High-throughput assay for measuring p53 ubiquitylation by MDM2.

2077 The activity of p53 tumor suppressor protein is regulated by MDM2, which targets p53 for ubiquitylation and subsequent degradation by proteasomes. MDM2 represents an attractive target for anti-cancer drug development. Inhibition of MDM2 ubiquitin ligase activity is expected to lead to increased growth arrest and apoptosis of p53-positive cancer cells resulting from metabolic stabilization of p53. We developed an in vitro assay for measuring MDM2-catalyzed ubiquitylation of p53 from human recombinant enzymes. Ubiquitylated p53 was detected using electrochemiluminescence on MSD’s SectorHTS™ Reader and Multi-Array Plates. The assay was formatted for high-throughput screening for potential MDM2 inhibitors in 384-well plates.