Comprehensive assessment of NR ligand polypharmacology by a multiplex reporter NR assay

• Published in: Scientific reports Vol. 12; no. 1; p. 3115
• Main Authors: Medvedev, Alexander, Moeser, Matt, Medvedeva, Liubov, Martsen, Elena, Granick, Alexander, Raines, Lydia, Gorman, Kristen, Lin, Benjamin, Zeng, Ming, Houck, Keith A., Makarov, Sergei S.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 24.02.2022; Nature Publishing Group; Nature Portfolio
• Subjects: 631/154/570; 631/1647/2017/1958; 631/45/776/812; 631/80/86/2363; 631/92/436/2387; 631/92/507; 631/92/613; Antagonists; Biological Assay - methods; Estrogens; Genes, Reporter - drug effects; Homology; Humanities and Social Sciences; Humans; Ligands; Mass Screening - methods; multidisciplinary; Nuclear receptors; Peroxisome proliferator-activated receptors; Polypharmacology - methods; Progestin; Protein Binding; Receptors, Cytoplasmic and Nuclear - drug effects; Receptors, Cytoplasmic and Nuclear - metabolism; Receptors, Cytoplasmic and Nuclear - physiology; Science; Science (multidisciplinary); Therapeutic targets; Transcription factors
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-022-07031-8

Nuclear receptors (NR) are ligand-modulated transcription factors that regulate multiple cell functions and thus represent excellent drug targets. However, due to a considerable NR structural homology, NR ligands often interact with multiple receptors. Here, we describe a multiplex reporter assay (the FACTORIAL NR) that enables parallel assessment of NR ligand activity across all 48 human NRs. The assay comprises one-hybrid GAL4-NR reporter modules transiently transfected into test cells. To evaluate the reporter activity, we assessed their RNA transcripts. We used a homogeneous RNA detection approach that afforded equal detection efficacy and permitted the multiplex detection in a single-well format. For validation, we examined a panel of selective NR ligands and polypharmacological agonists and antagonists of the progestin, estrogen, PPAR, ERR, and ROR receptors. The assay produced highly reproducible NR activity profiles (r > 0.96) permitting quantitative assessment of individual NR responses. The inferred EC50 values agreed with the published data. The assay showed excellent quality (<Z’>  = 0.73) and low variability ( = 7.2%). Furthermore, the assay permitted distinguishing direct and non-direct NR responses to ligands. Therefore, the FACTORIAL NR enables comprehensive evaluation of NR ligand polypharmacology.