JP2002528721A - Assays for nuclear receptor ligands - Google Patents

Abstract

  (57) [Summary] The present invention provides a novel nuclear receptor heterodimer and nuclear receptor-coactivator cofactor peptide assay for identifying nuclear receptor ligands using scintillation access and fluorescence resonance energy transfer (FRET). Including.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application by under 35 USC 119 (e) section filed October 23, 1998 U.S. Provisional Application No. 60 / 150,390; Dec. 23, 1998 No. 60 / 135,097 filed on May 19, 1999; and No. 60 / 134,836 filed on May 19, 1999.

[0002] the identification of ligands of background orphan nuclear receptors invention, traditionally, (if several ligands are known) reporter gene assay or cell-based
Any of the competitive binding assays have been used. Both transactivation and ligand binding assays, typically used to determine the activity of a candidate nuclear receptor activator, assess the effect of a test ligand on the isolated receptor. However, available evidence indicates that the majority of (1,2,8) known orphan nuclear receptors, including LXRs and FXR, have a common partner in dimer formation, RX
R (shown in 6) as a heterodimeric receptor complex. Recently, it has been demonstrated that certain synthetic RXR ligands exhibit preferential activation of the RXR heterodimer, and that this preference is determined by the receptor partner that binds to RXR (7). These findings suggested that binding of the ligand to the nuclear receptor, and therefore subsequent activation of the nuclear receptor, could be regulated by the state of receptor dimerization. In addition, microscopic reversibility suggests that ligand binding should modulate the affinity of the heterodimer. Therefore, the ability of a ligand to induce a change in the degree of receptor dimerization could be used as the basis for a novel assay to discover nuclear receptor ligands.

[0003] FXR was first identified from a rat liver cDNA library (8),
It is the orphan nuclear receptor most closely related to the insect ecdysone receptor. The ligand binding domain of the receptor has been cloned and expressed to support efforts to develop robust assays to identify new ligands. F
The availability of a ligand for XR will help elucidate the physiological role of this receptor. In addition, the information obtained will further increase the understanding of nuclear receptors as target groups.

[0004] For both FXR and LXR, basal levels of nuclear receptor-RXRα heterodimer formation are observed in the absence of added ligand. Ligands that promote heterodimer formation induce a concentration-dependent increase in the time-resolved fluorescent signal. Compounds that bind to the same degree to both the monomeric receptor and the receptor-RXRα heterodimer are not expected to give a change in signal, whereas ligands that bind preferentially to the monomeric receptor are: It would be expected to induce a concentration dependent decrease in the observed signal.

SUMMARY OF THE INVENTION The present invention encompasses a general approach to assay development for nuclear receptors using purified ligand binding domains. The general assay development concept has been extended to the development of in vitro assays that detect ligand binding by monitoring the changes in receptor heterodimerization induced by the ligand.
This approach, in the attached example, provides a scintillation approach.
proximity) and homogeneous time-resolved fluorometry (HTRF; homogeneous teime-resolve)
d fluorescence), but is not limited to these methods.
Other marking and measurement techniques may be used. However, the use of a scintillation approach or HTRF offers a simpler and more practical methodology.

[0006] Another aspect of the invention is a novel nuclear receptor-peptide assay for identifying ligands. The assay uses fluorescence resonance energy transfer (FRET; fluorescen).
ce resonance energy transfer) and was used to test whether the candidate ligand binds to FXR. The FRET assay is based on the principle that a ligand induces a conformational change in a nuclear receptor that facilitates the interaction with coactivator proteins required for transcriptional activation.

[0007] In FRET, a fluorescent donor molecule transfers energy to an acceptor molecule, which is typically a fluorescent molecule, via a non-radioactive dipole-dipole interaction. FRET is a standard spectroscopic technique for measuring distances in the range of 10-70 °. When an energy transfer dependent on the distance R- ⁶ between the donor and the acceptor occurs, the fluorescence of the donor decreases and the fluorescence of the acceptor increases, ie sensitizes. FRET
Is frequently used in both polymer science and structural biology, and in recent years DNA,
It has been used to study macromolecular complexes of RNA and proteins. In addition, Mathis used a europium cryptate with a polychromatic allophycocanin to achieve a very large R ₀ of 90 ° (Mathis, G. (1993) Clin. Chem. 39, 1953-1959). ).

[0008] The assay of the present invention is a rapid and simple method for determining the ligand of a nuclear receptor, comprising a ligand binding domain of an isolated nuclear receptor, which may be accompanied by a marking component, and a marker. Contacting the component to be tested with a partner in the dimerization of the nuclear receptor ligand binding domain; measuring the interaction with the marking component to convert the component to be tested to heterodimerization. Deciding whether or not to qualify. The method of the present invention includes:
Various known markers, such as radioactive markers, may be used. The marker is
It may be a fluorescent dye. If the marker is radioactive, a scintillation approach may be used to measure the marker. If the marker used is a fluorescent dye, a homogeneous time-resolved fluorimetric method can be used to detect the marker. Other known marking and measurement techniques may be used depending on the marker. However, the markers need to be in proximity to indicate heterodimerization. That is, to show that the component to be tested functions as a ligand for the dimerization pair.

