Abstract 5386: Disrupting p53:Mdm2 and p53:Mdm4 - Comparative cell-based assays for drug screening

Abstract

Abstract The important interconnected protein-protein interactions between the tumor suppressor p53 and its regulatory binding partners Mdm2 and Mdm4 (= MdmX/ HdmX) are implicated in pathogenesis of various cancers. However, until now there is a lack of reversible cell-based assays for concurrent analysis for both PPIs: p53/Mdm2 and p53/Mdm4. Our aim was to develop two comparative assays based on the Fluorescent 2-Hybrid (F2H) assay principle, which enable side-by-side analysis of antagonistic compounds. The F2H assay is a fully reversible microscopy-assisted assay for the direct intracellular analysis of PPIs. It offers a fast and straight-forward readout: an interaction-dependent co-localization of two fluorescent signals at a defined spot in the nucleus of mammalian cells. With these assays we analyzed a set of newly developed stapled peptides as potent p53:Mdm2 and/ or p53:Mdm4 inhibitors. Live cell data generated by the F2H assays enabled us to discriminate and describe the peptides according to their ability to penetrate into the cells, their efficacy on the PPIs and their cytotoxic side effects in one single assay. In parallel we performed a pilot F2H screen with a sub-set or 20 small molecule compounds (including Nutlin-3) that exhibited activity against one or both interactions at various potencies in ELISAs before. We could identify 5 potent compounds, which were dramatically reducing the number of p53:Mdm2 interactions. However, none of the small molecules, but only the stapled peptides exhibited an intracellular activity on p53:Mdm4. Furthermore, we were able to expand these assays and to identify mutants of Mdm2 resistant to Nutlin inhibition. In summary, we show that the p53:Mdm2 and p53:Mdm4 F2H assays described here, - enable side-by-side analysis of substances' dual Mdm2-Mdm4 activity - are suitable for screening and testing various types of compounds, such as peptidic inhibitors or small molecules - concurrently provide initial data on compound cell-permeability and cytotoxicity - allow real-time visualization of PPI dynamics in living cells. Citation Format: Larisa Yurlova, Christopher J. Brown, Maarten Derks, Farid John Ghadessy, Ian Hickson, David P. Lane, Eberhard Krausz, Kourosh Zolghadr. Disrupting p53:Mdm2 and p53:Mdm4 - Comparative cell-based assays for drug screening. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5386. doi:10.1158/1538-7445.AM2014-5386