WO2002072874A1 - METHOD OF SCREENING HNF4α AGONIST - Google Patents

Abstract

A method of screening an HNF4α agonist which involves: (1) the step of bringing a test compound into contact with cells expressing an HNF4α gene and further expressing a reporter gene under the regulation of a gene promoter the expression of which is controlled by HNF4α, in the presence of an acyl CoA synthase inhibitor; and (2) the step of measuring the expression dose of the reporter gene in the above cells. According to this method, the activation of HNF4α originating in a substance contained in the screening system can be inhibited and thus an HNF4α agonist can be screened more accurately with lowered background.

Description

 Specification

 Screening method for HNF 4

 Technical field

 The present invention relates to a staring method for HNF 4 agoquest.

 Background art

 HNF 4a (hepatocyte nuclear factor 4α) is a factor that functions as a nuclear receptor having a ligand binding site and a DNA binding site, but has recently developed since a young age and is characterized by insufficient insulin secretion. It has been clarified that this is due to a genetic abnormality in SHN F4α, a diabetes with a strong family history (MODY: Maturity-onset type diabetes of the young). Since HNF 4a is considered to be involved in the maintenance and proliferation of knee] 3 cells, which are insulin-secreting cells, HNF4 agonist can promote the function improvement of knee] 3 cells It is considered that there is a possibility.

 HNF 4a; which exists as a dimer binds to the ligand and binds to the promoter region of the downstream gene, thereby promoting the transcription of the downstream gene and the subsequent translation of the protein. For example, it is known that HNF4 is located downstream of HNF4, and HNF4 present as a dimer binds to the promoter region of HNF1a by binding to a ligand, Promotes transcription of HNF1 from the region, leading to expression of HNF1. HNF-1 is a transcription factor found in the liver, and is known as an expression regulator of albumin, antitrypsin, fibrinogen, and the like. At present, expression is also confirmed in the knee, kidney, small intestine, etc., and it is suggested that particularly in the knee, expression of GLUT2 and other genes involved in insulin secretion and insulin itself may be regulated. ing.

It has been reported that the causative gene of MOD Y 3 is HNF 1a, and that the causative gene of MOD Y 1 is HNF 4a (Kazuya Yamagata et al., Mutation in hepatocyte nuclear factor 1 a gene in maturity- onset diabetes of the young (M0DY3) .Nature 384: 455-458 (1996), Kazuya Yamagata et al., Mutation in hepatocyte nuclear factor 4 gene in maturity-onset. Diabetes of the young (M0DY1). Nature 384: 458-460 (1996)).

 Furthermore, as a factor involved in the activation of HNF4, HNF3 is involved in the transcription of pdX-1 which is involved in the formation of the knee, and is involved in the maintenance and proliferation of knee cells (Kuo-Liang Wu et al., Hepatocyte nuclear factor 3 β is involved in pancreatic β -cell-specific transcription of the pdx-1 gene.Mol.Cell Biol.

 17 (10): 6002-6013 (1997))) and HNF 6, and these factors form a cascade.

 The HNF4agonist is useful not only for analyzing the function of HNF4α but also for analyzing the functions of other factors that constitute the HNF cascade. It may lead to the search for a compound that has a new point of action for a disease.

 Disclosure of the invention

 It is an object of the present invention to provide a method for screening HNF 4 agonists. In particular, it aims to provide HNF4α agonists useful as therapeutic agents for diabetes.

In view of the above problems, the present inventors have worked on the construction of a screening system for HNF4 rabbits. Based on the knowledge that HNF 4 (X binds to ligands in serum to activate transcription of genes downstream of it, establishes an Atsushi system that can evaluate the activity of HNF 4 α under serum-added culture conditions However, when the Atsey system was used to search for the HNF4α agonist, HNF4α was activated while the test cells were cultured in the serum-containing medium, and the background increased. The present inventors cannot accurately measure the HNF 4 agonist activity of the target test compound, so the present inventors have further conducted intensive studies and performed the screening in the presence of an acyl C ァ synthase inhibitor. The present inventors have found that it is possible to suppress the activation of HNF4α derived from a substance other than the test compound inherent in the screening system, that is, that the background can be reduced. Led to the formation. Soku Chi present invention is as follows. (1) (i) A cell that expresses the HNF4 gene and expresses the reporter gene under the control of the promoter of the gene whose expression level is regulated by the HNF4 gene is synthesized by acyl-COA. A method for screening an HNF4α agonist, comprising the steps of: contacting a test compound in the presence of an enzyme inhibitor; and (ii) measuring the expression of a reporter gene in the cell.

