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Crystal Structures of the Nuclear Receptor, Liver Receptor Homolog 1, Bound to Synthetic Agonists*

Open AccessPublished:September 30, 2016DOI:https://doi.org/10.1074/jbc.M116.753541

      Abstract

      Liver receptor homolog 1 (NR5A2, LRH-1) is an orphan nuclear hormone receptor that regulates diverse biological processes, including metabolism, proliferation, and the resolution of endoplasmic reticulum stress. Although preclinical and cellular studies demonstrate that LRH-1 has great potential as a therapeutic target for metabolic diseases and cancer, development of LRH-1 modulators has been difficult. Recently, systematic modifications to one of the few known chemical scaffolds capable of activating LRH-1 failed to improve efficacy substantially. Moreover, mechanisms through which LRH-1 is activated by synthetic ligands are entirely unknown. Here, we use x-ray crystallography and other structural methods to explore conformational changes and receptor-ligand interactions associated with LRH-1 activation by a set of related agonists. Unlike phospholipid LRH-1 ligands, these agonists bind deep in the pocket and do not interact with residues near the mouth nor do they expand the pocket like phospholipids. Unexpectedly, two closely related agonists with similar efficacies (GSK8470 and RJW100) exhibit completely different binding modes. The dramatic repositioning is influenced by a differential ability to establish stable face-to-face π-π-stacking with the LRH-1 residue His-390, as well as by a novel polar interaction mediated by the RJW100 hydroxyl group. The differing binding modes result in distinct mechanisms of action for the two agonists. Finally, we identify a network of conserved water molecules near the ligand-binding site that are important for activation by both agonists. This work reveals a previously unappreciated complexity associated with LRH-1 agonist development and offers insights into rational design strategies.

