D009687:Nuclear Proteins; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2