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WO2000025134A1 - Procede d'identification de ligands de recepteurs nucleaires - Google Patents

Procede d'identification de ligands de recepteurs nucleaires Download PDF

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Publication number
WO2000025134A1
WO2000025134A1 PCT/US1999/024956 US9924956W WO0025134A1 WO 2000025134 A1 WO2000025134 A1 WO 2000025134A1 US 9924956 W US9924956 W US 9924956W WO 0025134 A1 WO0025134 A1 WO 0025134A1
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Prior art keywords
nuclear receptor
component
marking
binding domain
interaction
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Steven Gerard Blanchard
Derek J. Parks
Julie Beth Stimmel
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Glaxo Group Ltd
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Glaxo Group Ltd
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Priority to AU12290/00A priority Critical patent/AU1229000A/en
Priority to EP99971090A priority patent/EP1137940A4/fr
Priority to JP2000578657A priority patent/JP2002528721A/ja
Publication of WO2000025134A1 publication Critical patent/WO2000025134A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins

Definitions

  • RXR is an orphan nuclear receptor initially identified from a rat liver cDNA library (8) that is most closely related to the insect ecdysone receptor.
  • the ligand binding domain of the receptor was cloned and expressed in support of an effort to develop a robust assay to identify a novel ligand.
  • the availability of ligands for FXR will aid in the elucidation of the physiological role of this receptor.
  • the information gained will further increase understanding of nuclear receptors as a target class.
  • the present invention includes a generic approach to assay development for nuclear receptors, utilizing purified ligand binding domains.
  • the concept of generic assay development has been extended to develop in vitro assays that detect ligand binding by monitoring ligand induced changes in receptor heterodimerization.
  • This approach has been demonstrated using both scintillation proximity and homogenous time-resolved fluorimetry (HTRF) in the accompanying examples but it is not restricted to these methods. Other marking and measuring techniques may also be used.
  • HTRF time-resolved fluorimetry
  • Another aspect of the invention is a new nuclear receptor-peptide assay for identifying ligands.
  • This assay utilizes fluorescence resonance energy transfer (FRET) and was used to test whether putative ligands bound to FXR.
  • FRET fluorescence resonance energy transfer
  • the FRET assay is based upon the principle that ligands induce conformational changes in nuclear receptors that facilitate interactions with coactivator proteins required for transcriptional activation.
  • FRET a fluorescent donor molecule transfers energy via a non-radiative dipole-dipole interaction to an acceptor molecule (which is usually a fluorescent molecule).
  • FRET is a standard spectroscopic technique for measuring distances in the 10-70A range.
  • Fig. 1 As shown in Fig 1 , ligand binding to LXR ⁇ measured by modulation of LXR ⁇ :RXR heterodimer formation.
  • Fig.2. shows ligand binding to FXR measured by modulation of
  • a method for the rapid and simple determination of a ligand for a nuclear receptor which comprises contacting a component to be tested with an isolated nuclear receptor ligand binding domain which may be associated with a marking component, and a dimerization partner for the nuclear receptor ligand binding domain which is also associated with a marker; and measuring the interaction between the marking components to determine whether the component to be tested modifies heterodimerization.
  • markers may be used in the process of the present invention such as radioactive markers.
  • the marker could also be a fluorescent dye. When the marker is radioactive, scintillation proximity may be used to measure the marker. When the marker used is a fluorescent dye, homogenous time-resolved fluorimetry may be used to detect the marker.
  • Other known marking and measuring techniques may be used depending on the marker. However, the markers need to be in close proximity to indicate heterodimerization. That is, to indicate that the component to be tested functions as a ligand for the dimerization pair.