RXRs, PPARα, PPAR such as RXRα, RXRβ, and RXRγ
γ, and PPARs such as PPARδ, LXRα, β, ERa, ERβ, CA
Rα, HNF4 α, β, γ, NGFIB α, β, γ, PXR, PHR, EA
R-1, EAR-2, TR, RAR, and ERRs are examples of nuclear receptors that can be used as dimerization partners and / or as nuclear receptors in the methods of the invention. RXRa is illustrated in several examples. Any nuclear receptor can be selected for use in the assay. Additional dimerization partners readily available in the assay may be known or later discovered. The dimerization partner and the nuclear receptor ligand binding domain are preferably recombinant proteins and are preferably expressed in bacteria.

[0010] The method for rapidly determining the ligand of a nuclear receptor comprises the steps of:
Contacting an isolated nuclear receptor ligand binding domain with a first marking component and a heterodimerizing partner of a nuclear receptor ligand binding domain with a second marking component; Measuring the effect to determine whether said component to be tested modifies heterodimerization.

The first marking component can be a radioactive marker, and the second marking component (ie, the second marker) can be SPA beads. The interaction of the markers in this case is determined by the scintillation approach. Alternatively, the first marking component can be a first fluorescent dye that emits at a fluorescent wavelength that excites the second marking component, which can be a second fluorescent dye. The interaction of the markers in this case is determined by homogeneous time-resolved fluorometry.

[0012] In each case, the interaction of the marking components involves the testing of the signal generated by the combination of the heterodimer partner, the isolated nuclear receptor binding domain, and the component to be tested. It is determined by comparing the signal generated by the combination of the heterodimer partner and the isolated nuclear receptor ligand binding domain in the absence of the component to be processed.

Example 1 This example describes the use of ligand-mediated heterodimerization to quantify binding of a ligand to the nuclear receptor, liver X receptor (LXR).

Liver X receptor alpha (LXRα) is an orphan nuclear receptor originally identified from a rat liver cDNA library (1). Human LXRα (2) and LXRβ (3) have also been identified. The ligand binding domains of these receptors have been cloned and expressed to support efforts to develop powerful assays to identify new ligands. Oxysterols, including 24 (S), 25-epoxycholesterol, have been identified as weak activators of these receptors (4,5). The availability of more potent and selective ligands for LXRs will help elucidate the physiological role of these receptors. In addition, the information obtained will further increase the understanding of nuclear receptors as target groups.

This example describes the use of ligand-mediated heterodimerization to quantify ligand binding to the nuclear receptor, liver X receptor beta (LXRβ). The method comprises the steps of: (a) producing a purified LXR expressed by a bacterium;
The ability of β with RXRα, the ligand binding domain (LBD), to mediate heterodimerization is measured. Detection of associated LBDs is performed by time-resolved fluorometry (TRF)
Measure using After labeling the purified LBD of LXRβ with biotin, it is mixed with a stoichiometric amount of europium-labeled streptavidin (Wallac Inc). RXRα
The purified LBD is labeled with ^{CY5 TM.} Equimolar amounts of each modified LBD are mixed and allowed to equilibrate at least one hour before addition to the sample in which a variable or constant concentration of affinity is to be determined. After equilibration, the time-resolved fluorescent signal is quantified using a fluorescent plate reader. From the plot of fluorescence versus the concentration of test compound added, the affinity of the test compound is estimated.

A basal level of LXRβ: LXRα heterodimer formation is observed in the absence of added ligand. Ligands that promote heterodimer formation are:
Induces a concentration-dependent increase in the time-resolved fluorescent signal. Monomer LXRβ
And LXRβ: RXRα Heterodimers are expected to give no change in signal, whereas ligands that bind preferentially to the monomeric receptor are likely to bind to the observed signal. It would be expected to induce a concentration dependent decrease.

Methods and Materials Pre-preparation: The human LXRβ ligand binding domain (LXRβ LBD; Genbank Accession No. U07132, amino acids 185-461) was obtained from E. coli. coli BL2
In one strain (DE3), it was expressed as an amino-terminal polyhistidine-tagged fusion protein. Expression was under the control of an IPTG-inducible T7 promoter. The recombinant protein and the DNA encoding the modified polyhistidine tag were subcloned into the expression vector pRSETa (Invitrogen).

