Home » Key Scientific Articles » The Fluorescent Two-Hybrid Assay to Screen for Protein-Protein Interaction Inhibitors in Live Cells: Targeting the Interaction of p53 with Mdm2 and Mdm4.

The Fluorescent Two-Hybrid Assay to Screen for Protein-Protein Interaction Inhibitors in Live Cells: Targeting the Interaction of p53 with Mdm2 and Mdm4.

Yurlova L, Derks M, Buchfellner A, Hickson I, Janssen M, Morrison D, Stansfield I, Brown CJ, Ghadessy FJ, Lane DP, Rothbauer U, Zolghadr K, Krausz E.

J Biomol Screen. 2014;19(4):516-25.

ChromoTek GmbH, Planegg/Martinsried, Germany.

 

Abstract

 

Protein-protein interactions (PPIs) are attractive but challenging targets for drug discovery. To overcome numerous limitations of the currently available cell-based PPI assays, we have recently established a fully reversible microscopy-assisted fluorescent two-hybrid (F2H)assay. The F2H assay offers a fast and straightforward readout: an interaction-dependent co-localization of two distinguishable fluorescentsignals at a defined spot in the nucleus of mammalian cells. We developed two reversible F2H assays for the interactions between the tumor suppressor p53 and its negative regulators, Mdm2 and Mdm4. We then performed a pilot F2H screen with a subset of compounds, including small molecules (such as Nutlin-3) and stapled peptides. We identified five cell-penetrating compounds as potent p53-Mdm2inhibitors. However, none exhibited intracellular activity on p53-Mdm4. Live cell data generated by the F2H assays enable the characterization of stapled peptides based on their ability to penetrate cells and disrupt p53-Mdm2 interaction as well as p53-Mdm4interaction. Here, we show that the F2H assays enable side-by-side analysis of substances’ dual Mdm2-Mdm4 activity. In addition, they are suitable for testing various types of compounds (e.g., small molecules and peptidic inhibitors) and concurrently provide initial data on cellular toxicity. Furthermore, F2H assays readily allow real-time visualization of PPI dynamics in living cells.

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Additional Information:

Fluorescence images show bait and prey interaction in the cellular F2H assay.

Upper row: GFP-bait forms a bright green spot in the nucleus of F2H cells. RFP-prey interacts with the GFP-bait and forms a co-localizing red spot. Lower row: Upon incubation with an inhibitor red spot disappears, red signal is disperse => interaction is disrupted.

 

The Fluorescent Two-Hybrid Assay to Screen for Protein-Protein Inte