  • RXRs such as RXR ⁇ , RXR ⁇ , and RXRy
  • PPARs such as PPAR ⁇ , PPARy, and PPAR ⁇
  • LXR ⁇ , ⁇ , ER ⁇ , ER ⁇ , CAR ⁇ , HNF4 ⁇ , ⁇ , ⁇ , NGFIB ⁇ , ⁇ , ⁇ , PXR, PHR, EAR-1 , EAR-2, TR, RAR, and ERRs are examples of nuclear receptors that may be used as the dimerization partners and/or the nuclear receptors in the process of the present invention.
  • RXR ⁇ is exemplified in some of the examples. Any nuclear receptor can be selected for use in an assay. More dimerization partners may be known or later discovered which can readily be utilized in the assay. It is preferable that the dimerization partners and the nuclear receptor ligand binding domains are recombinant proteins and preferably are bacterial expressed.
  • This method for the rapid determination of a ligand for a nuclear receptor comprises contacting a component to be tested with an isolated nuclear receptor ligand binding domain which is associated with a first marking component, and a heterodimeric partner for the nuclear receptor ligand binding domain associated with a second marking component, and measuring the interaction between the marking components to determine whether the component to be tested modifies hetero-dimerization.
  • the first marking component may be a radioactive marker and the second marking component (or second marker) may be a SPA bead.
  • the interaction of the markers in this case is determined by scintillation proximity.
  • the first marking component may be a first fluorescent dye emitting at an emitting wavelength which excites the second marking component which may be a second fluorescent dye.
  • the interaction of the markers in this case is determined by homogenous time-resolved fluorimetry.
  • the interaction of the marking components in either case is measured by comparing a signal produced by a combination of the heterodimeric partner, the isolated nuclear receptor binding domain and the component to be tested with a signal produced by a combination of the heterodimeric partner and the isolated nuclear receptor ligand binding domain in the absence of the component to be tested.
  • Liver X receptor alpha (LXR ⁇ ) is an orphan nuclear receptor initially identified from a rat liver cDNA library (1 ). Human LXR ⁇ (2) and LXR ⁇ (3) have also been identified. The ligand binding domains of these receptors were cloned and expressed in support of an effort to develop a robust assay to identify a novel ligand. Oxysterols, including 24(S),25- epoxycholesterol have been identified as weak activators for these receptors (4,5). The availability of more potent and selective ligands for the LXRs may aid in the elucidation of the physiological role(s) of these receptors. In addition, the information gained will further increase understanding of nuclear receptors as a target class.
  • LXR ⁇ nuclear receptor Liver X Receptor beta
  • the method measures the ability of putative ligands to mediate the heterodimerization between the purified bacterial expressed LXR ⁇ , and RXR ⁇ , ligand binding domains (LBD). Detection of the associated LBD's are measured by time resolved fluorimetry (TRF).
  • TRF time resolved fluorimetry
  • the purified LBD of LXR ⁇ is labeled with biotin then mixed with stoichiometric amounts of europium labeled streptavidin (Wallac Inc).
  • the purified LBD of RXR ⁇ is labeled with CY5 Tm .
  • Equimolar amounts of each modified LBD are mixed together and allowed to equilibrate for at least one hour prior to the addition to either variable or constant concentrations of the sample for which the affinity is to be determined.
  • the time-resolved fluorescent signal is quantitated using a fluorescent plate reader.
  • the affinity of the test compound is estimated from a plot of fluorescence versus concentration of test compound added. A basal level of LXR ⁇ :RXR ⁇ heterodimer formation is observed in the absence of added ligand. Ligands that promote heterodimer formation induce a concentration-dependent increase in time-resolved fluorescent signal.
  • LXR ⁇ LBD Human LXR ⁇ Ligand Binding Domain
  • Genbank accession number U 07132, amino acids 185-461 was expressed in E.coli strain BL21 (DE3) as an amino-terminal polyhistidine tagged fusion protein. Expression was under the control of an IPTG inducible T7 promoter. DNA encoding this recombinant protein and a modified polyhistidine tag was subcloned into the expression vector pRSETa (Invitrogen).
