Nuclear Receptors Have Distinct Affinities for Coactivators: Characterization by Fluorescence Resonance Energy Transfer

Zhou, Gaochao; Cummings, Richard; Li, Ying; Mitra, Sudha W.; Wilkinson, Hilary A.; Elbrecht, Alex; Hermes, Jeffrey D.; Schaeffer, James M.; Smith, Roy G.; Moller, David E.

doi:10.1210/mend.12.10.0176

Cited by 119 publications

(56 citation statements)

References 43 publications

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“…To identify new FXR agonists that could serve as the starting point for medicinal chemistry optimization, we developed a high-throughput assay to screen for ligandinduced coactivator recruitment to FXR that was based on homogeneous time-resolved fluorescence (HTRF) (26). The FXR HTRF assay is similar to assays that were developed in-house for the estrogen and PPAR receptors (27,28). The assay was used to screen a library of approximately one million compounds and identified a potent agonist designated MFA-1 [17␤-(4-hydroxybenzoyl)androsta-3,5-diene-3-carboxylic acid] (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Identification of a potent synthetic FXR agonist with an unexpected mode of binding and activation

Soisson

Parthasarathy²,

Adams³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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The farnesoid X receptor (FXR), a member of the nuclear hormone receptor family, plays important roles in the regulation of bile acid and cholesterol homeostasis, glucose metabolism, and insulin sensitivity. There is intense interest in understanding the mechanisms of FXR regulation and in developing pharmaceutically suitable synthetic FXR ligands that might be used to treat metabolic syndrome. We report here the identification of a potent FXR agonist (MFA-1) and the elucidation of the structure of this ligand in ternary complex with the human receptor and a coactivator peptide fragment using x-ray crystallography at 1.9-Å resolution. The steroid ring system of MFA-1 binds with its D ring-facing helix 12 (AF-2) in a manner reminiscent of hormone binding to classical steroid hormone receptors and the reverse of the pose adopted by naturally occurring bile acids when bound to FXR. This binding mode appears to be driven by the presence of a carboxylate on MFA-1 that is situated to make a salt-bridge interaction with an arginine residue in the FXR-binding pocket that is normally used to neutralize bound bile acids. Receptor activation by MFA-1 differs from that by bile acids in that it relies on direct interactions between the ligand and residues in helices 11 and 12 and only indirectly involves a protonated histidine that is part of the activation trigger. The structure of the FXR:MFA-1 complex differs significantly from that of the complex with a structurally distinct agonist, fexaramine, highlighting the inherent plasticity of the receptor.NR1H4 ͉ bile acid receptor ͉ nuclear receptor ͉ x-ray crystallography T he farnesoid X receptor (FXR) plays key roles in regulating cholesterol and bile acid homeostasis (1-4). Central to this function is the ability of bile acid-activated FXR to downregulate the expression of Cyp7a (1, 5, 6), the rate-limiting step in the liver for the conversion of free cholesterol to bile acids, and the up-regulation of the bile salt excretion pump (BSEP), which functions to pump excess bile acids into the gall bladder for eventual fecal excretion (7). Treatment of ob/ob and db/db mice with the synthetic FXR agonist GW4064 significantly improves hypercholesterolemia (8) and lowers free fatty acids (9, 10) and triglyceride levels (11). Additional research has shown that fxr Ϫ/Ϫ mice show insulin resistance and impaired glucose tolerance (8,10,12), and that expression of constitutively active FXR in the liver results in hypoglycemia (10). Similarly, treatment with GW4064 increases insulin sensitivity in ob/ob and db/db mice (8, 10). As such, FXR agonists may have utility in treating metabolic syndrome, a clustering of cardiovascular risk factors characterized by dyslipidemia (elevated triglyceride and low HDL levels), insulin resistance, and poor glucose regulation. Several excellent reviews have summarized the current state of FXR understanding (13,14) and help build the case for the development of FXR modulators for the treatment of diabetes and metabolic syndrome (15).FXR belongs to the lar...

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Section: Resultsmentioning

confidence: 99%

“…The HTRF-based coactivator recruitment assay was performed essentially as described in ref. 28; specific details related to the FXR assay can be found in SI Text.…”

Section: Methodsmentioning

confidence: 99%

Identification of a potent synthetic FXR agonist with an unexpected mode of binding and activation

Soisson

Parthasarathy²,

Adams³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…To date, several in vitro assays have been developed for screening ER ligands by using either purified ER␣ protein or ER isolated from cell lysates (18)(19)(20)(21). Limited fluorescence-based assays (22) have been developed to measure receptor conformational changes (23) and recruitment of coactivator peptides (22,24,25) in the full-length hER␣ within cell culture (26). Other assays have been designed to study the effects of synthetic ligands on ER transcription through the activation of downstream target genes (27).…”

Section: Discussionmentioning

confidence: 99%

An intramolecular folding sensor for imaging estrogen receptor–ligand interactions

Paulmurugan

Gambhir

2006

Proc. Natl. Acad. Sci. U.S.A.

