First BRET‐based screening assay performed in budding yeast leads to the discovery of CDK5/p25 interaction inhibitors

Abstract

The protein kinase CDK5 (cyclin-dependent kinase 5) is activated through its association with a cyclin-like protein p35 or p39. In pathological conditions (such as Alzheimer's disease and various other neuropathies), truncation of p35 leads to the appearance of the p25 protein. The interaction of p25 with CDK5 up-regulates the kinase activity and modifies the substrate specificity. ATP-mimetic inhibitors of CDK5 have already been developed. However, the lack of selectivity of such inhibitors is often a matter of concern. An alternative approach can be used to identify highly specific inhibitors that disrupt protein interactions involving protein kinases. We have developed a bioluminescence resonance energy transfer (BRET)-based screening assay in yeast to discover protein-protein interaction inhibitors (P2I2). Here, we present the first use of BRET in yeast for the screening of small molecule libraries. This screening campaign led to the discovery of one molecule that prevents the interaction between CDK5 and p25, thus inhibiting the protein kinase activity. This molecule may give rise to high-specificity drug candidates.

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). …”

Tampering with Cell Division by Using Small‐Molecule Inhibitors of CDK–CKS Protein Interactions

“…This phenomenon was observed with other small-molecule inhibitors of protein-protein interactions. [11,23] We used this protein interaction assay to test chemical analogues. For each of the two confirmed hits, we identified an active and an inactive analogue, which we named A1/B1 and A2/B2, respectively (Scheme 1 and Figure 2 B).…”

“…[9,10] A first proof of concept of CDK inhibition by a small molecule targeting a protein-protein interaction involved the discovery of a molecule that prevents complex formation between CDK5 and its activating protein p25, thereby efficiently disrupting kinase activity. [11] Our objective here was to target protein-protein interactions that regulate some of the functions of a subset of CDKs, without being strictly necessary for their enzymatic activity. We opted for the interactions between CDK and CKS proteins.…”

High‐Throughput Screening of Marine Resources

“…As a consequence, several technologies have been developed, including yeast twohybrid (Y2H), reverse yeast two-hybrid (Rezwan and Auerbach, 2012), bacteria two-hybrid (Stynen et al , 2012), Mammalian Protein-Protein Interaction Trap (MAPPIT) , reverse MAPPIT (Lemmens et al , 2006), bimolecular fluorescence complementation (BiFC), and FRET-and BRETbased methods (Ciruela, 2008;Corbel et al , 2011).…”