  • This lysate was loaded onto a column (6 x 8 cm) packed with Sepharose (Ni ++ charged) Chelation resin (Pharmacia) and pre-equilibrated with TBS pH 8.5/ 50mM imidazole. After washing to baseline absorbance with equilibration buffer, the column was developed with a linear gradient of 50 to 275 mM imidazole in TBS, pH 8.5. Column fractions were pooled and dialyzed against TBS, pH 8.5, containing 5% 1 ,2-propanediol, 5mM DTT and 0.5mM EDTA.
  • the protein sample was concentrated using Centri-prep 10K (Amicon) and subjected to size exclusion, using a column (3 x 90 cm) packed with Sepharose S-75 resin (Pharmacia) pre-equilibrated with TBS, pH 8.5, containing 5% 1 ,2-propanediol, 5mM DTT and 0.5mM EDTA.
  • Biotinylation of LXR ⁇ was concentrated using Centri-prep 10K (Amicon) and subjected to size exclusion, using a column (3 x 90 cm) packed with Sepharose S-75 resin (Pharmacia) pre-equilibrated with TBS, pH 8.5, containing 5% 1 ,2-propanediol, 5mM DTT and 0.5mM EDTA.
  • LXR ⁇ LBD was desalted/buffer exchanged using PD-10 gel filtration columns into PBS [100mM Na Phosphate, pH 7.2, 150 mM NaCI].
  • LXR ⁇ LBD was diluted to approximately 10 ⁇ M in PBS and five-fold molar excess of NHS-LC-Biotin (Pierce) was added in a minimal volume of PBS. This solution was incubated with gentle mixing for 30 minutes at room temperature. The biotinylation modification reaction was stopped by the addition of 2000x molar excess of Tris-HCI, pH 8.
  • the modified LXR ⁇ LBD was dialyzed against 4 buffer changes, each of at least 50 volumes, PBS containing 5mM DTT 2mM EDTA and 2% sucrose.
  • the biotinylated LXR ⁇ LBD was subjected to mass spectrometric analysis to reveal the extent of modification by the biotinylation reagent. In general, approximately 95% of the protein had at least a single site of biotinylation; and the overall extent of biotinylation followed a normal distribution of multiple sites, ranging from one to nine.
  • RXR ⁇ LBD Human Retinoid X Receptor alpha Ligand Binding Domains RXR-alpha LBD (amino acids 225-462) was expressed in E. coli strain BL21 (DE3) as an amino-terminal polyHistidine tagged fusion protein. Expression was under the control of an IPTG inducible T7 promoter. DNA encoding this recombinant protein and a modified histidine tag was subcloned into the expression vector pRSETa (Invitrogen). The sequence used in the construction of RXR-alpha LBD was derived from Genbank accession number X52773.
  • This lysate was loaded onto a column (3 x 8 cm) packed with Sepharose [Ni ++ charged] Chelation resin (Pharmacia) and pre-equilibrated with TBS, pH 7.2, containing 50mM imidazole. After washing to baseline absorbance, the column was developed with a linear gradient of 50 to 500 mM imidazole in TBS, pH 7.2. Column fractions were pooled and dialyzed against TBS, pH 7.2, containing 5 mM DTT and .5mM EDTA. After dialysis the sample was concentrated using
  • RXR ⁇ LBD Purified RXR ⁇ LBD was diluted to approximately 10 ⁇ M in PBS and approximately five-fold molar excess of Cy5TM monofunctional reactive dye [NHS ester] (Amersham Life Sciences) was added in a minimal volume of PBS. This solution was incubated in the dark with mixing for 30 minutes at ambient room temperature (approximately 23°C). The modification reaction was stopped by the addition of an excess of Tris-HCI, pH 8. Fluorescent dye modified RXR ⁇ LBD was dialyzed at 4°C, with minimal exposure to light, against 4 buffer changes, each of at least 50 volumes, PBS containing 5mM DTT, 2mM EDTA, and 2% (w/v) sucrose. Aliquots were frozen on dry ice and stored at -80°C.
  • Assay Buffer 50 mM KCI, 0.1 mg/mL BSA, 10 mM DTT and 50 mM Tris (pH 8)
  • the stock buffer is made by dissolving 2.853g Tris base, 4.167 g Tris hydrochloride, 3.73 g KCI, and 0.1 g fatty acid free bovine serum albumin, in 1 L of deionized water. The pH is checked and adjusted to 8.0, if necessary, before adjusting to final volume. 0.154 g of solid DTT is added per 100 mL of buffer just before the start of an experiment.