At 25 ° C. in Rich PO ₄ medium containing 0.1 mg / mL ampicillin,
A 10 liter fermentation batch is grown for 12 hours, cooled to 9 ° C., and OD ₆₀₀ = 1
The temperature was maintained for 36 hours until a density of 4. At this cell density, 0
. Add 25 mM IPTG and add 24 hours 9 until final OD ₆₀₀ is 16.
Induction proceeded at ° C. Cells were collected by centrifugation (20 minutes, 3500 g, 4 ° C.) and the concentrated cell slurry was stored at −8 ° C. in PBS.

Purification of the Receptor Ligand Binding Domain Normally, 30-40 g of cell paste (corresponding to a 2-3 liter fermentation batch) is mixed with 300-400 mL of TBS, pH 8.5 (25 mM Tris, 150 mM).
(mM NaCl). The cells were lysed by passing through a homogenizer (Rannie) three times, and cell debris was removed by centrifugation (30 minutes, 20,000 g, 4 ° C.).
The clarified supernatant is filtered through a coarse prefilter and TBS containing 500 mM imidazole, pH 8.5, so that the final concentration of imidazole is 50 mM.
Was added. This lysate was loaded onto a column (6 × 8 cm) packed with Sepharose (Ni ⁺⁺ charged) Chelation resin (Pharmacia) and TBS pH 8.5 /
Pre-equilibrated with 50 mM imidazole. After washing to baseline absorbance using equilibration buffer, the column was developed with a linear gradient of 50 to 275 mM imidazole in TBS, pH 8.5. Column fractions are pooled and 5%
T containing 1,2 propanediol, 5 mM DTT and 0.5 mM EDTA
It was dialyzed against BS, pH 8.5. The protein samples were concentrated using Centri-prep 10K (Amicon) and Sepharose S-75 resin pre-equilibrated with TBS pH 8.5 containing 5% 1,2 propanediol, 5 mM DTT and 0.5 mM EDTA ( Pharmacia) using a column (3 x 90 cm)
Subjected to size exclusion.

LXRβ biotinylated PBS [100 mM sodium phosphate, pH 7.2, 150 mM NaCl
The purified LXRβ, LBD was subjected to desalting / buffer exchange using the PD-10 gel filtration column in [1]. LXRβ LBD is diluted in PBS to about 10 μM and a 5-fold molar excess of NHS-LC-biotin (Pierce) is added to a minimal amount of PBS.
Added inside. The solution was incubated with gentle mixing at room temperature for 30 minutes. The biotinylation modification reaction was performed using a 2000 × molar excess of Tris-HCl, pH
Stopped by the addition of 8. The modified LXRβ LBD was dialyzed with four buffer exchanges of at least 50 volumes each of PBS containing 5 mM DTT, 2 mM EDTA, and 2% sucrose. Biotinylated LXRβ LBD was subjected to mass spectrometry to determine the extent of modification by the biotinylated reagent. Generally, about 95% of the proteins had at least one biotinylation site: the total amount of biotinylation ranged from 1 to 9 and followed a normal distribution of multiple sites.

RXRα LBD human retinoid X receptor alpha ligand binding domain: E. coli BL21 strain (DE3), as an amino-terminal polyhistidine-tagged fusion protein, RXR-alpha LBD (amino acids 225-462)
Was expressed. Expression was under the control of an IPTG-inducible T7 promoter. The DNA encoding this recombinant protein and the modified polyhistidine tag was subcloned into the expression vector pRSETa (Invitrogen). RX
The sequence used for the construction of R-alpha LBD is Genbank accession number X527
73.

At 25 ° C. in Rich PO ₄ medium containing 0.1 mg / mL ampicillin,
A 10 liter fermentation batch is grown for 12 hours, cooled to 9 ° C., and OD ₆₀₀ = 1
The temperature was maintained for 36 hours until a density of 4. At this cell density, 0
. Add 25 mL of IPTG and add 24 hours 9 until final OD ₆₀₀ is 16.
Induction proceeded at ° C. Cells were collected by centrifugation (20 minutes, 3500 g, 4 ° C.) and the concentrated cell slurry was stored at −8 ° C. in PBS.

Protein Purification Normally, 40-50 g of frozen cell paste (corresponding to a 2-3 liter fermentation batch) is thawed and 300 mL of TBS, pH 7.2 (25 mM Tris
, 150 mM NaCl). The cells were lysed by passing through a homogenizer (Rannie) three times, and cell debris was removed by centrifugation (30 minutes, 20,000 g, 4 ° C.). The clarified supernatant is filtered through a coarse pre-filter and TB containing 500 mM imidazole is brought to a final concentration of 50 mM imidazole.
S, pH 7.2 was added. Sepharose (Ni ⁺⁺ charged) Chelation resin (Phar
This lysate was loaded onto a column (3 x 8 cm) packed with
Pre-equilibrated with TBS pH 7.2 containing M 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. Pool column fractions and pool 5m
It was dialyzed against TBS containing M DTT and 0.5 mM EDTA, pH 7.2. After dialysis, the sample was concentrated using Centri-prep 10K (Amicon) and using a column (3 × 90 cm) packed with Sepharose S-75 resin (Pharmacia) pre-equilibrated with the same buffer. And size exclusion.

