Study of nuclear receptor dynamics by BRET
Nuclear receptors (NRs) are ligand-dependent transcription factors that control a wide variety of biological processes in human physiology, which has made them preferred pharmacological targets for many diseases. One such receptor, the estrogen receptor alpha (ERα), can activate cell proliferation in some sections of the mammary epithelium while another, the retinoic acid receptor alpha (RARα), can cause growth arrest and cellular differentiation. Signalling by these receptors can be altered in breast cancer, contributing to tumorigenesis. ERα can be blocked by antiestrogens (AEs) in the clinical setting to inhibit tumor cell proliferation. However, attempts to activate the RARα pathway with retinoids have not proven beneficial in clinical trials. This may result from the lack of specificity of the tested ligands for RARa and/or from their activity only in a subset of breast tumors.  Since nuclear receptors form homo- and heterodimers, we sought to develop novel pharmacological assays to study the activity of specific receptor-dimer complexes, their dynamics and quaternary structure. We report here a new FRET technique dubbed BRET with fluorescence enhanced by combined transfers (BRETFect) that can detect the formation of ternary nuclear receptor complexes. BRETFect can monitor the activation of ERα-ERβ heterodimers and highlights an allosteric mechanism of activation that each receptor exerts on its dimer partner. Use of BRETFect in combination with PCA-BRET has allowed us to observe the formation of functional ERα multimers in live cells for the first time. The formation of multimers is favored by AEs which induce receptor degradation, and may underlie their specific properties. These assays are a net improvement over the classical luciferase-reporter experiment as they deliver real-time receptor specific information with no interference by other process such as transcription and translation. Using these BRET assays we developed a new class of NR hybrid ligands that can modulate ER or RAR activity and inhibit HDACs. This has allowed for the development of promising new bifunctional molecules such as the RAR-agonist/HDACi hybrid molecule TTNN-HA. In conclusion, the work presented here brings new insight in NR dynamics and quaternary structure and offers novel tools to study their mechanism of action or design new modulators of NR activity such as hybrid AE-HDACis and Retinoid-HDACis.
FRET BRET Nuclear receptors Pharmacology estrogen Retinoic acid