  • Each well to be assayed contained a previously prepared solution of CY5 TM RXR ⁇ and Europium labeled LXR and the desired concentration of test samples or controls (100 ⁇ L total volume). In general, the total volume was held constant by varying the concentration and volume of premixed receptors to compensate for any changes in the volume of a particular set of samples. The plates were incubated for at least 2 hours at room temperature and the fluorescent signal determined in a Wallac Victor Multilabel Fluorescence Reader. Data Reduction:
  • F sample is the signal observed in a particular sample well
  • F tota is the signal observed in the presence of control inhibitor
  • is the count rate observed in the presence of no ligand.
  • were averages of the corresponding control wells included on every plate.
  • the data were first normalized to % of control using eq. (1 ).
  • a plot of C L the % of control observed at ligand concentration L, versus ligand concentration, L was constructed.
  • the data were fit to equation (2) to obtain best-fit parameters for the EC 50 , F max and ' basal-
  • F max the maximal amplitude observed at saturating ligand concentrations, can be either a positive or negative value.
  • the sign of this parameter indicates whether a particular test compound favors binding to the LXR:RXR complex (positive F max ) or to either of the component receptors in a non-heterodimeric state (negative F max ).
  • both F max and F basa) are expressed in units of % of a standard compound.
  • This example describes the use of ligand mediated heterodimerization to quantify ligand binding to the nuclear receptor Farnasoid X Receptor (FXR).
  • the method measures the ability of putative ligands to mediate the heterodimerization between the purified bacterial expressed FXR and RXR ⁇ ligand binding domains (LBD). Detection of the associated LBD's are measured by time resolved fluorimetry (TRF).
  • TRF time resolved fluorimetry
  • the purified LBD of FXR is labeled with biotin then mixed with stoichiometric amounts of europium labeled streptavidin (Wallac Inc).
  • the purified LBD of RXR ⁇ is labeled with CY5TM Equimolar amounts of each modified LBD are mixed together and allowed to equilibrate for at least 1 hour prior to the addition to either variable or constant concentrations of the sample for which the affinity is to be determined. After equilibration, the time-resolved fluorescent signal is quantitated using a fluorescent plate reader.
  • the affinity of the test compound is estimated from a plot of fluorescence versus concentration of test compound added.
  • FXR ⁇ LBD Human Famasoid X Receptor alpha Ligand Binding Domain Human FXR ⁇ Ligand Binding Domain
  • cell paste (equivalent to 2-3 liters of the fermentation batch) was resuspended in 200-250 mL TBS, pH 7.2 (25mM Tris, 150 mM NaCI). Cells were lysed by passing 3 times through a French Press and cell debris was removed by centrifugation (30 minutes, 20,000g, 4°C). The cleared supernatant was filtered through course pre-filters, and TBS, pH 7.2, containing 500 mM imidazole was added to obtain a final imidazole concentration of 50mM.
  • This lysate was loaded onto a column (6 x 8 cm) packed with Sepharose [Ni ++ charged] Chelation resin (Pharmacia) and pre- equilibrated with TBS pH 7.2/ 50mM imidazole. After washing to baseline absorbance with equilibration buffer, the column was washed with one column volume of TBS pH 7.2 containing 90mM imidazole. FXR ⁇ LBD was eluted directly with 365 mM imidazole. Column fractions were pooled and dialyzed against TBS, pH 7.2, containing 0.5mM EDTA and 5mM DTT.
  • the dialyzed protein sample was concentrated using Centri-prep 10 K (Amicon) and subjected to size exclusion, using a column (3 x 90 cm) packed with Sepharose S-75 resin (Pharmacia) pre-equilibrated with TBS, pH 7.2, containing 0.5mM EDTA and 5mM DTT.
  • Biotinylation of FXR Purified FXR ⁇ LBD was desalted/buffer exchanged using PD-10 gel filtration columns into PBS [100mM NaPhosphate, pH 7.2, 150mM NaCI].
  • FXR ⁇ LBD was diluted to approximately 10 ⁇ M in PBS and five-fold molar excess of NHS-LC-Biotin (Pierce) was added in a minimal volume of PBS. This solution was incubated with gentle mixing for 30 minutes at room temperature. The biotinylation modification reaction was stopped by the addition of 2000x molar excess of Tris-HCI, pH 8. The modified FXR ⁇ LBD was dialyzed against 4 buffer changes, each of at least 50 volumes, PBS containing 5mM DTT, 2mM EDTA and 2% sucrose. The biotinylated FXR ⁇ LBD was subjected to mass spectrometric analysis to reveal the extent of modification by the biotinylation reagent. In general, approximately 95% of the protein had at least a single site of biotinylation; and the overall extent of biotinylation followed a normal distribution of multiple sites, ranging from one to nine.