 (2) The screening method according to the above (1), wherein the promoter of the gene whose expression level is regulated by the HNF4 promoter is the HNF1 promoter.

 (3) Acryl C ο Α Synthase inhibitor is triaxine. The screening method according to the above (1) or (2).

 (4) The screening method according to any one of the above (1) to (3), wherein the HNF 4 hagiost is a remedy for diabetes.

 (5) An HNF 4 agonist obtained by the screening method described in (1) above.

 BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a graph showing the effect of triaxin C on HNF4a activity in HNF4α-expressing cells transfected with an HNF4α expression vector and in control cells not expressing HNF4a.

 FIG. 2 is a graph showing the effect of triaxin C on HNF4α activity in previously cloned HNF4α-expressing cells.

 Detailed description of the invention

In the present invention, “HNF 4 agonist” refers to a substance that enhances HNF 4 activity, more specifically, HNF 4 to a promoter of a gene whose expression level is regulated by HNF 4α. A substance having an action of enhancing the binding ability of α is intended. An example is a substance that increases the ability of HNF4α to bind to the HNF1α promoter. Similarly, "HNF 4 a activity" as used herein, HNF 4 _a is bonded to the promoter of the gene whose expression level by HNF 4 a is adjusted, followed by the gene downstream of said promoter Machines that promote transcription and expression Noh is intended. For example, the action of HNF4a, which binds to the HNF1α promoter and subsequently promotes transcription and expression of genes downstream of the HNFla motor.

Used in subscription-learning method of the present invention, express HNF 4 _alpha gene and HNF 4 Hiniyotte cells expressing the reporter gene to control under the promoter of the gene whose expression level is regulated, There is no particular limitation so long as it expresses HNF4a and expresses a reporter protein under the control of a promoter of a gene whose expression level is regulated by HNF4. And can be prepared by appropriately combining them. More specifically, (1) an HNF4 expression vector (described below) and a vector having a DNA encoding a reporter protein under the control of a promoter of a gene whose expression level is regulated by HNF4a (hereinafter referred to as “ A cell that expresses HNF4 and a reporter protein transiently, and (2) a high-expressing HNF4α-expressing cell (described later), which is obtained by introducing a reporter vector: At the time of the assay, a cell or the like obtained by introducing a reporter vector is used.

 Here, various known promoters can be used as the promoter of the gene whose expression level is regulated by HNF4. Specific examples include HNF1 promoter, ap'o CIII promoter, pyruvate kinase-L (PKL) promoter, PEPCK promoter and the like. Preferably, an HNF1a promoter which is usually present downstream of the HNF4 gene and whose expression is regulated by HNF4α is used.

Cloning of HNF4α high-expressing cells can be performed by appropriately combining techniques known in the art. Specifically, a transformant obtained by introducing an HNF4a expression vector having a selectable marker gene (described later) is screened with the selectable marker, and further, a reporter vector is introduced and its HNF4α activity is determined. Is measured to obtain cells with high HNF4α activity, that is, cells with high expression of HNF4α. HNF 4 alpha expression vector used in the present invention, for example, the HNF 4 Hiniyotte HNF 4 Fei downstream of its promoter promoter of the gene whose expression level is regulated can function in a host of functions such as HNF 1 _alpha promoter There is no particular limitation as long as it has the encoding DNA. Specifically, promoters capable of functioning in various eukaryotic cells, preferably mammalian cells (eg, cytomegalovirus promoter 1, SV40-derived early promoter, Rous sarcoma virus promoter, Moroni-mouse leukemia virus (MoMu LV) -derived long terminal repeat, virus promoter region such as adenovirus-derived early promoter), DNA encoding HNF4a, and the transcription termination signal of the gene, namely terminator It contains a central region. Preferably, for the purpose of transformant selection (particularly when preparing HNF4a high expression cells in advance), a selectable marker gene (a gene conferring resistance to a drug such as neomycin, zeocin, hygromycin, Auxotrophic mutation, etc.). If the HNF4α-encoding DN does not contain an initiation codon and a termination codon, the initiation codon (ATG) and the termination codon (TAG, TGA, TAA) are placed downstream of the promoter region and the terminator, respectively. A vector containing one region upstream is preferably used.

