KR100784859B1 - Fluorescence polarization-based method for analysis of Farnesoid X Receptor-ligand complex interaction and for screening inhibitors against the Farnesoid X Receptor-ligand complex binding - Google Patents

Abstract

The present invention relates to a method for analyzing the interaction of a panesoid X receptor-ligand complex using fluorescence polarization and a method for screening binding inhibitors, and specifically, between a panesoid X receptor (FXR) and a fluorescently labeled ligand using fluorescence polarization. Interaction analysis method and binding inhibitory candidates were added to samples containing fluorescently labeled ligands and Panesoid X receptors, and fluorescent fluorescence was measured to confirm competitive binding of ligands to inhibitors of binding to Panesoid X receptors. The present invention relates to a method for screening a binding inhibitor of a panesoid X receptor and a ligand complex. The interaction analysis method and binding inhibitor search method of the present invention can be effectively applied to the ultra-fast search using the well plate is very useful for the development of hyperlipidemia therapeutics.

Panesoid X Receptor, Fluorescence Polarization, Ligand

Description

Fluorescence polarization-based method for analysis of Farnesoid X Receptor-ligand complex interaction and for screening inhibitors against the Farnesoid X Receptor-ligand complex binding}

1 is a diagram showing the structure of the kenodioxy cholic acid (CDCA) and the fluorescent dye dye fluorescein-labeled kenodioxy cholic acid (CDCA-F),

Figure 2 is a view showing the result of measuring the fluorescence polarization while increasing the concentration of Panesoid X receptor (FXR) in a certain amount of fluorescein,

Figure 3 measures the increase in fluorescence polarization due to the production of FXR-CDCA-F complex while increasing the concentration of Panesoid X receptor (FXR) in a certain amount of fluorescein-labeled kenodioxycholic acid (CDCA-F) The drawing is shown,

4 is a diagram for measuring the fluorescence polarization while increasing the concentration of estrogen-related receptor alpha (ERRα) in a certain amount of fluorescein-labeled kenodioxycholic acid (CDCA-F),

FIG. 5 shows FXR-CDCA- with increasing concentrations of kenodioxycholic acid (CDCA) in a sample containing panesoid X receptor (FXR) and fluorescein-labeled kenodioxycholic acid (CDCA-F). Figure showing the results of measuring the change in fluorescence polarization of the F complex,

6 is a well plate fluorescence analyzer Victor using a 96 well plate, Panesoid X receptor (FXR, ●), estrogen-associated receptor alpha (ERRα, ○), and structural androstane receptor (CAR, It is a figure measuring the fluorescence polarization while increasing the concentration of ▲).

The present invention relates to a method for analyzing the interaction of a panesoid X receptor-ligand complex using fluorescent polarization and a method for screening a binding inhibitor, and more particularly, a panesoid X receptor (FXR) and a fluorescent label using fluorescent polarization. Method of analysis of interaction between the ligands and the inhibitory candidates were added to the sample containing the fluorescently labeled ligands and the Panesoid X receptor, and the fluorescence polarization was measured to competitively bind the ligands with the inhibitory candidates to the Panesoid X receptor. The present invention relates to a method for screening a binding inhibitor of a panesoid X receptor and a ligand complex.

The study of the effect of bile acid on the binding of the Panesoid X receptor to the coactivator is mainly carried out using radioisotopes, surface plasmon resonance (SPR), and fluorescence. It is made by the method and the like.

The radioisotope method is a method of measuring radioactive isotope-labeled ligands, nuclear receptor proteins, and coactivators by mixing and binding, followed by electrophoresis.

The method using surface plasmon resonance (SPR) is to fix the co-activator to the sensor chip, and to measure the change in refractive index due to binding by flowing a solution pre-mixed with the nuclear receptor protein to the sensor while varying the ligand concentration.

In the fluorescent method, a co-activator and a nuclear receptor protein are labeled with a fluorescent substance, and when they are combined, a fluorescent energy transfer assay and a co-activator are labeled with a fluorescent dye to transfer fluorescence energy from one side to another. And a method for measuring the binding of the auxiliary active agent with fluorescence polarization.

Here, the fluorescence polarization measurement method using fluorescence polarization is devised by Perrin, which is a method of measuring the fluorescence polarization generated when the excitation of the fluorescent molecules in the solution by the polarization. When the fluorescent molecules are excited and maintained in a stable state, fluorescence polarization is generated. When the fluorescent molecules are excited, the fluorescent molecules are rotated by the Brownian motion to emit fluorescence in a plane different from the excitation plane. This principle is to be lost.