Biotinylation of Human Retinoid X Receptor Ligand Binding Domain Biotinylation of purified RXRα LBD was carried out in the same manner as described for LXRβ LBD.

[0025] As will be labeled approximately 10μM in ^{CY5 TM} of RXR [alpha, dilute the purified RXR [alpha LBD in PBS,
Approximately 5-fold molar excess of Cy5 ^TM monofunctional reactive dye [NHS ester] (Amer
sham Life Sciences) was added to a minimal amount of PBS. 3 at ambient room temperature (about 23 ° C)
The solution was incubated in the dark with mixing for 0 minutes. Excess TrisH
The modification reaction was stopped by the addition of Cl, pH 8. 5m with minimal exposure
RXRα LBD modified with fluorescent dye was dialyzed at 4 ° C. with 4 buffer exchanges of at least 50 volumes each of PBS containing M DTT, 2 mM EDTA, and 2% (w / v) sucrose. Freeze aliquots on dry ice
Stored at 80 ° C.

Preparation of CY5 ^™ -RXRα: Streptavidin- (Europium Chelate) -L XRβ Complex Equimolar concentrations of biotinylated LXRβ and streptavidin-conjugated europium chelate in assay buffer containing 10 mM DTT for at least 10 minutes Was incubated. To this solution, a ^{Cy5 TM} labeled RXRα equimolar concentration was added to equilibrate for at least 30 minutes. Then, for example, Titertek Multidrop 38
Utilizing 4, the premixed receptor was added to the compound plate all at once.

[0027] Materials: ^CY5 TM -RXRα and europium labeled streptavidin LXRβ complex assay buffer: 50mM KCl, 0.1mg / mL BSA , 10mM
DTT and 50 mM Tris (pH 8). The storage buffer was 2.853 g of Tr
is base, 4.167 g Tris hydrochloride, 3.73 g KCl, and 0.1 g
It is made by dissolving fatty acid-free bovine serum albumin in 1 L of deionized water. Check pH and if necessary adjust to 8.0 before adjusting to final volume. Immediately before the start of the experiment, add 0.154 g of solid DTT per 100 mL of buffer.

BSA, no fatty acid DTT KCl Europium-labeled streptavidin: (Wallac CR28-100) Tris-HCl 96-well plate: polypropylene for intermediate dilution (Costar # 3794) and clear bottom white SPA plate for assay (Costar # 3) 3632) or Black Poly
Any of the filtronics plates (UP350 PSB).

Method: Experimental Details: Each well to be assayed contains a solution of a previously prepared CY5 ^™ RXRα, Europium-labeled LXR, and a test sample or control at the desired concentration (
(100 μL total). In general, the total volume was kept constant by changing the concentration and volume of the premixed receptor to compensate for any change in the volume of a sample group. The plates were incubated at room temperature for at least 2 hours and the fluorescence signal was measured in a Wallac Victor Multilabel Fluorescence Reader.

Data Conversion: For each concentration assay, the results for each test well were reported as eq. 1. And expressed as% C of the control.

[0031]

(Equation 1)

Here, F _sample is a signal observed in a certain sample well, F _total is a signal observed in the presence of a control inhibitor,
F _basal is the number of counts observed in the absence of ligand. The values used for F _sid and F _basal are the average values of the corresponding control wells contained in all plates.

For a dose response response assay, first, eq. Using (1), standardized to control%. _CL (% relative to the control observed at ligand concentration L)
A) A plot of ligand concentration L vs. ligand was made. This data was fitted to equation (2) to obtain the optimal parameters for EC ₅₀ , F _max and F _basal .

[0034]

(Equation 2)

Note that F _max (the maximum amplitude observed at saturated ligand concentration) can be either positive or negative. The sign of this parameter indicates that one test compound is LXR:
Indicates whether it prefers binding to the RXR complex (positive F _max ) or binds to any of the component receptors in a non-heterodimeric state (negative F _max ). Furthermore, both _Fmax and _Fbasal are expressed in units of% relative to the standard compound.

Results Both the magnitude and sign of Fmax (the magnitude of maximum ligand induction) must be considered. Note that the definition of the maximum response observed for 24 (S), 25-epoxycholesterol = 100% is arbitrarily assigned. Normalization is merely to allow comparison of the values obtained for different compounds on different days and / or with different fluorescent plate meters. The results are shown in FIG.

Example 2 Measurement of ligand binding to the farnasoid X receptor: retinoid X receptor heterodimer using time-resolved fluorometry.

This example describes the use of ligand-mediated heterodimerization to quantify binding of a ligand to the nuclear receptor, farnasoid X receptor (FXR).