 When an animal cell is used as a host, the expression vector preferably further contains an enhancer sequence, untranslated regions on the 5, 5 and 3 'sides of HNF4, a polyadenylation site, and the like.

 The DNA encoding HNF4ο; contained in the expression vector may be a DNA known per se, for example, the sequence described in Gene (1994), 147: 269-272.

(Fig. 2 in the same document). Specifically, the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing, an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, added or modified in the amino acid sequence, or a fragment of the amino acid sequence ( However, the protein comprising the mutated amino acid sequence or the fragment is a DNA that encodes a DNA-binding domain and a ligand-binding domain. here The term “functionally” means that the ability to bind to a ligand and the ability to bind to a promoter are maintained, and that the ability to bind to a promoter is increased by an agost, and the transcription of a gene downstream of the promoter is increased. · It means that the expression can be promoted. More preferably, the DNA encoding HNF4 contained in the expression vector is a DNA comprising the nucleotide sequence represented by nucleotide numbers 20 to 1417 in the nucleotide sequence represented by SEQ ID NO: 1 in the Sequence Listing, DNA containing a base sequence in which one or several bases have been deleted, substituted, inserted or added, or a DN consisting of the base sequence

The fragment of A (the mutant base sequence or the protein encoded by the fragment functionally has a DNA binding domain and a ligand binding domain (described above)).

 Particularly preferably, DN encoding the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing

A, a DNA comprising the nucleotide sequence represented by nucleotide numbers 20 to 1417 in the nucleotide sequence represented by SEQ ID NO: 1 in the Sequence Listing.

The reporter vector used in the present invention is a vector having a reporter gene under the control (downstream) of a promoter of a gene whose expression level is regulated by HNF4, wherein a dimeric HNF4 is added to the promoter. By binding, a reporter gene downstream thereof can be expressed. The reporter vector is simply constructed by using a reporter gene expression vector and a promoter (a promoter of a gene whose expression level is regulated by HNF4α) available in the art. Downstream, it can be constructed by ligating a DNA encoding a reporter gene by a conventional method. Such promoters include, for example, the HNF1α promoter, and such HNF1α promoter regions are known in the art, for example, Kaisaki, PJ et al., Mutations in the Hepatocyte Nuclear Factor-1 CK Gene in MODY and Early-Onset NIDDM. Diabetes 46: 528-535 (1997). The promoter region has a nucleotide sequence in the nucleotide sequence as long as it does not adversely affect the binding of HNF4 and its promoter activity. It may be a DNA containing a base sequence in which one or several bases are deleted, substituted, inserted or added, or a DNA fragment consisting of the base sequence. For example, a reporter vector containing a luciferase gene as a reporter gene under the control of the HNF1α promoter is commercially available (eg, Promega).

 As long as it does not adversely affect the expression of HNF40; and the expression of the reporter protein, construct the HNF4α expression cassette and the reporter protein expression cassette so that they are on the same vector. Can be.

 As the reporter gene used in the present invention, those commonly used in the art can be used. Specifically, firefly luciferase gene, chloramphenicol acetyl acetyltransferase (CAT) gene, green 'fluorescein' protein (GFP) gene, β-galata tosidase (/ 3-Gal) gene, and the like.

 Preferably, in addition to the reporter vector for the above experiment, a second reporter vector (hereinafter also referred to as a control reporter vector) serving as an internal standard is cotransfected to standardize the experimental results. The vector for normalization contains the reporter gene under the transcriptional control of a promoter that is insensitive to the treatment performed on the reporter vector for the experiment. Specific examples of such Atsushi systems include a reporter vector for an experiment constructed to contain a firefly luciferase gene under the control of the HNF1 promoter, and a control reporter vector such as thymidine kinase. An example is a system that uses a construct constructed to contain the Mycobacterium luciferase gene under the control of a promoter, SV40 early promoter, and the like. Both reporter vectors are cotransfected.

As a method for introducing the above-described various vectors into a host cell, a method usually used in this field is used. Specific examples include a calcium phosphate coprecipitation method, a protoplast fusion method, a microinjection method, an electoral poration method, and a lipofection method. Each vector D to be introduced into host cells (described below) The amount of NA varies depending on the cell used, the vector used, the transfection method used, and the like, and optimal conditions can be set as appropriate. Usually, in the lipofection method using lipofectamine, HNF4α expression vector 0.2 to 2.5 / ig per 1 to ⁴ × 10 ⁴ cells, repo ^ "ter vector 0.2 to 2.5. 5 μg, 0.25 to 2.5 ng of the control reporter vector are introduced, and the control vector without the HNF4α expression cassette used as a control is used for the assay. It is used in an amount according to the HNF4α expression vector.