This fluorescence polarization method has been recognized as a powerful and unique method for the interpretation of intermolecular interactions. The method is a direct analysis method that does not require solid phase separation, and can measure the intermolecular interaction of fluorescently labeled biomaterials with high sensitivity by using a principle in which the fluorescence polarization is different depending on the size of the molecule. Therefore, fluorescence polarization also increases when molecular weight increases due to binding between biological materials, and fluorescence polarization decreases when molecular weight decreases due to dissociation or decomposition. Thus, the interaction between biological materials can be analyzed using this principle.

Cholesterol is a component of cell membrane, nerve cell myelin, and lipoprotein. It is used as a source of steroid hormones and bile acids. However, cholesterol in the body causes atherosclerosis. Cholesterol homeostasis is regulated by methods such as ingestion, biosynthesis in vivo and conversion to bile acids. Recently, cholesterol 7a-hydroxylase (Cyp7a), which is involved in the conversion to bile acids, is a rate limiting enzyme for bile acid biosynthesis, controlled by the panesoid X receptor and the panesoid X receptor It has been reported to be activated by the binding of oxycholic acid. Therefore, the panesoid X receptor-kenodioxycholic acid complex binds with a co-activator to suppress the expression of cholesterol 7a-hydroxylase to regulate the amount of bile acid by feedback regulation in a manner that inhibits the synthesis of bile acids.

Therefore, the Panesoid X receptor has emerged as a target drug to remove or regulate cholesterol in the body, and to date, although it is important to measure the binding of the Panesoid X receptor to the ligand, Panesoid X The results of direct measurement and analysis of the binding of receptors to ligand molecules or the use of inhibitors have not been published.

Accordingly, the present inventors synthesized a fluorescent probe molecule having a fluorescent dye (fluorescein) attached to the main bile acid, kenodioxycholic acid, reacted with a panesoid X receptor, and measured fluorescence polarization with a fluorescence spectrometer to quantitatively analyze their binding. Developed a method.

This method can be used to measure the direct binding strength of the panesoid X receptor and the ligand kenodioxycholic acid, and through the competition reaction targeting the panesoid X receptor-kenodioxycholic acid complex, Panesoid X The present invention has been completed by confirming that it can be used to select a substance that binds to a receptor. In addition, the specific binding between the panesoid X receptor-kenodioxycholic acid was confirmed by the measurement of the fluorescence polarization using a well plate to confirm that a high-speed search of this assay was possible.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for analyzing interaction of a panesoid X receptor-ligand complex and a method for screening a binding inhibitor using fluorescence polarization.

The present invention provides a method for analyzing interaction between a panesoid X receptor (FXR) and a ligand using fluorescence polarization.

In addition, the present invention adds a binding inhibitory candidate to a sample containing a fluorescently labeled ligand and a Panesoid X receptor, and confirms the competitive binding of the candidate binding inhibitor to a ligand for the Panesoid X receptor by measuring the fluorescence polarization. Thus, the present invention provides a method for screening a binding inhibitor of a panesoid X receptor and a ligand complex.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for analyzing interaction between a panesoid X receptor (FXR) and a ligand using fluorescence polarization.

The ligand is preferably kenodioxycholic acid (CDCA, see FIG. 1).

Specifically, the assay method measures the binding polarity of the complex by measuring the fluorescence polarization of the panedoid X receptor and the kenodioxycholic acid (CDCA-F, see FIG. 1) complex (FXR-CDCA-F) labeled with a fluorescent dye. By determining

The fluorescent dye is preferably fluorescein.

The binding constant can be obtained by calculating from caladidagraph using the following equations (1) and (2).

                                                                      (Equation 1)

FP: Fluorescence polarization of the sample

FP ₀ : Fluorescence polarization when [FXR] = 0

FP _max : Fluorescence polarization when all CDCA-F formed FXR-CDCA-complex

[FXR] ₀ : FXR concentration in the sample

[CDCA-F] ₀ : The concentration of CDCA-F in the sample

K _d : Coupling constant between FXR and CDCA-F

                                                                       (Equation 2)

[CDCA]: concentration of CDCA in the sample

K _D : Coupling constant between FXR and CDCA

On the other hand, by measuring the fluorescence polarization by using a commercial fluorescence polarizer while increasing the concentration of FXR in the buffer solution containing fluorescein it can be seen that the fluorescein does not specifically bind to the FXR (see Fig. 2). ). Next, when the fluorescein-labeled kenodioxycholic acid is added while increasing the concentration of the FXR to measure the fluorescence polarization, the fluorescence polarization also increases as the concentration of the FXR increases (see FIG. 3). An increase in the degree of polarization may confirm that the concentration of the FXR-CDCA-F complex is increased.

