The bioluminescence resonance energy transfer (BRET) method is based on resonance energy transfer between a light-emitting enzyme and a fluorescent acceptor. Since its first description in 1999, several versions of BRET have been described using different substrates and energy donor/acceptor couples. Today, BRET is considered as one of the most versatile techniques for studying the dynamics of protein-protein interactions in living cells. Various studies have applied BRET-based assays to screen new receptor ligands and inhibitors of disease-related-proteases. Inhibitors of protein-protein interactions are likely to become a new major class of therapeutic drugs, and BRET technology is expected to play an important role in the identification of such compounds. This review describes the original BRET-based methodology, more recent variants, and potential applications to drug screening.

Electrochemical impedance spectroscopy was tested to monitor the cell attachment and the biofilm proliferation in order to identify characteristic events induced on the metal surface by Gram-negative (Pseudomonas aeruginosa PAO1) and Gram-positive (Bacillus subtilis) bacteria strains. Electrochemical impedance spectra of AISI 304 electrodes during cell attachment and initial biofilm growth for both strains were obtained. It can be observed that the resistance increases gradually with the culture time and decreases with the biofilm detachment. So, the applicability of electric cell-substrate impedance sensing (ECIS) for studying the attachment and spreading of cells on a metal surface has been demonstrated. The biofilm formation was also characterized by the use of scanning electron microscopy and confocal laser scanning microscopy and COMSTAT image analysis. The electrochemical results roughly agree with the microscope image observations. The ECIS technique used in this study was used for continuous real-time monitoring of the initial bacterial adhesion and the biofilm growth. It provides a simple and non-expensive electrochemical method for in vitro assessment of the presence of biofilms on metal surfaces.

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.

Cyclin-dependent kinase 5 (CDK5) is a multifunctional enzyme that plays numerous roles, notably in brain development. CDK5 is activated through its association with the activators, p35 and p39, rather than by cyclins. Proteolytic procession of the N-terminal part of its activators has been linked to Alzheimer's disease and various other neuropathies. The interaction with the proteolytic product p25 prolongs CDK5 activation and modifies the substrate specificity. In order to discover small-molecule inhibitors of the interaction between CDK5 and p25, we have used a bioluminescence resonance energy transfer (BRET)-based screening assay. Among the 1,760 compounds screened, the generic drug tamoxifen has been identified. The inhibition of the CDK5 activity by tamoxifen was notably validated by monitoring the phosphorylation state of tau protein. The study of the molecular mechanism of inhibition indicates that tamoxifen interacts with p25 to block the CDK5/p25 interaction and pave the way for new treatments of tauopathies.