[0039] The method comprises the steps of: (a) purifying FXR expressed by a bacterium;
The ability to mediate heterodimerization with the Ra ligand binding domain (LBD) is measured. Detection of associated LBDs is measured by time-resolved fluorometry (TRF). After labeling the purified LBD of FXR with biotin, it is mixed with a stoichiometric amount of europium-labeled streptavidin (Wallac Inc). Purified LBD of RXRα
It is labeled with ^{CY5 TM.} Equimolar amounts of each modified LBD are mixed and allowed to equilibrate at least one hour before addition to the sample in which a variable or constant concentration of affinity is to be determined. After equilibration, the time-resolved fluorescent signal is quantified using a fluorescent plate reader. From the plot of fluorescence versus the concentration of test compound added, the affinity of the test compound is estimated.

A basal level of FXR: RXR heterodimer formation is observed in the absence of added ligand. Ligands that promote heterodimer formation induce a concentration-dependent increase in the time-resolved fluorescent signal. Monomers FXR and F
Compounds that bind to the same extent to both XR: RXRα heterodimers are not expected to give a change in signal, whereas ligands that bind preferentially to the monomeric receptor will show the observed signal concentration It would be expected to result in reduced dependence.

Methods and Materials Preparation: Human Farnasoid X Receptor Alpha Ligand Binding Domain The human FXRα ligand binding domain (FXRα LBD) is coli
It was expressed as an amino-terminal polyhistidine-tagged fusion protein in strain BL21 (DE3). Expression was under the control of an IPTG-inducible T7 promoter. The DNA encoding the recombinant protein was subcloned into the expression vector pRSET-A (Invitrogen). The coding sequence for human FXRα LBD is Gen
It was derived from bank accession number U68233 (amino acids 222-472).

In Rich PO ₄ medium containing 0.1 mg / mL ampicillin, at 25 ° C., 1
A 0 liter fermentation batch is grown for 12 hours, cooled to 9 ° C., and OD ₆₀₀ = 14
The temperature was maintained for 36 hours until a density of. At this cell density, 0.
25 mM IPTG was added and induction was allowed to proceed at 9 ° C. for 24 hours until the final OD ₆₀₀ was 16. Collect cells by centrifugation (20 minutes, 3500 g, 4 ° C.)
The concentrated cell slurry was stored at -8 ° C in PBS.

Purification of the Receptor Ligand Binding Domain Normally, 30-40 g of cell paste (corresponding to a 2-3 liter fermentation batch) is treated with 200-250 mL of TBS, pH 7.2 (25 mM Tris, 150 mM).
(mM NaCl). The cells were lysed by passing through a French press three times, and cell debris was removed by centrifugation (30 minutes, 20,000 g, 4 ° C.). The clarified supernatant was filtered through a coarse pre-filter, and TBS containing 500 mM imidazole, pH 7.2 was added so that the final concentration of imidazole was 50 mM. Sephar
A column (6 × 8) packed with ose (Ni ⁺⁺ charged) Chelation resin (Pharmacia)
cm), the lysate was loaded and pre-equilibrated with TBS pH 7.2 / 50 mM imidazole. After washing with the equilibration buffer to the absorbance at the baseline, the column was washed with 1 column equivalent of TBS, pH 7.2 containing 90 mM imidazole. FXRαLBD eluted directly with 365 mM imidazole. Column fractions were pooled and TBS containing 0.5 mM EDTA and 5 mM DTT, pH
Dialyzed against 7.2. The dialyzed protein sample was concentrated using Centri-prep 10K (Amicon) and the column packed with Sepharose S-75 resin (Pharmacia) pre-equilibrated with TBS pH 7.2 containing 0.5 mM EDTA and 5 mM DTT (Pharmacia). (3 × 90 cm) was used for size exclusion.

FXR biotinylated PBS [100 mM sodium phosphate, pH 7.2, 150 mM NaCl
The purified FXRα LBD was desalted / buffer-exchanged using a PD-10 gel filtration column in []. FXRα LBD is diluted in PBS to about 10 μM and a 5-fold molar excess of NHS-LC-biotin (Pierce) is added to a minimal amount of PBS.
Added to The solution was incubated with gentle mixing at room temperature for 30 minutes. The biotinylation modification reaction was performed with a 2000 × molar excess of Tris-HCl, pH 8
Was stopped by the addition of The modified FXRa LBD was dialyzed by buffer exchange with at least 50 volumes each of PBS containing 5 mM DTT, 2 mM EDTA and 2% sucrose, four times each. Biotinylated FXRa LBD was subjected to mass spectrometry to determine the extent of modification by the biotinylation reagent. Generally, about 95% of the proteins had at least one biotinylation site: the total amount of biotinylation ranged from 1 to 9 and followed a normal distribution of multiple sites.

