The BRET technology and its application to screening assays

Abstract

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.

Modulation of Glucagon-like Peptide-1 (GLP-1) Potency by Endocannabinoid-like Lipids Represents a Novel Mode of Regulating GLP-1 Receptor Signaling

“…The real-time intracellular cAMP assay used in our study is an enhanced BRET2 (24) assay that has better separation of the energy emitted by the donor and acceptor (22,33) and yields data comparable with that obtained by other previously reported methods (22,33). This enhanced assay has the advantage of being able to monitor intracellular cAMP production (not cAMP accumulation) in the absence of phosphodiesterase inhibition (e.g.…”

“…Construction of RG-cAMP Sensor-To reduce BRET background and obtain better separation of the donor and acceptor energy emission peaks, we replaced the energy donor and acceptor in the early version Epac1 cAMP biosensor (22,23) with Rluc8 (20) and GFP2, respectively, for use in enhanced BRET2 (24) (Fig. 1, A-C).…”

“…Adenylyl cyclase inhibitor MDL-12,330A hydrochloride, puromycin, G418, and 2-OG were from Sigma-Aldrich. OEA, oleic acid (OA), and all other fatty acids and lipids were from Cayman (Ann Arbor, MI Construction of RG-cAMP Sensor-We replaced the energy donor and acceptor of the previously described Epac1 biosensor (22,23) with Rluc8 and GFP2, respectively, for enhanced bioluminescence resonance energy transfer 2 (BRET2) (24). Primers used to generate the fusion protein construct are shown in Table 1.…”

“…We probed the potential conformation of GLP-1(7-36) amide based on its susceptibility to trypsin digestion. The two trypsin cleavage products of GLP-1 amide are an inactive GLP-1 (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) and a partially active GLP-1(7-34) (Fig. 8A) (32).…”

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

“…The use of bioluminescence resonance energy transfer, or BRET, holds promise to fulfill at least some of the criteria for a simple and reliable cell-based assay to test the effect of compounds on disrupting or even modifying a PPI [3][4]. The BRET is based on fusing the Renilla luciferase (Rluc, energy donor) and YFP (acceptor) to either of two interacting proteins.…”