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Strategies for high-throughput analysis of interactions between various hormones and drugs with the estrogen receptor (ER) are crucial for accelerating the understanding of ER biology and pharmacology. Through careful analyses of the crystal structures of the human ER (hER) ligand-binding domain (hER-LBD) in complex with different ligands, we hypothesized that the hER-LBD intramolecular folding pattern could be used to distinguish ER agonists from selective ER modulators and pure antiestrogens. We therefore constructed and validated intramolecular folding sensors encoding various hER-LBD fusion proteins that could lead to split Renilla͞firefly luciferase reporter complementation in the presence of the appropriate ligands. A mutant hER-LBD with low affinity for circulating estradiol was also identified for imaging in living subjects. Cells stably expressing the intramolecular folding sensors expressing wild-type and mutant hER-LBD were used for imaging ligand-induced intramolecular folding in living mice. This is the first hER-LBD intramolecular folding sensor suited for high-throughput quantitative analysis of interactions between hER with hormones and drugs using cell lysates, intact cells, and molecular imaging of small living subjects. The strategies developed can also be extended to study and image other important protein intramolecular folding systems.complementation ͉ optical imaging ͉ split reporters E strogens are responsible for the growth, development, and maintenance of the reproductive, skeletal, neuronal, and immune systems as well as and other systems. The physiological effects of these hormones are mediated by the estrogen receptor (ER), which is a ligand-inducible nuclear transcription factor (1). In the classical pathway of steroid hormone action, 17␤-estradiol (E2), hormones, and a variety of other estrogens bind to the ligandbinding domain (LBD) of ER and lead to its dimerization and subsequent binding to a specific regulatory sequence in the promoters of ER target genes known as the estrogen response elements (2, 3), which then trigger activation or repression of many downstream target genes (4). The deficiency or excess of estrogens can lead to various pathological conditions including osteoporosis and breast carcinomas (5), making ER a major cellular therapeutic target.Elegant crystallographic studies with ER-LBD have shown that conformation of helix 12 (H12) is critical in responses observed with various ER ligands (4, 6, 7). The conformation of H12 behaves as a ''molecular switch'' that either prevents or enhances ER from binding to an array of coactivator proteins, which then activates transcription of many downstream estrogen-regulated genes responsible for cell growth. Given the critical role of H12 in ER signaling, we reasoned that it may be feasible to develop an intramolecular ER folding sensor with specific split reporter complementation patterns to study ligand pharmacology based directly on the conformational changes of H12 in response to different ligands (Fig. 1).We used a spli...

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“…A complete description of this assay has been published elsewhere (16). Briefly, 198 l of reaction mixture (100 mM HEPES, 125 mM KF, 0.125% (w/v) CHAPS, 0.05% dry milk, 5 nM GST-hPPAR␣LBD, 2 nM anti-GST-(Eu)K, 10 nM biotin-CBP-(1-453), and 20 nM SA/XL665) were added to each well followed by the addition of 2 l of Me 2 SO or compound 1 (in Me 2 SO) in appropriate wells.…”

Section: Methodsmentioning

confidence: 99%

Differential Gene Regulation in Human Versus Rodent Hepatocytes by Peroxisome Proliferator-activated Receptor (PPAR) α

Lawrence¹,

Liu²,

Chen³

et al. 2001

Journal of Biological Chemistry

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170

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We compared the ability of rat and human hepatocytes to respond to fenofibric acid and a novel potent phenylacetic acid peroxisome proliferator-activated receptor (PPAR) ␣ agonist (compound 1). Fatty acyl-CoA oxidase (FACO) activity and mRNA were increased after treatment with either fenofibric acid or compound 1 in rat hepatocytes. In addition, apolipoprotein CIII mRNA was decreased by both fenofibric acid and compound 1 in rat hepatocytes. Both agonists decreased apolipoprotein CIII mRNA in human hepatocytes; however, very little change in FACO activity or mRNA was observed.

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Nuclear Receptors Have Distinct Affinities for Coactivators: Characterization by Fluorescence Resonance Energy Transfer

Cited by 119 publications

References 43 publications

Identification of a potent synthetic FXR agonist with an unexpected mode of binding and activation

Identification of a potent synthetic FXR agonist with an unexpected mode of binding and activation

An intramolecular folding sensor for imaging estrogen receptor–ligand interactions

Differential Gene Regulation in Human Versus Rodent Hepatocytes by Peroxisome Proliferator-activated Receptor (PPAR) α

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