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Updated: Jan 2, 2026
Counting Proteins in Single Cells with Addressable Droplet Microarrays
Published on: July 6, 2018
Simultaneous measurement of p53:Mdm2 and p53:Mdm4 protein-protein interactions in whole cells using fluorescence
Y Frosi1, K Inoue2, Siti Radhiah Ramlan1
1p53 Laboratory, A*STAR (Agency for Science, Technology and Research), 8A Biomedical Grove, #06-04/05, Neuros/Immunos, Singapore, 138648, Singapore.
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.
Insights
We developed FLUOPPI, a novel assay to simultaneously measure Mdm2:p53 and Mdm4:p53 interactions. This tool aids in discovering cancer therapeutics targeting these critical protein-protein interactions.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Mdm2 and Mdm4 overexpression inactivate p53, a tumor suppressor, in various human cancers.
- This inactivation occurs through mechanisms including inhibition of transcriptional activity, enhanced protein degradation, and impaired nuclear import.
- Targeting Mdm2:p53 and Mdm4:p53 interactions is a key strategy in anti-cancer drug development.
Purpose of the Study:
- To develop and validate a novel assay, Fluorescent Protein-Protein Interaction-visualization (FLUOPPI), for simultaneous measurement of Mdm2:p53 and Mdm4:p53 interactions.
- To assess the relative efficacy of p53 peptide helix mimetic molecules against these two distinct protein-protein interactions (PPIs).
- To enable the efficient identification of compounds with specific or bispecific inhibitory activity against Mdm2/Mdm4:p53 interactions.
Main Methods:
- Development of a bimodal FLUOPPI system for simultaneous detection of two distinct PPIs.
- Characterization of compounds using the FLUOPPI assay to determine their specificity (monospecific for Mdm2 or bispecific for Mdm2/Mdm4).
- Validation of the assay's reliability in differentiating specific from non-specific disruption of protein complexes through normalization.
Main Results:
- The bimodal FLUOPPI system successfully enabled simultaneous measurement of Mdm2:p53 and Mdm4:p53 interactions.
- Compounds were characterized as either monospecific for Mdm2 or bispecific for both Mdm2 and Mdm4.
- The FLUOPPI assay demonstrated reliable differentiation between specific and non-specific compound effects.
Conclusions:
- The developed FLUOPPI methodology provides a robust platform for evaluating compounds targeting Mdm2:p53 and Mdm4:p53 interactions.
- This assay enhances the efficiency of identifying potential anti-cancer therapeutics with desired specificity profiles.
- FLUOPPI facilitates the discovery of compounds that selectively inhibit single PPIs or broadly target multiple related PPIs.
