Development and implementation of a cell-based assay to discover agonists of the nuclear receptor REV-ERBα

Abstract

The nuclear receptors are transcription factors involved in the regulation of a variety of physiological processes whose activity can be modulated by binding to relevant small molecule ligands. Their dysfunction has been shown to play a role in disease states such as diabetes, cancer, inflammatory diseases, and hormonal resistance ailments, which makes them interesting targets for drug discovery. The nuclear receptor REV-ERBα is involved in regulating the circadian rhythm and metabolism. Its natural ligand is heme and there is significant interest in identifying novel synthetic modulators to serve as tools to characterize its function and to serve as drugs in treating metabolic disorders. To do so, we established a mammalian cell-based two-hybrid assay system capable of measuring the interaction between REV-ERBα and its co-repressor, nuclear co-repressor 1. This assay was validated to industry standard criteria and was used to screen a subset of the LOPAC ®1280 library and 29568 compounds from a diverse compound library. Profiling of the primary hits in a panel of counter and selectivity assays confirmed that REV-ERBα activity can be modulated pharmacologically and chemical scaffolds have been identified for optimization.

Recent advances in modulators of circadian rhythms: an update and perspective

“…Compound 24 showed a high selectivity over 20 nuclear receptors, which can reverse the degradation of REV-ERBa protein mediated by inflammatory stimuli. Subsequently, Gul et al 76 established a mammalian cell-based two-hybrid assay system and found compound 25 as a novel agonist of REV-ERB. In addition, three other compounds against REV-ERB, compounds 26-28 (Figure 4), were found using this method.…”

Combinatorial regulation of hepatic cytoplasmic signaling and nuclear transcriptional events by the OGT/REV-ERBα complex

“…Heme binding to REV-ERBα ( K d 2–4 µM, ref. 15 ) is prevented by mutating H602 into F ( 16 ). REV-ERBα H602F displayed a decreased transcriptional repressive activity ( Fig.…”

An optimized protocol with a stepwise approach to identify specific nuclear receptor ligands from cultured mammalian cells

“…2014 N/A pSG5-GAL4(DBD)-hPXR(LBD) Lemaire et al. 2006 N/A pM (Gal4 empty) Clontech Cat# PT3119-5 pGAL4-hCAR1 Hering et al. 2018 N/A p(UAS) 4 -tk-SEAP This article N/A p-RL (CMV) [renilla luciferase normalization vector] Promega Cat# E2261 p-EGFP-hCAR1 GeneCopoeia Cat# EX-A0964-M29 pGal4-Rev-erbα Hering et al.…”

“…Finally, to minimize off-target detection effects, we describe the use of mammalian one- (or two-) hybrid systems. For complete details on the use and execution of this protocol, please refer to Hering et al. (2018) .…”

“…For complete details on the use and execution of this protocol, please refer to Hering et al. (2018) .…”