Confirmation of high-throughput screening data and novel mechanistic insights into FXR-xenobiotic interactions by orthogonal assays

Abstract

Toxicology in the 21st Century (Tox21) is a federal collaboration employing a high-throughput robotic screening system to test 10,000 environmental chemicals. One of the primary goals of the program is prioritizing toxicity evaluations through in vitro high-throughput screening (HTS) assays for large numbers of chemicals already in commercial use for which little or no toxicity data is available. Within the Tox21 screening program, disruption in nuclear receptor (NR) signaling represents a particular area of interest. Given the role of NR’s in modulating a wide range of biological processes, alterations of their activity can have profound biological impacts. Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that has demonstrated importance in bile acid homeostasis, glucose metabolism, lipid homeostasis and hepatic regeneration. In this study, we re-evaluated 24 FXR agonists and antagonists identified through Tox21 using select orthogonal assays. In transient transactivation assays, 7/8 putative agonists and 4/4 putative inactive compounds were confirmed. Likewise, we confirmed 9/12 antagonists tested. Using a mammalian two hybrid approach we demonstrate that both FXR agonists and antagonists facilitate FXRα-coregulator interactions suggesting that differential coregulator recruitment may mediate activation/repression of FXRα mediated transcription. Additionally, we tested the ability of select FXR agonists and antagonists to facilitate hepatic transcription of FXR gene targets Shp and Bsep in a teleost (Medaka) model. Through application of in vitro cell-based assays, in silico modeling and in vivo gene expressions, we demonstrated the molecular complexity of FXR:ligand interactions and confirmed the ability of diverse ligands to modulate FXRα, facilitate differential coregulator recruitment and activate/repress receptor-mediated transcription. Overall, we suggest a multiplicative approach to assessment of nuclear receptor function may facilitate a greater understanding of the biological and mechanistic complexities of nuclear receptor activities and further our ability to interpret broad HTS outcomes.