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Development of a Robust Direct Binding Assay for Phospholipid‐Sensing NR5A Nuclear Receptors
Author(s) -
D'Agostino Emma Hope,
Cornelison Jeffery,
Mays Suzanne,
Flynn Autumn,
Jui Nathan,
Ortlund Eric
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.634.3
Subject(s) - ligand binding assay , nuclear receptor , chemistry , receptor , small molecule , ligand (biochemistry) , binding site , agonist , biochemistry , in vitro , fluorescence anisotropy , phospholipid , biophysics , biology , transcription factor , gene , membrane
The human NR5A nuclear receptors, steroidogenic factor‐1 (SF‐1, NR5A1) and liver receptor homolog‐1 (LRH‐1, NR5A2), are phospholipid‐sensing regulators of steroidogenesis, development, and metabolism. Their control of such diverse biological processes renders them attractive pharmacological targets for the treatment of several cancers and metabolic diseases such as obesity, type II diabetes, and non‐alcoholic fatty liver disease. However, the evaluation of candidate endogenous ligands and the development of small molecule modulators have been hindered by the lack of a robust direct binding assay. Using a structure‐guided approach, we recently developed a potent NR5A agonist (EC 50 of 15 nM) targeting the ligand‐binding pocket. We have leveraged this molecule to create a high‐affinity fluorescent probe that binds SF‐1 and LRH‐1 with affinites of 12 nM and 1 nM, respectively. This probe has enabled the creation of a fluorescence polarization (FP)‐based competition assay suitable for both synthetic small molecules and mammalian phospholipids. FP is solution‐based, allowing proteins to retain their native conformation, and the competition format of the assay ensures that binding will only be detected in the ligand‐binding pocket. We have found that for a set of 25 LRH‐1 agonists derived from the previously‐reported RJW100 scaffold, affinities determined by FP correlate with potencies in a luciferase reporter assay, suggesting that in vitro binding affinity may be predictive of in‐cell activity for this class of molecule. Moreover, binding affinities reported for other NR5A agonists harboring different chemical scaffolds agree with our FP‐based measurements. Finally, we show that the previously‐reported dilauroylphosphatidylcholine agonist binds LRH‐1 with a K i of 80 nM, suggesting that this assay may be used to evaluate candidate endogenous ligands for these receptors. Support or Funding Information R01 DK115213 02EHD has additional support from the National Science Foundation Graduate Research Fellowship and the Emory University NIH‐5T32GM008367‐27. SGM has additional support from F31 DK111171 03. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .