Using BRET to study chemical compound‐induced disruptions of the p53‐HDM2 interactions in live cells

Abstract

Modification of protein‐protein interactions (PPIs) holds promise for novel rational drug design. Disrupting or modifying protein interactions offers new challenges in terms of chemical compound libraries and techniques for compound validation. As proteins interact with several partners in different allosteric conformation in a pathological and tissue‐specific fashion, it is difficult to predict the in vivo effect of PPI acting compounds identified by in vitro screening assays. It is therefore desirable to develop techniques that rapidly allow cell‐based validation of protein interacting compounds. The binding of the p53 tumor suppressor to the HDM2 E3 ubiquitin ligase is important for controlling p53 activity, and several compounds, such as Nutlin‐3, have been designed to bind a hydrophobic pocket in the N‐terminus of HDM2 to prevent the interaction with p53 to stabilize and activate downstream p53 pathways. We have used the p53‐HDM2 interaction as a model system to explore the bioluminescence resonance energy transfer (BRET) technique for validating compounds that disrupt PPIs in living cells.

Setting Up a Bioluminescence Resonance Energy Transfer High throughput Screening Assay to Search for Protein/Protein Interaction Inhibitors in Mammalian Cells

“…Indeed, BRET superiority was shown by its ability to monitor PPI using endogenous level of protein expression (Couturier and Jockers, 2003; Pfleger and Eidne, 2003) and its consequent application to various live cell screening assays (Pfleger et al, 2007; Bacart et al, 2008; Kocan and Pfleger, 2011). Finally, using this method to screen for P2I2 is further supported as BRET is prone to disruption or modulation by co-expression of untagged interacting partner (Bacart et al, 2008; Ayoub and Pfleger, 2010; Kulahin et al, 2011) and by incubation with inhibitory peptides (Granier et al, 2004; Harikumar et al, 2006; Jarry et al, 2010) or inhibitory chemical compounds (Mazars and Fåhraeus, 2010; Corbel et al, 2011). …”

“…Rluc2 or Rluc8, mutants of Rluc with higher stability and quantum yield (Loening et al, 2006), greatly increased BRET1 signal (Kocan et al, 2008; Kamal et al, 2009; Schelshorn et al, 2012). Recently, BRET1 was used to develop two P2I2 screening assays (Mazars and Fåhraeus, 2010; Corbel et al, 2011). …”

“…However, another study found the opposite (Dacres et al, 2009). To date, only BRET1-based P2I2 screening assays have been described and showed the feasibility of this approach (Mazars and Fåhraeus, 2010; Corbel et al, 2011). BRET1 seems to be nowadays the best suited BRET method to develop P2I2 screening assays until proven otherwise.…”

“…An example using transitory transfection has shown that a known inhibitory compound was active in this assay (Mazars and Fåhraeus, 2010), however no hits based on this assay has been further published. On the contrary, a successful P2I2 screening assay using yeast stably expressing donor and acceptor respectively in an inducible and constitutive way has led to identification of chemical hits able to prevent the interaction between human cdk5 and p25 (Corbel et al, 2011).…”

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

“…10 Small changes in the positioning of the donor and acceptor, however, result in signal decline, which does not necessarily reflect a splitting of the PPI under investigation. [9][10][11] Finally, microscopy-based methods have been introduced that take advantage of subcellular protein redistribution. The use of such assays is restricted to proteins that undergo subcellular translocation on interaction or are engineered to do so.…”