      Introduction

      Liver receptor homolog 1 (LRH-1; NR5A2) is a nuclear hormone receptor (NR)
      The abbreviations used are: NR, nuclear hormone receptor; PDB, Protein Data Bank; PL, phospholipid; HDX, hydrogen-deuterium exchange; MDS, molecular dynamics simulation; LBD, ligand binding domain; DLPC, dilauroylphosphatidylcholine; PIP3, phosphatidylinositol 3,4,5-triphosphate; AFS, activation function surface; TEV, tobacco etch virus; DSF, differential scanning fluorimetry.
      that controls expression of a diverse set of genes important both in normal physiology and disease. In addition to a vital role during development (
      • Gu P.
      • Goodwin B.
      • Chung A.C.
      • Xu X.
      • Wheeler D.A.
      • Price R.R.
      • Galardi C.
      • Peng L.
      • Latour A.M.
      • Koller B.H.
      • Gossen J.
      • Kliewer S.A.
      • Cooney A.J.
      Orphan nuclear receptor LRH-1 is required to maintain Oct4 expression at the epiblast stage of embryonic development.
      ,
      • Wagner R.T.
      • Xu X.
      • Yi F.
      • Merrill B.J.
      • Cooney A.J.
      Canonical Wnt/β-catenin regulation of liver receptor homolog-1 mediates pluripotency gene expression.
      ), LRH-1 regulates many genes related to metabolism, proliferation, and cell survival. In the liver, LRH-1 regulates bile acid biosynthesis (
      • Lu T.T.
      • Makishima M.
      • Repa J.J.
      • Schoonjans K.
      • Kerr T.A.
      • Auwerx J.
      • Mangelsdorf D.J.
      Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors.
      ) and reverse cholesterol transport (
      • Stein S.
      • Oosterveer M.H.
      • Mataki C.
      • Xu P.
      • Lemos V.
      • Havinga R.
      • Dittner C.
      • Ryu D.
      • Menzies K.J.
      • Wang X.
      • Perino A.
      • Houten S.M.
      • Melchior F.
      • Schoonjans K.
      SUMOylation-dependent LRH-1/PROX1 interaction promotes atherosclerosis by decreasing hepatic reverse cholesterol transport.
      ,
      • Schoonjans K.
      • Annicotte J.-S.
      • Huby T.
      • Botrugno O.A.
      • Fayard E.
      • Ueda Y.
      • Chapman J.
      • Auwerx J.
      Liver receptor homolog 1 controls the expression of the scavenger receptor class B type I.
      ), affecting hepatic and circulating cholesterol levels. Glucose metabolism is also regulated by LRH-1 at several points, including GLUT-4-mediated transport (
      • Bolado-Carrancio A.
      • Riancho J.A.
      • Sainz J.
      • Rodríguez-Rey J.C.
      Activation of nuclear receptor NR5A2 increases Glut4 expression and glucose metabolism in muscle cells.
      ) and glucose phosphorylation, the latter of which is essential for proper postprandial glucose sensing, flux through glycolysis and glycogenesis pathways, and de novo lipogenesis (
      • Oosterveer M.H.
      • Mataki C.
      • Yamamoto H.
      • Harach T.
      • Moullan N.
      • van Dijk T.H.
      • Ayuso E.
      • Bosch F.
      • Postic C.
      • Groen A.K.
      • Auwerx J.
      • Schoonjans K.
      LRH-1-dependent glucose sensing determines intermediary metabolism in liver.
      ). LRH-1 is a key mediator of the cell stress response through control of genes involved in the hepatic acute phase response (
      • Venteclef N.
      • Smith J.C.
      • Goodwin B.
      • Delerive P.
      Liver receptor homolog 1 is a negative regulator of the hepatic acute-phase response.
      ) and in the cytoprotective resolution of endoplasmic reticulum stress (
      • Mamrosh J.L.
      • Lee J.M.
      • Wagner M.
      • Stambrook P.J.
      • Whitby R.J.
      • Sifers R.N.
      • Wu S.P.
      • Tsai M.J.
      • Demayo F.J.
      • Moore D.D.
      Nuclear receptor LRH-1/NR5A2 is required and targetable for liver endoplasmic reticulum stress resolution.
      ). Additionally, LRH-1 can be aberrantly overexpressed in certain cancers and can promote tumor growth through estrogen receptor and β-catenin signaling (
      • Bayrer J.R.
      • Mukkamala S.
      • Sablin E.P.
      • Webb P.
      • Fletterick R.J.
      Silencing LRH-1 in colon cancer cell lines impairs proliferation and alters gene expression programs.
      • Bianco S.
      • Brunelle M.
      • Jangal M.
      • Magnani L.
      • Gévry N.
      LRH-1 governs vital transcriptional programs in endocrine-sensitive and -resistant breast cancer cells.