In addition, the binding of the other nuclear receptor protein, estrogen related receptor alpha (ERRα), to determine if CDCA-F specifically binds to FXR, the protein is bound to CDCA-F. It can be confirmed that no (see Fig. 4).

In addition, the present invention adds a binding inhibitory candidate to a sample containing a fluorescently labeled ligand and a Panesoid X receptor, and confirms the competitive binding of the candidate binding inhibitor to a ligand for the Panesoid X receptor by measuring the fluorescence polarization. Thus, the present invention provides a method for screening a binding inhibitor of a panesoid X receptor and a ligand complex.

The fluorescently labeled ligand is fluorescein labeled kenodioxycholic acid (CDCA-F).

For example, the reaction of kenodioxycholic acid to the panesoid X receptor and ligand complex and the measurement of fluorescence polarization result in the kenodioxycholic acid reducing the fluorescence polarization of the panesoid X receptor and ligand complex (see FIG. 5). It can be confirmed that the substance inhibits the formation of the complex.

In addition, using a well plate to increase the concentration of the panesoid X receptor (FXR), estrogen-related receptor alpha (ERRα), structural androstane receptor (CAR) protein in a certain concentration of CDCA-F together with the fluorescence polarization The high-speed search can be used to confirm the specificity of each protein (see FIG. 6). In particular, the screening method of the present invention can be used to search for candidates for treating hyperlipidemia.

Therefore, the method for analyzing the interaction of the panesoid X receptor-ligand complex and the binding inhibitor detection method using the fluorescence polarization of the present invention can be used to develop a therapeutic agent for a disease having specificity for the complex of the panesoid X receptor-ligand. In addition, it is economical because the ultra-fast search method is used as a method for determining the binding constant of the panesoid X receptor-ligand.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of FXR Protein

The human farnesoid X receptor ligand binding domain (FXR LBD, amino acid sequences 245-476) labeled with histidine at the N-terminus inserts the gene into a modified vector of pET 21b (Novagen). To prepare a plasmidE. coli It was transformed into BL21 (DE3) and mass-expressed. Cells were inoculated in LB medium containing 50 μg / mL of ampicillin at 37 ° C. until the absorbance was 0.5, followed by 0.5 mM isopropyl-beta-di-thiogalactopyranoside (isopropyl). -β-D-thiogalactopyranoside (IPTG) was induced and incubated at 18 ° C for 15 hours. The recovered cells were buffered (50 mM Tris-HCl), pH 8.0, 500 mM sodium chloride (NaCl), 10 mM imidazole, 10% glycerol, 2 mM 2-mercaptoethanol (2-mercaptoethanol)) and destroyed by sonication at 4 ° C. The cell extract was centrifuged at 12,000 rpm for 40 minutes to purify the supernatant by two steps of chromatography. First, metal-chelate chromatography was performed on the N-terminal histidine label using Ni-NTA resin. Next, HiLoad 26/60 superdex 200 prep, previously equilibrated with buffer solution (50 mM Tris-HCl, pH 8.0, 62.5 mM sodium chloride (NaCl), 1 mM dithiothreitol) Gel filtration chromatography was performed using a -grade column (Amersham-Pharmacia). Purity of the purified protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and concentrated to a concentration of 11 mg / ml.

Example 2 CDCA-F Preparation

We prepared CDCA-F (see FIG. 1; kenoxidiooxylic acid labeled with fluorescein) to be used in the binding experiment. First, 24 mg (= 60 μM) of fluorescein isothiocyanate (FITC) was dissolved in 2 mL of methanol and 40 mg (= 300 μM) of ethylenediamine (EDA) were methanol containing 10 mL / L of triethylamine. It was dissolved in 10 mL. Stirring the EDA solution, the FITC solution was added dropwise, and then reacted at room temperature for one hour. After filtration with a filter to remove the precipitate, the filter was washed with 10 mL methanol and the resulting fluorescein thiocabamylethylenediamine solution was dried at room temperature.

5 mL of 20 mg (= 200 μM) N-hydroxysuccinimide (NHS), 77 mg (= 400 μM) 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) 4.2 mg (= 10 μM) of kenodioxycholic acid was dissolved in 500 μL of a solution dissolved in dimethylformamide (DMF). 4.5 mg (= 10 μM) of fluorescein thiocarbamyl ethylenediamine was added to the solution, followed by overnight reaction at room temperature, followed by separation using thin layer chromatography (TLC). TLC was carried out in dichloromethane: methanol (6: 1, v / v) and extracted with 0.5 mL methanol using Bio-Spin Disposable Chromatography Columns (BIO-RAD). It was. The purified CDCA-F confirmed the molecular weight by MALDI-TOF.