  • RXR ⁇ LBD was prepared and labeled with CY5 Tm in accordance with the procedures set forth in example 1.
  • Assay Buffer 50 mM KCI, 0. 1 mg/mL BSA, 10 mM DTT, and 50 mM Tris
  • the stock buffer is made by dissolving 2.853g Tris base, 4.167 g Tris hydrochloride, 3.73 g KCI, and 0. 1 g fatty acid free bovine serum albumin, in 1 L of deionized water. The pH is checked and adjusted to 8.0, if necessary, before adjusting to final volume. 0.154 g of solid DTT is added per 100 mL of buffer just before the start of an experiment.
  • 96 well plates polypropylene for intermediate dilutions (Costar #3794) and either a clear-bottomed white SPA plates (Costar #3632) or a black Polyfiltronics plate (UP350 PSB) for assays.
  • FRET fluorescence resonance energy transfer
  • the ability of ligand to induce changes in the degree of this complex was then used as a basis for an inventive assay for the discovery of nuclear receptor ligands. Certain sequences of the cofactor may only be required to interact with the nuclear receptor.
  • SRC-1 and CBP were synthesized and tested in HTRF and Biacore to determine the best sequences to use.
  • the peptide, CPSSHSSLTERHKILHRLLQEGSPS-CONH 2 (SEQ ID NO.:1 ), i.e., SRC-1 (LCD2,676-700) was used in screening efforts with FXR and this forms a further aspect of this invention.
  • Coactivator proteins interact with nuclear receptors in a ligand-dependent manner and augment transcription (9).
  • a short amphipathic ⁇ - helical domain that includes the amino acid motif LXXLL (L is Leu and X is any other amino acid) serves as the interaction interface between these coactivator molecules and the ligand-dependent activation function (AF-2) located in the COOH-terminus of the nuclear receptor LBD (10).
  • FRET fluorescence resonance energy transfer
  • Human FXR LBD was prepared and fluorescently labeled as described in Example 2.
  • the LBD of human FXR was labeled with the fluorophore allophycocyanin and incubated with a peptide derived from the second LXXLL (SEQ ID NO.:1 ) motif of SRC1 (amino acids 676 to 700) that was labeled with europium chelate.
  • the FRET ligand-sensing assay was performed by incubating 10 nM of the biotinylated FXR LBD that was labeled with streptavidin-conjugated allophycocyanin (Molecular Probes) and 10 nM of the SRC1 peptide [amino acids 676 to 700, 5'-biotin-
  • Biotinylated SRC-1 (LDC2,676-700):Biotin-CPSSHSSLTERHKILHRLL- QEGSPS-CONH 2 (SynPEP) Assay Buffer: 50 mM KCI, 2mM EDTA, 0.1 mg/mL BSA, 10 mM DTT, and 50 mM Tris (pH 8).
  • the stock buffer is made by dissolving 2.853g Tris base, 4.167 g Tris hydrochloride, 3.73 g KCI, 0.74 g EDTA (disodium salt, dihydrate) and 0.1 g fatty acid free bovine serum albumin, in 1 L of deionized water. The pH is checked and adjusted to 8.0, if necessary, before adjusting to final volume. 0.154 g of solid DTT is added per 100 mL of buffer just before the start of an experiment. BSA, fatty acid free DTT
  • 96 well plates polypropylene for intermediate dilutions (Costar #3794) and either a clear-bottomed white SPA plates (Costar #3632) or a black Polyfiltronics plate (UP350 PSB) for assays.
  • Ligands increased the interaction between FXR and the SRC1 peptide as determined with time-resolved FRET. Dose response analysis showed that the ligands increased the amount of SRC1 peptide bound to the FXR
  • nuclear receptor coactivator peptide means a peptide whose affinity for the receptor is changed in the presence of ligand and which has a LXXLL motif.
  • coactivator peptides useful for ligand identification by this method that have been demonstrated to interact with FXR include SRC-1 and those listed below:
  • sequences of peptides 1 and 4 appear in a number of coactivators, hence the multiple names.
  • the "B” in the sequences stands for biotinylated, which is a modification that allows attachment of the peptide during the analysis.
  • the abbreviations used in the examples are included below.