[0045] in accordance with the procedure shown in RXR [alpha LBD Example 1, to prepare a RXR [alpha LBD, labeled with ^{CY5 TM.}

Preparation of CY5 ^™ -RXRα: Streptavidin- (Europium Chelate) -FXR Complex Equimolar concentrations of biotinylated FXR and streptavidin-conjugated europium in assay buffer containing 10 mM DTT for at least 10 minutes. The chelate was incubated. To this solution, a ^{Cy5 TM} labeled RXRα equimolar concentration was added to equilibrate for at least 30 minutes. Then, for example, Titertek Multidrop 38
Utilizing 4, the premixed receptor was added to the compound plate all at once.

Materials: Assay buffer: 50 mM KCl, 0.1 mg / mL BSA, 10 mM
DTT and 50 mM Tris (pH 8). The storage buffer was 2.853 g of Tr
is base, 4.167 g Tris hydrochloride, 3.73 g KCl, and 0.1 g
It is made by dissolving fatty acid-free bovine serum albumin in 1 L of deionized water. Check pH and if necessary adjust to 8.0 before adjusting to final volume. Immediately before the start of the experiment, add 0.154 g of solid DTT per 100 mL of buffer.

BSA, fatty acid free DTT KCl europium labeled streptavidin: (Wallac CR28-100) Tris HCl 96-well plate: polypropylene for intermediate dilution (Costar # 3794) and clear bottom white SPA plate for assay (Costar # 3) 3632) or Black Poly
filtronics plate (UP350 PSB).

Method: The method, data conversion and interpretation of the results are as described in Example 1 and the results are shown in FIG.

Example 3 A novel fluorescence resonance energy transfer (FRET) assay, described further below, was used to test whether a candidate ligand binds to a nuclear receptor (using FXR as an example). This FRET assay is based on the principle that the ligand induces a conformational change in the nuclear receptor that facilitates the interaction with the nuclear receptor coactivator protein required for transcriptional activation. In one example, the ligand-binding domain of FXR labeled with fluorophore allophycocyanin (APC) is combined with a peptide containing the nuclear receptor interaction domain from the SRC-1 coactivator labeled with europium cryptate. Incubated.

[0051] Both transactivation and ligand binding assays, typically used to determine the activity of a candidate nuclear receptor activator, measure the effect of the test ligand on the isolated receptor. evaluate. However, most of the known orphan nuclear receptors interact with cofactor or coactivator proteins as a complex. Thus, the present invention suggests that binding of a ligand to a nuclear receptor may be regulated by complexing the receptor with a cofactor peptide. Subsequently, the ability of the ligand to induce varying degrees of this complex was used as the basis for a new assay for the discovery of nuclear receptor ligands. Certain sequences of cofactors may be necessary only to interact with nuclear receptors. To determine the most suitable sequence for use, various sequences of the two cofactor proteins SRC-1 and CBP were synthesized and HTRF
And tested in Biacore. Screening trials using FXR, peptide CPSSHSSLTERHKILHRLLQEGSPS-CONH ₂ (SEQ ID NO: 1), i.e. using SRC-1 a (LCD2,676-700). this is,
It constitutes a further aspect of the present invention.

The co-activator protein interacts with the nuclear receptor depending on the ligand,
Increase transcription (9). An amino acid motif LXXLL (L is leucine, X
Are any other amino acids), a short amphipathic α-helix domain comprising these coactivator molecules and a ligand-dependent activation function (COOH-terminal) located at the COOH terminus of nuclear receptor LBD. AF-2; Acts as an interface for interaction with activation function-2) (10). FXR prefers binding of coactivator
In order to test whether the ligand induces the conformation of the protein, the fluorescence resonance energy was monitored to monitor the allosteric interaction between a peptide based on the sequence of the steroid receptor coactivator 1 (SRC1) and the receptor. Metastasis (
A cell-free ligand detection assay utilizing FRET) was established. The use of FRET to monitor the formation of macromolecular complexes, especially in immunoassays (11), is well established and this detection method has recently been used to characterize ligand binding to nuclear receptors. (12).

[0053] Human FXR LBD was prepared and fluorescently labeled as described in Example 2.
The LBD of human FXR is labeled with a fluorophore allophycocyanin and the second L of SRC1 (amino acids 676-700) labeled with a europium chelate
Incubated with the peptide from the XXLL (SEQ ID NO: 1) motif.
In the presence of the test compound, 50 mM Tris pH 8, 50 mM KCl, 0.1
10 nM biotinylated FXR LBD labeled with streptavidin-conjugated allophycocyanin (Molecular Probes) in streptavidin-conjugated europium chelate (mg / mL BSA, 1 mM EDTA, and 10 mM DTT)
Wallac) -labeled 10 nM SRC1 peptide [amino acids 676 to 70
0,5'-biotin-CPSSHSSLTERHKILHRLLQEGSPS-
The FRET ligand detection assay was performed by incubating CONH ₂ ] (SynPEP) at 22 ° C. for 2 hours. Data was collected using a Wallac Victor ^™ fluorescence reader in time-resolved mode. The relative fluorescence was measured at 665 nM and data conversion was performed as described in Example 1.