      ,
      • Chand A.L.
      • Herridge K.A.
      • Thompson E.W.
      • Clyne C.D.
      The orphan nuclear receptor LRH-1 promotes breast cancer motility and invasion.
      ,
      • Clyne C.D.
      • Kovacic A.
      • Speed C.J.
      • Zhou J.
      • Pezzi V.
      • Simpson E.R.
      Regulation of aromatase expression by the nuclear receptor LRH-1 in adipose tissue.
      ,
      • Lai C.F.
      • Flach K.D.
      • Alexi X.
      • Fox S.P.
      • Ottaviani S.
      • Thiruchelvam P.T.
      • Kyle F.J.
      • Thomas R.S.
      • Launchbury R.
      • Hua H.
      • Callaghan H.B.
      • Carroll J.S.
      • Charles Coombes R.
      • Zwart W.
      • Buluwela L.
      • Ali S.
      Co-regulated gene expression by oestrogen receptor α and liver receptor homolog-1 is a feature of the oestrogen response in breast cancer cells.
      ,
      • Thiruchelvam P.T.
      • Lai C.F.
      • Hua H.
      • Thomas R.S.
      • Hurtado A.
      • Hudson W.
      • Bayly A.R.
      • Kyle F.J.
      • Periyasamy M.
      • Photiou A.
      • Spivey A.C.
      • Ortlund E.A.
      • Whitby R.J.
      • Carroll J.S.
      • Coombes R.C.
      • et al.
      The liver receptor homolog-1 regulates estrogen receptor expression in breast cancer cells.
      • Lin Q.
      • Aihara A.
      • Chung W.
      • Li Y.
      • Huang Z.
      • Chen X.
      • Weng S.
      • Carlson R.I.
      • Wands J.R.
      • Dong X.
      LRH1 as a driving factor in pancreatic cancer growth.
      ).
      Considering the breadth and significance of these physiological effects, LRH-1 modulators are highly desired as potential therapeutic agents. Chemical modulators would also be extremely useful as tools to dissect complex or temporal aspects of LRH-1 biology. However, development of LRH-1-targeted compounds has been challenging, due in part to a lipophilic binding pocket that becomes occupied with bacterial phospholipids (PL) in recombinant protein. Very few small molecules are able to displace these PL in library screens. Moreover, ligand-mediated regulation of LRH-1 is poorly understood. Endogenous ligands for LRH-1 are unknown, but exogenous administration of dilauroylphosphatidylcholine (DLPC) (phosphatidylcholine 12:0/12:0) activates LRH-1 and has profound anti-diabetic effects in vivo, which are absent in a liver-specific LRH-1 knock-out mouse (
      • Lee J.M.
      • Lee Y.K.
      • Mamrosh J.L.
      • Busby S.A.
      • Griffin P.R.
      • Pathak M.C.
      • Ortlund E.A.
      • Moore D.D.
      A nuclear-receptor-dependent phosphatidylcholine pathway with antidiabetic effects.
      ). In addition to phosphatidylcholines, the signaling PL phosphatidylinositol 3,4,5-triphosphate (PIP3) binds LRH-1 (
      • Sablin E.P.
      • Blind R.D.
      • Uthayaruban R.
      • Chiu H.J.
      • Deacon A.M.
      • Das D.
      • Ingraham H.A.
      • Fletterick R.J.
      Structure of liver receptor homolog-1 (NR5A2) with PIP hormone bound in the ligand binding pocket.
      ,
      • Krylova I.N.
      • Sablin E.P.
      • Moore J.
      • Xu R.X.
      • Waitt G.M.
      • MacKay J.A.
      • Juzumiene D.
      • Bynum J.M.
      • Madauss K.
      • Montana V.
      • Lebedeva L.
      • Suzawa M.
      • Williams J.D.
      • Williams S.P.
      • Guy R.K.
      • et al.
      Structural analyses reveal phosphatidyl inositols as ligands for the NR5 orphan receptors SF-1 and LRH-1.
      ), although downstream effects of this interaction have yet to be determined.
      Typically, NRs are activated by a ligand-induced conformational change, which promotes recruitment of co-activator proteins to the activation function surface (AFS) in the ligand binding domain (LBD) to drive transcription. Our structural studies with DLPC have shown that, contrary to the canonical model of NR activation, LRH-1 relies on small conformational fluctuations to recruit co-activator or co-repressor proteins. These occur mainly in the AFS (composed of portions of H3, H4, and the AF-H in the LBD) as well as in the H6/β-sheet region at a distal portion of the LBD (
      • Musille P.M.
      • Pathak M.C.
      • Lauer J.L.
      • Hudson W.H.
      • Griffin P.R.
      • Ortlund E.A.
      Antidiabetic phospholipid-nuclear receptor complex reveals the mechanism for phospholipid-driven gene regulation.