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Abstract

L'invention concerne de nouveaux procédés d'identification de peptides de récepteurs-coactivateurs nucléaires et d'hétérodimères de récepteurs nucléaires, ce qui permet d'identifier des ligands de récepteurs nucléaires au moyen de techniques de proximité de scintillation et de transfert d'énergie de résonance de fluorescence (FRET).
PCT/US1999/024956 1998-10-23 1999-10-22 Procede d'identification de ligands de recepteurs nucleaires Ceased WO2000025134A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU12290/00A AU1229000A (en) 1998-10-23 1999-10-22 Assays for ligands for nuclear receptors
EP99971090A EP1137940A4 (fr) 1998-10-23 1999-10-22 Procede d'identification de ligands de recepteurs nucleaires
JP2000578657A JP2002528721A (ja) 1998-10-23 1999-10-22 核内受容体のリガンド用のアッセイ

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US10539098P 1998-10-23 1998-10-23
US13509798P 1998-12-23 1998-12-23
US60/135,097 1998-12-23
US13483699P 1999-05-19 1999-05-19
US60/105,390 1999-05-19
US60/134,836 1999-05-19

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6639078B1 (en) 1998-12-23 2003-10-28 Smithkline Beecham Corporation Assays for ligands for nuclear receptors
WO2002077229A3 (fr) * 2001-03-01 2003-12-04 Lion Bioscience Ag Nouveaux cofacteurs du recepteur alpha x du foie et techniques d'utilisation
US6987121B2 (en) 2002-04-25 2006-01-17 Smithkline Beecham Corporation Compositions and methods for hepatoprotection and treatment of cholestasis
US7115640B2 (en) 2001-12-21 2006-10-03 X-Ceptor Therapeutics, Inc. Heterocyclic modulators of nuclear receptors
US7138390B2 (en) 2001-03-12 2006-11-21 Intercept Pharmaceuticals Steroids as agonists for FXR
WO2010004052A3 (fr) * 2008-07-11 2010-04-15 Medizinische Universität Innsbruck Agonistes de nr2f6 destinés à l'immunosuppression
US7994352B2 (en) 2005-05-19 2011-08-09 Intercept Pharmaceuticals, Inc. Process for preparing 3a(β)-7a(β)-dihydroxy-6a(β)-alkyl-5β-cholanic acid
US7998986B2 (en) 2001-12-21 2011-08-16 Exelixis Patent Company Llc Modulators of LXR
US8013001B2 (en) 2001-12-21 2011-09-06 Exelixis, Inc. Modulators of LXR
US8114862B2 (en) 2008-11-19 2012-02-14 Intercept Pharmaceuticals, Inc. TGR5 modulators and methods of use thereof
US8410083B2 (en) 2007-01-19 2013-04-02 Intercept Pharmaceuticals, Inc. 23-substituted bile acids as TGR5 modulators and methods of use thereof
US8568997B2 (en) 2006-11-10 2013-10-29 Dimerix Bioscience Pty Ltd. Detection system and uses therefor