Preparation of Streptavidin- (Europium Chelate) -SRC1: Streptavidin- (APC) -FXR Complex Biotinylated SRC-1 (LDC2, 676-700) in an assay buffer containing 10 mM DTT for at least 30 minutes. ) The peptide was incubated with a 化学 stoichiometric amount of streptavidin-conjugated europium chelate. 10 mM
A second solution of a stoichiometric amount of biotinylated FXR and streptavidin-conjugated APC was incubated in assay buffer containing DTT for at least 30 minutes.
Subsequently, each solution was blocked with a 5-fold molar excess of biotin and equilibrated for at least 15 minutes. The labeled receptor and the labeled peptide are mixed and again mixed with at least 3
After equilibration for 0 min, they were added to the compound plate all at once using, for example, a Titertek Multidrop 384.

Materials: APC labeled streptavidin FXR and europium labeled streptavidin SRC-1 (LDC2, 676-700) SRC-1 (LDC2, 676-700: SynPEP) APC labeled streptavidin FXR and europium labeled streptavidin SRC-1 (LDC2, 676-700) Biotinylated human farnasoid X receptor LBD: Biotinylated SRC-1 (LDC2, 676-700): Biotin-CPSSH
_{SSLTERHKILHRLLQEGSPS-CONH} 2] (SynPEP) Assay Buffer: 50mM KCl, 2mMEDTA, 0.1mg / mL B
SA, 10 mM DTT and 50 mM Tris (pH 8). The storage buffer is 2
. 853 g Tris base, 4.167 g Tris hydrochloride, 3.73 g KC
1. Prepare by dissolving 0.74 g EDTA (disodium salt dihydrate) and 0.1 g fatty acid-free bovine serum albumin in 1 L deionized water. Check pH and if necessary adjust to 8.0 before adjusting to final volume. Immediately before the start of the experiment, add 0.154 g of solid DTT per 100 mL of buffer.

BSA, fatty acid-free DTT EDTA, disodium salt dihydrate KCl allophycocyanin-labeled streptavidin: (Molecular Probes S-86
8) Europium labeled streptavidin: (Wallac CR28-100) Tris HCl 96-well plate: polypropylene for intermediate dilution (Costar # 3794) and clear bottom white SPA plate for assay (Costar # 3632) or black Poly
Any of the filtronics plates (UP350 PSB).

Results Ligands increased the interaction between FXR and SRCl peptide as measured by time-resolved FRET. Dose response analysis showed that the ligand increased the amount of SRCl peptide bound to FXR LBD. A typical saturating concentration response curve characteristic of a specific interaction was observed.

The “nuclear receptor activation cofactor peptide” used in the claims of the assay is
, In the presence of a ligand, the affinity for the receptor is changed and the LXXLL motif is
Has a peptide. The method has been shown to interact with FXR.
Examples of coactivator peptides useful for identifying ligands by SRC include:
Listed: B-QEQLSPKKKENNALLRYLDLDDDPS-CONH₂(
SEQ ID NO: 2), ACTR (734-758), RAC3 (724-748), S
RC-3 (724-748), AIB1 (724-748), pCIP (716
To 740) 2. B-QEPVSPKKKENALLRYLLLDKDDTKD-CONH₂(
2. SEQ ID NO: 3), TIF2 (732-756) B-GSTHGTSLKEKHKILHRRLQDSSSPVD-CONH ₂ (SEQ ID NO: 4), TIF2 (676-702) B-SNMHGSLLQEKHRILHKLLQNGNSPAE-CONH ₂ (SEQ ID NO: 5), pCIP (664-690), RAC3 (671-697)
, ACTR (681 to 707) and AIB1 (671 to 697).

Since the sequences of peptides 1 and 4 appear in many coactivators, there are various names. "B" in the sequence indicates biotinylation, a modification to which the peptide attaches during analysis.

The abbreviations used in the above examples are shown below. ATCR thyroid hormone and retinoid receptor activator AIB1 APC allophycocyanin APMSF amplified in breast cancer p-amidinophenylmethylsulfonyl fluoride, HCl Bestatin [(2S, 3R) -3-amino-2-hydroxy-
4-phenylbutanoyl] -leucine BSA bovine serum albumin CHAPS (3- [3-cholamidopropyl] dimethylammonio) -1
-Propane sulfonate CPM Counts per minute DMSO Dimethyl sulfoxide DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid FXR Farnasoid X receptor IBTG Isopropyl-β-D-thiogalactopyranoside LBD Ligand binding domain LXR Liver X receptor OD ₆₀₀ Absorbance at ₆₀₀ nm PBS Phosphate buffered saline [100 mM sodium phosphate, pH 7.2, 50 mM NaCl] pCIP Co-Integrator protein RAC Receptor activation co-factor RPM Rotation per minute RXR Retinoid X receptor SA-APC streptavidin-crosslinked allophycocyanin SPA scintillation access assay SRC steroid receptor cofactor TIF transcriptional intermediate Tris Tris (hydro Shimechiru) aminomethane

The following references are specified and the entire disclosure of each is incorporated herein by reference.