      ,
      • Musille P.M.
      • Kossmann B.R.
      • Kohn J.A.
      • Ivanov I.
      • Ortlund E.A.
      Unexpected allosteric network contributes to LRH-1 coregulator selectivity.
      ). Flexibility in the H6/β-sheet region is required for activation by PLs (
      • Musille P.M.
      • Pathak M.C.
      • Lauer J.L.
      • Hudson W.H.
      • Griffin P.R.
      • Ortlund E.A.
      Antidiabetic phospholipid-nuclear receptor complex reveals the mechanism for phospholipid-driven gene regulation.
      ). Mechanisms through which LRH-1 is activated by synthetic ligands have not been explored but are likely quite different, given the differing structural composition of synthetic versus PL ligands.
      There are very few known chemical scaffolds capable of activating LRH-1 above basal levels, the best studied of which are the cis-bicyclo[3.3.0]-octenes discovered by Whitby et al. (
      • Whitby R.J.
      • Dixon S.
      • Maloney P.R.
      • Delerive P.
      • Goodwin B.J.
      • Parks D.J.
      • Willson T.M.
      Identification of small molecule agonists of the orphan nuclear receptors liver receptor homolog-1 and steroidogenic factor-1.
      ,
      • Whitby R.J.
      • Stec J.
      • Blind R.D.
      • Dixon S.
      • Leesnitzer L.M.
      • Orband-Miller L.A.
      • Williams S.P.
      • Willson T.M.
      • Xu R.
      • Zuercher W.J.
      • Cai F.
      • Ingraham H.A.
      Small molecule agonists of the orphan nuclear receptors steroidogenic factor-1 (SF-1, NR5A1) and liver receptor homologue-1 (LRH-1, NR5A2).
      ). The first compound described with this scaffold, named GSK8470 (Fig. 1), was somewhat effective but was acid-labile (
      • Whitby R.J.
      • Stec J.
      • Blind R.D.
      • Dixon S.
      • Leesnitzer L.M.
      • Orband-Miller L.A.
      • Williams S.P.
      • Willson T.M.
      • Xu R.
      • Zuercher W.J.
      • Cai F.
      • Ingraham H.A.
      Small molecule agonists of the orphan nuclear receptors steroidogenic factor-1 (SF-1, NR5A1) and liver receptor homologue-1 (LRH-1, NR5A2).
      ). Substitution of the aniline group improved compound stability, and the GSK8470-LRH-1 crystal structure provided the basis for an extensive structure-activity relationship study (
      • Whitby R.J.
      • Stec J.
      • Blind R.D.
      • Dixon S.
      • Leesnitzer L.M.
      • Orband-Miller L.A.
      • Williams S.P.
      • Willson T.M.
      • Xu R.
      • Zuercher W.J.
      • Cai F.
      • Ingraham H.A.
      Small molecule agonists of the orphan nuclear receptors steroidogenic factor-1 (SF-1, NR5A1) and liver receptor homologue-1 (LRH-1, NR5A2).
      ). One of the major objectives of this study was to introduce functional groups near select polar residues within the predominantly hydrophobic pocket. The new lead compound produced from this study, named RJW100, contains an exo-hydroxyl group at the 1-position of the pentalene scaffold (indicated in red in Fig. 1) intended to interact with LRH-1 residues His-390 or Arg-393. In contrast, a diastereomer with endo stereochemistry (previously known as 24-endo, Fig. 1) was not predicted to be able to make these interactions due to the alternative conformation of the hydroxyl oxygen. The endo derivative was less active in biochemical assays, seeming to support this hypothesis. However, RJW100 was not much more potent or effective than GSK8470, and the study did not illuminate strategies for further improvement.
      Figure thumbnail gr1
      FIGURE 1Chemical structures of LRH-1 agonists. A, GSK8470, the parent compound. B, RJW100 enantiomers. C, RJW100 analog lacking the hydroxyl group (named 18a), assayed in .
      In this work, we present crystal structures of RJW100 and its endo-diastereomer bound to LRH-1. We demonstrate that these compounds bind quite differently than PLs and have distinct effects on protein dynamics compared with DLPC. Unexpectedly, these agonists also bind quite differently from the very closely related compound, GSK8470. We identify receptor-ligand interactions driving the repositioning and show that particular interactions are important for LRH-1 activation. These findings provide the first description of mechanisms involved in LRH-1 activation by synthetic molecules.

