A Dual Estrogen Receptor TR-FRET Assay for Simultaneous Measurement of Steroid Site Binding and Coactivator Recruitment

Abstract

The human estrogen receptors (hER) are members of the nuclear hormone receptor (NHR) superfamily and represent important drug targets for the pharmaceutical industry. Initially, ligand binding assays were used to identify novel ligands using receptors purified from native tissues. With the advent of molecular cloning techniques, cell-based transactivation assays have been the gold standard for many years of drug discovery. With the elucidation of the structural mechanisms underlying the activation of NHRs, cell-free assays with purified receptors have become important tools to directly assess different binding sites (e.g., the hormone binding site or the cofactor binding site). The available cell-free assays have so far facilitated the study of one binding site at a time. With the introduction of Terbium (Tb3+)–based time-resolved fluorescence energy transfer (TR-FRET), it has become possible to measure 2 different interactions within 1 test tube in parallel. The authors have applied this technology to develop a dual readout system for the simultaneous monitoring of steroid hormone site binding and cofactor peptide recruitment. They took advantage of a commercially available fluorescent tracer as an indicator for classical steroid site binding and designed a novel peptide derived from the peroxisome proliferator-activated receptor gamma coactivator-1a (PGC1a) as an indicator for functional agonistic behavior of a test compound. The established assay is able to differentiate between agonists, antagonists, partial agonists, and compounds binding to the cofactor recruitment site. The IC50 values obtained for a number of reference compounds in the multiplexed assay are in concordance with published data. The simple 1-step mix-and-measure protocol gives excellent quality and robustness and can be miniaturized to 5-µL volume.

In Vitro and in Vivo Molecular Imaging of Estrogen Receptor α and β Homo- and Heterodimerization: Exploration of New Modes of Receptor Regulation

“…Optical methods have previously been used to study ER homo-and heterodimerization in cell-free in vitro systems and in intact cells (24,29); however, none of these approaches could potentially be used to study the same process in living animals (31). In earlier work, we used fluorescence resonance energy transfer and excimer-emission methods to study the effect of ligand binding on the kinetic and thermodynamic stability of ER␣ homodimers in vitro and the additional stabilizing effect due to coactivator binding to ER-agonist complexes (19,32,33).…”

In Pursuit of Synthetic Modulators for the Orphan Retina-Specific Nuclear Receptor NR2E3

“…Historically, screening assays for smallmolecule modulators of nuclear hormone receptors are often based on ligand-sensitive interaction with transcriptional coactivators and corepressors. [31][32][33] It has been shown that apo-NR2E3 is capable of interacting with corepressors N-CoR, SMRT, and RetCOR. 22,26 Given that NR-corepressor interaction is agonist sensitive for the vast majority of NRs, we developed a cell-free HTS-compatible TR-FRET assay for small-molecule NR2E3 modulators, which is based on agonist-induced disruption of the interaction between GSTtagged apo-NR2E3 and MBP-tagged fragment of transcriptional corepressor RetCOR (Fig.…”

Cyclic Ketoximes as Estrogen Receptor β Selective Agonists

“…The use of a tr-FRET assay [16, 17] of this design is very useful to quantitatively probe the ability of new ligands to form a complex with the ER that is capable of recruiting coactivator, an important indicator of an active biological complex. This assay can be performed in two ways: coactivator titration into a preformed ER-ligand complex measures the affinity of the coactivator.…”