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

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.

Functional Display of Bioactive Peptides on the vGFP Scaffold.

“…p53 and Mdm2 have been used as a model interacting protein pair in the development of several biosensing technologies [20][21][22][23][24][25] . We have previously described a biosensor comprising b-lactamase coupled to an inhibitory protein (BLIP) in cis via a linker containing the M2 peptide sequence 26,27 .…”

Development of covalent inhibitors: Principle, design, and application in cancer

“…Mdm2 and Mdm4 are very important negative regulators of the p53 protein, and they interact with the p53 protein to inactivate it. 100 Inactivation of the p53 protein may result in malignant proliferation of tumor cells. [101][102][103] Therefore, it is a promising approach to inhibit p53-Mdm2 or p53-Mdm4 interactions.…”

“…The p53 protein is encoded by the p53 gene, which is a tumor suppressor gene and plays an important role in cell cycle proliferation, apoptosis promotion, and genomic stability maintenance. Mdm2 and Mdm4 are very important negative regulators of the p53 protein, and they interact with the p53 protein to inactivate it 100 . Inactivation of the p53 protein may result in malignant proliferation of tumor cells 101–103 .…”

Fluorescence Quantum Yields in Complex Environments from QM-MM TDDFT Simulations: The Case of Indole in Different Solvents

“…Among these systems, proteins stand out. − There are only a few amino acids in nature presenting fluorescence: tyrosine, phenylalanine, and tryptophan. The fluorescence spectra of these chromophores are used as indicators of structural changes, such as folding/unfolding, , binding with a substrate or a ligand, , protein–protein interaction, , etc. The most widely used is undoubtedly tryptophan , because its optical properties present a greater sensitivity to the microenvironment in which it is found.…”