      Discussion

      Although LRH-1 synthetic modulators are highly sought as pharmacological tools and as potential therapeutic agents, a limited understanding of ligand characteristics important for binding and activating LRH-1 has impeded agonist development. This work represents the first detailed exploration of structural mechanisms governing regulation of LRH-1 by synthetic ligands. Relative to the PL LRH-1 agonist, DLPC, the current best agonist (RR-RJW100) constricts the binding pocket and destabilizes portions of the AFS (FIGURE 1, FIGURE 2). In future studies, it will be interesting to investigate the causes of this latter effect, because stabilization of the AFS may facilitate co-activator binding, leading to greater potency or efficacy. Alternatively, analogs designed to enhance the AFS destabilization may be effective antagonists or inverse agonists.
      In a previous study, RJW100 was the most effective of a large series of GSK8470 derivatives but still only modestly increased LRH-1 activation (
      • Whitby R.J.
      • Stec J.
      • Blind R.D.
      • Dixon S.
      • Leesnitzer L.M.
      • Orband-Miller L.A.
      • Williams S.P.
      • Willson T.M.
      • Xu R.
      • Zuercher W.J.
      • Cai F.
      • Ingraham H.A.
      Small molecule agonists of the orphan nuclear receptors steroidogenic factor-1 (SF-1, NR5A1) and liver receptor homologue-1 (LRH-1, NR5A2).
      ). Indeed, we find that these two agonists are statistically indistinguishable in luciferase reporter assays measuring LRH-1 activity (Fig. 8D). Given the similarities in structures and efficacies for these ligands, we expected them to utilize similar mechanisms of action; however, this is not the case. Our crystal structure reveals a dramatically different binding mode for RR-RJW100 compared with GSK8470 (Fig. 4). Although this was surprising, it is not unreasonable, considering that LRH-1 has a very large hydrophobic binding pocket and that these agonists are also quite hydrophobic, filling only 37% of the available space (excluding waters). It is possible that many of the GSK8470 analogs investigated in the previous structure-activity relationship study adopt a variety of different conformations. This seems to be the case in our docking studies with these ligands; multiple very different binding modes with similar energies are predicted (data not shown). Importantly, however, the repositioning of RR-RJW100 in our structure appears to be driven by particular interactions, because SR-RJW100 assumes a very similar pose (Fig. 5). This occurs despite the fact that the SR derivative exhibits signs of motion in our crystal structure, with significant disorder in the tail of the ligand and higher relative B-factors than RR-RJW100.
      A major factor driving repositioning of the RJW100 isomers was the hydrogen bonding interaction made by the hydroxyl group. Although the contact with residue Thr-352 is indirect, it is mediated by a water molecule that is part of a network of waters found in every published LRH-1 crystal structure (with the exception of PDB 4DOR, in which a major portion of the ligand-binding pocket is disordered (
      • Musille P.M.
      • Pathak M.C.
      • Lauer J.L.
      • Hudson W.H.
      • Griffin P.R.
      • Ortlund E.A.
      Antidiabetic phospholipid-nuclear receptor complex reveals the mechanism for phospholipid-driven gene regulation.
      )). The existence of conserved water molecules, as well as their participation in ligand binding, has been described (
      • Ogata K.
      • Wodak S.J.
      Conserved water molecules in MHC class-I molecules and their structural and functional roles.
      ,
      • Barillari C.
      • Taylor J.
      • Viner R.
      • Essex J.W.
      Classification of water molecules in protein binding sites.
      • Klebe G.
      Applying thermodynamic profiling in lead finding and optimization.
      ). Thus, this interaction could serve as an anchor point to secure the compound in a predictable orientation, enabling the targeting of desired parts of the binding pocket via strategic addition of substituents to the ligand's scaffold. Moreover, replacing the RJW100 hydroxyl group with a larger polar moiety may allow direct contact with Thr-352, leading to a stronger interaction. This strategy is being actively explored in our laboratory.
      The role of the Thr-352 interaction in LRH-1 activation by RR-RJW100 was demonstrated through the marked loss of activation by this compound when this residue was mutated (Fig. 8). In addition, an RJW100 analog lacking a hydroxyl group and thus unable to make this was a poor activator. Unexpectedly, the T352V mutation also resulted in a loss of activity for GSK8470, although this compound does not interact with the Thr-352-coordinated water molecule. However, we show that the T352V mutation weakens GSK8470's interaction with His-390, perhaps via destabilization of the conserved water network (Fig. 8, G and H). This could be responsible for the loss of activity of GSK8470 when Thr-352 is mutated.
      It has been hypothesized in in silico studies that π-π-stacking with residue His-390 is critical for activation of LRH-1 by this ligand class (
      • Lalit M.
      • Gangwal R.P.
      • Dhoke G.V.
      • Damre M.V.
      • Khandelwal K.
      • Sangamwar A.T.
      A combined pharmacophore modeling, 3D-QSAR and molecular docking study of substituted bicyclo-[3.3.0]oct-2-enes as liver receptor homolog-1 (LRH-1) agonists.
      ); however, this had not been explicitly tested. We find that this is the case for GSK8470, which stably interacts with His-390 via face-to-face π-π-stacking. Interestingly, although mutation of His-390 to alanine ablated LRH-1 activation by GSK8470, it had no effect on RR-RJW100-mediated activation (Fig. 8). We show that the RR-RJW100 interaction with His-390 is much less stable than that of GSK8470 and is mediated by a different phenyl ring (Fig. 7). Substitution of the GSK8470 aniline group with the styrene appears to have had the unexpected effect of making face-to-face stacking with His-390 less favorable (Fig. 9). This, combined with the favorable water-mediated interaction with Thr-352, influences the positioning of RJW100.
      Together, these findings reveal that the interaction of small molecule agonists with LRH-1 is more complex than originally supposed. Not only do these agonists affect receptor conformation differently from PL ligands, but they also exhibit an unexpected variability in binding modes. This work has uncovered some of the molecular interactions responsible for both positioning and activation of two very similar agonists, which provide insights into strategies to improve the design of LRH-1-targeted compounds.

      Author Contributions

      S. G. M. and E. A. O. conceived the studies. S. G. M. purified protein-ligand complexes, determined crystal structures, and conducted DSF and cellular assays. C. D. O. performed and analyzed molecular dynamics simulations. D. G. and P. R. G. conducted HDX and analyzed results. R. J. W., J. S., M. C. D., A. R. F., and N. T. J. synthesized GSK8470, RJW100, and analogs. S. G. M., C. D. O., R. J. W., N. T. J., D. G., P. R. G., and E. A. O. wrote the manuscript.

      Acknowledgments

      We thank Sally Bloodworth (University of Southampton, United Kingdom) for assistance with synthetic chemistry, Bradley Kossmann and Dr. Ivaylo Ivanov (Georgia State University) for helpful discussions about MDS, and Dr. Kay Diederichs (Universität Konstanz) for advice in model refinement.

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