[0062]

[Table 1]

[0063]

[Table 2]

[Brief description of the drawings]

FIG. 1. As shown in FIG. 1, binding of a ligand to LXRβ is determined by LXRβ: RX
It was measured by modulation of R heterodimer formation.

FIG. 2. As shown in FIG. 2, ligand binding to FXR was measured by modulation of FXR: RXR heterodimer formation.

──────────────────────────────────────────────────続 き Continued on the front page (31) Priority claim number 60 / 134,836 (32) Priority date May 19, 1999 (May 19, 1999) (33) Priority claim country United States (US) ( 81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, R, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (71) Applicant Glaxo Wellcome House, Berkeley Avenue Greenford, Middlesex UB6. Great Brita in (72) Inventor Blanchard, Stephen Gerald 27709 Research Triang, North Carolina, United States Park, P.O.Box 13398, Five Moore Drive, GlaxoSmith Klein (72) Inventor Parks, Direk Jay, United States, North Carolina 27709 Research Triangle Park, P.O. Box 13398, Five Moore Drive, GlaxoSmith Klein (72) Inventor Stimmer, Julie Beth United States of America, 27709 Research Triangle Park, P.O.Box 13398, Five Moore Drive, GlaxoSmith Klein F-term (reference) 4H045 AA10 AA30 BA10 BA18 CA40 DA50 EA50 FA73 FA74 GA22 HA03

Claims (16)

[Claims] Claims: 1. A method for rapidly determining a ligand for a nuclear receptor, comprising the steps of: (a) selecting a component to be tested from an isolated nuclear receptor ligand binding domain with a first marking component; Contacting a nuclear receptor activation cofactor peptide with a marking component, and measuring the interaction between the marking components, wherein the component to be tested is the nuclear receptor ligand binding domain and the nuclear Determining whether to modify the binding to the internal receptor co-activator peptide. 2. The method of claim 1, wherein said first marking component is a radioactive marker and said second marking component is SPA beads. 3. The method of claim 1, wherein the first marking component is a first fluorescent dye that emits at a fluorescent wavelength that excites the second marking component, which is a second fluorescent dye. 4. The method of claim 1, wherein said nuclear receptor is a farnasoid X receptor ligand binding domain. 5. The method according to claim 1, wherein the nuclear receptor activation cofactor peptide is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. 6. The method of claim 2, wherein said interaction of said marker is determined by a scintillation approach. 7. The method of claim 3, wherein said interaction of said marker is determined by homogeneous time-resolved fluorometry. 8. The interaction of the marking component wherein the signal produced by the combination of the nuclear receptor activator cofactor peptide, the isolated nuclear receptor binding domain, and the component to be tested. Measured in the absence of the component to be tested by comparing the signal produced by the combination of the nuclear receptor coactivator peptide and the isolated nuclear receptor ligand binding domain. Item 1. The method of Item 1. 9. A method for identifying a compound for treating a disease or disorder controlled by FXR, comprising the step of determining whether the compound interacts directly with FXR, Wherein the compound is a compound for treatment. 10. A method for rapidly determining a ligand for a nuclear receptor, comprising: a component to be tested comprising: an isolated nuclear receptor ligand binding domain with a first marking component; Contacting a heterodimer partner of the nuclear receptor ligand binding domain with two marking components; determining the interaction between the marking components to determine whether the component to be tested modulates heterodimerization. Determining and a method comprising: 11. The method of claim 10, wherein said first marking component is a radioactive marker and said second marking component is SPA beads. 12. The method of claim 10, wherein the first marking component is a first fluorescent dye that emits at a fluorescent wavelength that excites the second marking component, which is a second fluorescent dye. 13. The heterodimer partner is RXR, PPAR,
LXRα, LXRβ, ERα, ERβ, CARα, HNF4, NGFIB, PX
11. The method of claim 10, wherein the method is R, PHR, EAR-1, EAR-2, TR, RAR, ERR, or RAR. 14. The method of claim 11, wherein said marker interaction is determined by scintillation access. 15. The method of claim 12, wherein said marker interaction is determined by homogeneous time-resolved fluorimetry. 16. The method wherein said interaction of said marking component is caused by a combination of said heterodimer partner, said isolated nuclear receptor binding domain, and said component to be tested. 2. The method of claim 1, wherein the signal is measured by comparing the signal generated by the combination of the heterodimer partner and the isolated nuclear receptor ligand binding domain in the absence of a component.
0 method.