WO2014028749A2 (fr) 2012-08-15 2014-02-20 Boston Medical Center Corporation Production de globules rouges et de plaquettes à partir de cellules souches
US8796249B2 (en) 2008-07-30 2014-08-05 Intercept Pharmaceuticals, Inc. TGR5 modulators and methods of use thereof
US9238673B2 (en) 2012-06-19 2016-01-19 Intercept Pharmaceuticals, Inc. Preparation and uses of obeticholic acid
US9314450B2 (en) 2011-01-11 2016-04-19 Dimerix Bioscience Pty Ltd. Combination therapy
US9498484B2 (en) 2004-03-12 2016-11-22 Intercept Pharmaceuticals, Inc. Treatment of fibrosis using FXR ligands
US9982008B2 (en) 2012-06-19 2018-05-29 Intercept Pharmaceuticals, Inc. Preparation and uses of obeticholic acid
US10987362B2 (en) 2004-03-12 2021-04-27 Intercept Pharmaceuticals, Inc. Treatment of fibrosis using FXR ligands

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JP4803976B2 (ja) * 2003-07-09 2011-10-26 独立行政法人科学技術振興機構 細胞内ｉｐ３測定用分子センサー

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4568649A (en) * 1983-02-22 1986-02-04 Immunex Corporation Immediate ligand detection assay
US5670360A (en) * 1992-03-25 1997-09-23 Novo Nordisk A/S Mammalian receptors for glucagon-like-peptide-1 (GLP-1), corresponding DNA and recombinant expression systems, and screening assays for GLP-1 agonists and enhancers
US5776699A (en) * 1995-09-01 1998-07-07 Allergan, Inc. Method of identifying negative hormone and/or antagonist activities

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6005086A (en) * 1995-01-13 1999-12-21 The Salk Institute For Biological Studies Farnesoid activated receptor polypeptides, and nucleic acid encoding the same
EP1019059A4 (fr) * 1997-01-24 2004-01-14 Univ California UTILISATION D'ACTIVATEURS DE FXR, PPARalpha ET LXRalpha DANS LE BUT DE RESTAURER LA FONCTION DE BARRIERE, DE FAVORISER LA DIFFERENCIATION EPIDERMIQUE ET D'INHIBER LA PROLIFERATION EPIDERMIQUE
JP2001519525A (ja) * 1997-10-07 2001-10-23 メルク エンド カムパニー インコーポレーテッド Ｆｒｅｔを用いる核内受容体リガンドに関するアッセイ
US6410245B1 (en) * 1998-04-01 2002-06-25 Affymax, Inc. Compositions and methods for detecting ligand-dependent nuclear receptor and coactivator interactions

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4568649A (en) * 1983-02-22 1986-02-04 Immunex Corporation Immediate ligand detection assay
US5670360A (en) * 1992-03-25 1997-09-23 Novo Nordisk A/S Mammalian receptors for glucagon-like-peptide-1 (GLP-1), corresponding DNA and recombinant expression systems, and screening assays for GLP-1 agonists and enhancers
US5776699A (en) * 1995-09-01 1998-07-07 Allergan, Inc. Method of identifying negative hormone and/or antagonist activities

