Simultaneous measurement of p53:Mdm2 and p53:Mdm4 protein-protein interactions in whole cells using fluorescence labelled foci

Y Frosi,Siti Radhiah Ramlan,C J Brown,T Watanabe,D P Lane,K Inoue

doi:10.1038/s41598-019-54123-z

Y Frosi, Siti Radhiah Ramlan + Show 4 more

Open Access

https://doi.org/10.1038/s41598-019-54123-z

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Abstract

In this report we describe the development of a Fluorescent Protein-Protein Interaction-visualization (FLUOPPI) to enable the simultaneous measurement of both Mdm2:p53 and Mdm4:p53 interactions in order to assess the relative efficiencies of mimetic molecules of the p53 peptide helix against both PPIs. Mdm2 and Mdm4 overexpression frequently leads to the inactivation of non-mutated p53 in human cancers, via inhibition of its transcriptional activity, enhancing its degradation by the proteasome or by preventing its nuclear import. Development of inhibitors to disrupt the binding of one or both of these protein interactions have been the subject of intensive pharmaceutical development for anti-cancer therapies. Using the bimodal FLUOPPI system we have characterised compounds that were either monospecific for Mdm2 or bispecific for both Mdm2 and Mdm4. We have also demonstrated that the FLUOPPI assay can reliably differentiate between specific and non-specific disruption of these protein complexes via accurate assessment and normalization to the cell population under measurement. We envision that this methodology will increase the efficiency of identifying compounds that are either specific against a single PPI from a closely related family of interactions or compounds that interact across multiple related PPI pairs, depending on which is more desirable.

Highlights

These range from methodologies that utilize techniques such as fluorescent lifetime measurements[13], fluorescence/bioluminescence resonance energy transfer (BRET)[14], protein complementation assays (PCA)[15], yeast two hybrid (Y2H)[16] and cellular localization assays[10,11]
The FLUOPPI39 (MBL) technology is an imaged based assay system that offers several advantages over other plate based reading methodologies (e.g. protein complementation assays (PCA) and florescence resonance energy transfer (FRET)): (1) allows single cell analysis, (2) detect rare events and (3) enables the characterization of other cellular properties such as morphology and localization that can expedite the elimination of false positives
Neither Nutlin 3A nor RO-5963 caused any observable stabilization or induction of p53 after 1 hr or 24 hours of treatment. These results demonstrate that the disruption of the fluorescent foci in the two Fluorescent Protein-Protein Interaction-visualization (FLUOPPI) PPI assays is due to direct antagonism by both Nutlin 3A and RO-5963

Summary

Introduction

Intracellular protein-protein-interactions (PPIs) represent a wide class of potential drug targets across several important disease areas such as cancer and infectious diseases[1]. Addition of either Nutlin 3A or RO-5963 (at respective concentrations of 12.5 μM or 50 μM) onto CHO-K1 cells transiently transfected with the ASH-p53:AG-Mdm[2] PPI system for 24 hours, resulted in disruption of the fluorescent foci demonstrating that foci formation is reversible and dependent on the Mdm2:p53 interaction (Fig. 1D).

Results

Conclusion