Example 3 Analysis of Coupling Constants (Dissociation Constants) of CDCA-F and FXR

The present inventors have prepared CDCA-F prepared in <Example 2> and <Example 1> The binding constants (dissociation constants) were analyzed by measuring the fluorescence polarization which changed when the protein-ligand complex was formed using FXR. Fluorescence polarization was measured using a fluorometer (manufactured by Perkin-Elmer), the slit size was set to 5 nm, the integration time was set to 2 seconds. The buffer solution contained 50 mM Tris, 100 mM sodium chloride, 1 mM dithiothitol (DTT) and used a solution whose pH was adjusted to 8.0 with 1N hydrochloric acid. First, in order to determine whether the fluorescent dye labeled on CDCA binds to FXR, the fluorescence polarization was measured by increasing the concentration of FXR in the buffer solution containing 20 nM of fluorescein. It was confirmed that no specific binding to FXR (see Figure 2). Next, as a result of measuring the change in fluorescence polarization by increasing the concentration of FXR to CDCA-F, the increase in fluorescence polarization was observed as the concentration of FXR in solution was increased (see FIG. 3). It was found that the concentration of the -F complex was increased. Equations 1 and 2 were used to determine the binding constants between FXR and CDCA-F.

The binding constant between FXR and CDCA-F was 14 μM as calculated by the KaleidaGraph program using the graph obtained above (see FIG. 3). Next, to see if CDCA-F specifically binds to FXR, we examined the binding of other nuclear receptor proteins, estrogen-related receptor alpha (ERRα), which showed little binding to CDCA-F. (See FIG. 4). Therefore, it was found that the CDCA-F, a fluorescent probe molecule used in the present invention, specifically binds only to the binding site of FXR.

Example 4 Inhibition of FXR-CDCA-F Complex Formation by CDCA

In order to search for substances inhibiting the complex formation of FXR-CDCA-F, a sample containing 20 nM of CDCA-F and 20 μM of FXR was prepared under the conditions described in Example 3 . Herein, the fluorescence polarization was measured while increasing the concentration of CDCA to confirm the inhibition of FXR-CDCA-F complex formation by the competition reaction of CDCA (see FIG. 5). Using the above formula for calculating the binding constant, the binding constant of CDCA was calculated using the calender graph program, and 26 μM was obtained.

Fluorescence polarization analysis method using the fluorescent probe molecule of the present invention can easily search for substances that inhibit the binding of FXR and CDCA, and these substances are developed as candidates for inhibiting the formation of cholesterol, and also as therapeutic agents for diseases related thereto. Could be.

Finally, the fluorescence polarization was measured by increasing the concentration of protein in a certain concentration of CDCA-F for three different nuclear receptor proteins FXR, ERRα, and CAR using 96-well plates to investigate the possibility of ultrafast search. As a result, the increase in fluorescence polarization by the specific binding of CDCA-F and FXR was confirmed (see FIG. 6).

The method for analyzing interactions and binding inhibitors of the panesoid X receptor-ligand complex using the fluorescence polarization of the present invention may be performed by measuring the interaction between the panesoid X receptor-ligand and the detection of binding inhibitors by the change in the fluorescence polarization. It can be used economically because it is simple to analyze and can be automated in a manner using well plates.

Claims (10)

By using the Panesoid X receptor (FXR) and ethylenediamine as linkers, the fluorescence polarization of the fluorescein labeled ligand Kenodioxycholic acid complex (CDCA-F) is measured to determine the binding constant of the complex. Analysis method of interaction between FXR and ligand. delete delete delete The analysis method according to claim 1, wherein the binding constant is obtained by calculating from a calender graph using Equations 1 and 2 below.

                                                                     (Equation 1) FP: Fluorescence polarization of the sample FP ₀ : Fluorescence polarization when [FXR] = 0 FP _max : Fluorescence polarization when all CDCA-F formed FXR-CDCA-complex [FXR] ₀ : FXR concentration in the sample [CDCA-F] ₀ : The concentration of CDCA-F in the sample K _d : Coupling constant between FXR and CDCA-F

                                                                       (Equation 2) [CDCA]: concentration of CDCA in the sample K _D : Coupling constant between FXR and CDCA Using ethylenediamine as a linker, a candidate inhibitor was added to a sample containing a kenodioxycholic acid complex (CDCA-F), a ligand of a fluorescein-labeled panesoid X receptor, and a panesoid X receptor, A method for screening a binding inhibitor of a panesoid X receptor and a ligand conjugate, characterized in that the binding of the ligand to the panesoid X receptor is inhibited by measuring the degree of polarization. delete 7. The method of claim 6, wherein the screening method is used to search for a candidate for treatment of hyperlipidemia. delete delete

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