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MANGELSDORF D.J.: "The RXR Heterodimers and Orphan Receptors", CELL, vol. 83, 15 December 1995 (1995-12-15), pages 841 - 850, XP002922898 *
MUKHERJEE R.: "Sensitization of Diabetic and Obese Mice to Insulin by Retinoid X Receptor Agaonists", NATURE, vol. 386, 27 March 1997 (1997-03-27), pages 407 - 410, XP002922899 *
See also references of EP1137940A4 *

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WO2002077229A3 (fr) * 2001-03-01 2003-12-04 Lion Bioscience Ag Nouveaux cofacteurs du recepteur alpha x du foie et techniques d'utilisation
US8058267B2 (en) 2001-03-12 2011-11-15 Intercept Pharmaceuticals, Inc. Steroids as agonists for FXR
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US8013001B2 (en) 2001-12-21 2011-09-06 Exelixis, Inc. Modulators of LXR
US7998986B2 (en) 2001-12-21 2011-08-16 Exelixis Patent Company Llc Modulators of LXR
US7115640B2 (en) 2001-12-21 2006-10-03 X-Ceptor Therapeutics, Inc. Heterocyclic modulators of nuclear receptors
US6987121B2 (en) 2002-04-25 2006-01-17 Smithkline Beecham Corporation Compositions and methods for hepatoprotection and treatment of cholestasis
US10258633B2 (en) 2004-03-12 2019-04-16 Intercept Pharmaceuticals, Inc. Treatment of fibrosis using FXR ligands
US9498484B2 (en) 2004-03-12 2016-11-22 Intercept Pharmaceuticals, Inc. Treatment of fibrosis using FXR ligands
US10987362B2 (en) 2004-03-12 2021-04-27 Intercept Pharmaceuticals, Inc. Treatment of fibrosis using FXR ligands
US7994352B2 (en) 2005-05-19 2011-08-09 Intercept Pharmaceuticals, Inc. Process for preparing 3a(β)-7a(β)-dihydroxy-6a(β)-alkyl-5β-cholanic acid
US8568997B2 (en) 2006-11-10 2013-10-29 Dimerix Bioscience Pty Ltd. Detection system and uses therefor
US8410083B2 (en) 2007-01-19 2013-04-02 Intercept Pharmaceuticals, Inc. 23-substituted bile acids as TGR5 modulators and methods of use thereof
US9243027B2 (en) 2007-01-19 2016-01-26 Intercept Pharmaceuticals, Inc. TGR5 modulators and methods of use thereof
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US8796249B2 (en) 2008-07-30 2014-08-05 Intercept Pharmaceuticals, Inc. TGR5 modulators and methods of use thereof
US9540414B2 (en) 2008-07-30 2017-01-10 Intercept Pharmaceuticals, Inc. TGR5 modulators and methods of use thereof
US8445472B2 (en) 2008-11-19 2013-05-21 Intercept Pharmaceuticals, Inc. TGR5 modulators and methods of use thereof
US9650409B2 (en) 2008-11-19 2017-05-16 Intercept Pharmaceuticals, Inc. TGR5 modulators and methods of use thereof
US8114862B2 (en) 2008-11-19 2012-02-14 Intercept Pharmaceuticals, Inc. TGR5 modulators and methods of use thereof
US10058555B2 (en) 2011-01-11 2018-08-28 Dimerix Bioscience Pty Ltd. Combination therapy
US9314450B2 (en) 2011-01-11 2016-04-19 Dimerix Bioscience Pty Ltd. Combination therapy
US10525038B2 (en) 2011-01-11 2020-01-07 Dimerix Bioscience Pty Ltd. Combination therapy
US11382896B2 (en) 2011-01-11 2022-07-12 Dimerix Bioscience Pty Ltd. Method for treating inflammatory disorders
US12083102B2 (en) 2011-01-11 2024-09-10 Dimerix Bioscience Pty Ltd. Method for treating inflammatory disorders
US10047117B2 (en) 2012-06-19 2018-08-14 Intercept Pharmaceuticals, Inc. Preparation and uses of obeticholic acid
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EP1137940A1 (fr) 2001-10-04

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