Development of a Versatile and Sensitive Direct Ligand Binding Assay for Human NR5A Nuclear Receptors

Abstract

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 (EC50 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 Ki 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.

Enantiomer-specific activities of an LRH-1 and SF-1 dual agonist

“…Previous coregulator recruitment studies indicate that both RJW100 enantiomers bind LRH-1 and SF-1 34 , but ligand binding affinities have not been measured. To determine binding affinities, we utilized a fluorescence polarization-based, equilibrium ligand binding assay in which a fluorescein-labelled probe is displaced by unlabelled competing ligands 39 . Differences in probe affinity for LRH-1 and SF-1 were calculated prior to the competition assays and are accounted for in the equation for fitting the competition data (details in the methods section and our previous publication) 39 .…”

“…To determine binding affinities, we utilized a fluorescence polarization-based, equilibrium ligand binding assay in which a fluorescein-labelled probe is displaced by unlabelled competing ligands 39 . Differences in probe affinity for LRH-1 and SF-1 were calculated prior to the competition assays and are accounted for in the equation for fitting the competition data (details in the methods section and our previous publication) 39 . Both enantiomers outcompete the probe, indicating that they bind the receptors in the binding pockets (Fig.…”

“…With RR-RJW100, the configuration of the π-π stacking and hydrogen-bonding sites is complementary to the shape of the pocket, allowing an extremely stable binding mode. Understanding this has been vital for LRH-1 agonist development, enabling the discovery of potent and efficacious new lead agonists with a robust structure–activity relationship 18 , 37 , 39 . Likewise, the novel binding poses of SS-RJW100 present opportunities to target other regions of the binding pocket.…”

A novel phospholipid mimetic targeting LRH-1 ameliorates colitis

“…Targeting LRH-1 in UC is a particularly intriguing strategy, as its activation suppresses inflammation locally in the gut. 41,52 UC is commonly treated with corticosteroids, anti-TNFa therapies, or other systemic immunosuppressants that often cause adverse effects associated with immunosuppression in tissues other than the colon 53 . Thus, LRH-1 agonists may offer a more precise approach to UC treatment, reducing the potential for on-target, adverse effects in other tissues.…”

Development of a new class of liver receptor homolog-1 (LRH-1) agonists by photoredox conjugate addition

“…We tested the biological activity of this simplified 6HP series using a fluorescence polarization (FP) competition ligand binding assay recently developed in our lab 25 and a luciferase reporter assay to measure LRH-1 transcriptional activity. We assessed the binding affinity (Ki), in-cell potency (EC50), and efficacy (fold activation) of compounds 5 and 8, the direct analogs of 6HP-CA and 6N respectively, with the bridgehead group entirely removed.…”