Development of radioligand binding assays for REV-ERBα and REV-ERBβ

REV-ERBα and REV-ERBβ are atypical nuclear receptors that function as ligand-dependent repressors of transcription. They play critical roles in the regulation of the circadian rhythm, inflammation, and metabolism. The natural ligand for the REV-ERBs is heme, and synthetic ligands (both agonists and antagonists) have been designed and utilized to probe the potential clinical utility of targeting REV-ERBs for drug development. Although biochemical assays that can detect REV-ERB ligands have been developed based on protein-protein interactions, no classical fluorescent- or radioligand-binding assay has yet been developed that directly detects ligand binding. Here, we describe the development of the first radioligand binding assay (RLBA) using scintillation proximity assay (SPA) technology for both REV-ERBα and REV-ERBβ using labeled STL1267, a potent REV-ERBα/β agonist we recently described. ³H-STL1267 displayed saturable binding to the ligand binding domains of both REV-ERBα and REV-ERBβ with equilibrium dissociation constants (K_ds) of 392 nM and 202 nM, respectively. In competition radioligand binding assays, we used unlabeled STL1267 or the well-characterized first-generation REV-ERB agonist SR9009 as competitors to ³H-STL1267 binding. STL1267 displayed K_is for REV-ERBα and REV-ERBβ of 253 ± 30 nM and 98 ± 14 nM, respectively. As expected, SR9009 displayed considerably lower potency than STL1267, with a K_i of 692 ± 209 nM for REV-ERBα and 2546 ± 127 nM for REV-ERBβ. Although developing an RLBA has been challenging due to the lack of high-affinity ligands that can be used as probes, our results demonstrate the feasibility of such an assay for both receptors, providing a robust assay with utility for ligand/drug discovery.