 Such a control vector is equivalent to the HNF4α expression vector except that it does not have the HNF4α expression cassette.

 In the present invention, the HNF4α expression vector or control vector, and the host cell into which the reporter vector and the control reporter vector are introduced are not particularly limited as long as they are compatible with the vector to be used and can be transformed. Various cells such as natural cells or artificially established recombinant cells commonly used in the technical field of the present invention can be used. Preferably, mammalian cells, especially rat-derived cells, hamster-derived cells (CHO, BHK, etc.), mouse-derived cells (COP, L, C127, Sp2 / 0, NS-1, NIHT3, etc.) ), Monkey-derived cells (COS1, COS3, COS7, CV1, Vero, etc.) and human-derived cells (HeLa, diploid fibroblast-derived cells, myeloid cells, N ama 1 wa). More preferred are human or monkey-derived cells, especially COS cells or HeLa cells.

The present invention relates to cells expressing the HNF4α gene obtained according to the above-described method and expressing a reporter gene under the control of a promoter of a gene whose expression level is regulated by HNF4a (hereinafter simply referred to as cells). Preferably, in addition to the test cells, control cells that do not express HNF4α (ie, cells that are equivalent to the test cells except that they do not express ΝF4α) A control compound, which is obtained by introducing a control vector into a host cell (hereinafter, simply referred to as a control cell) in the presence of an acyl C Α synthase inhibitor. Including the step of performing The acyl CoA synthase inhibitor used in the present invention is an activity of an acyl CoA synthase that catalyzes the synthesis of fatty acyl-CoA thioester from long chain fatty acid and coenzyme A (CoA). No particular limitation is imposed as long as it inhibits the activity of the enzyme. For example, triaxin is used (Hiroshi Soeda, Satoshi Omura, IV Enzyme Inhibitors, “Fatty Acid Activating Enzyme Inhibitor, Triaxin”, protein. 38, No 11 (1993)). Triaxin is a natural product produced by actinomycetes, and four species, A, B, C and D, are currently reported. In the present invention, triaxin C having the following structural formula is preferably used.

 The amount of the acyl CoA synthase inhibitor used in the screening method of the present invention is not particularly limited as long as removal of the fatty acyl-CoA thioester from the screening system is achieved. It can be set appropriately depending on the cells used and the like. When triaxin C is used, it is usually added to a screening system at a concentration of Ι / M or more, preferably about 10 or more.

Ashiru presence of C o Alpha synthetase inhibitor, contact also the test cell and / or the test compound to Control cells, but are not particularly limited, the test compound, the HNF 4 _alpha Agonisuto action Is added to the screening system on a schedule suitable for testing. For example, the following method is exemplified. ·

After transfection of the reporter vector into the HNF4α high-expressing cells described above, the cells are replated again. After confirming that the seeded cells have adhered to the plate, add Triaxin C and the test compound. The adhesion varies depending on the state of the cells and the like, and usually requires 30 minutes to 1 hour, but is not particularly limited. Further, the contact can also be achieved by adding a culture solution containing a predetermined concentration of triaxin C to the cells after the transfusion treatment. The timing of addition of triaxin c and the test compound can be variously changed depending on the mode of addition, and specifically, after the completion of the transfusion to 2-3 hours.

As used herein, the term "test compound" refers to a compound that has been selected or synthesized for the purpose of examining the presence or absence of HNF4 agonist action, and has been reported to have another action besides a novel compound. It also includes certain known compounds. Compound was observed to have HNF 4 _alpha Agoyusuto action by subscription one Jung method of the invention, as above mentioned, is considered to diabetes, it is useful to particular瞵臓progenitor cells differentiate into睥0 cells It is expected to be used in cases of insulin-dependent and non-insulin-dependent diabetes with abnormal insulin secretion.

The method for measuring the expression of a reporter gene in the screening method of the present invention can be appropriately carried out using a method known in the art according to the reporter gene to be used. For example, if the reporter gene to be used is a firefly luciferase gene, beetle luciferin or the like is used as a substrate, if the reporter gene used is a mushroom luciferase gene, coelenterazine is used as a substrate, or if the 3-galactosidase gene is ONPG ( 0-nitrophenyl_] 3-D-galactopyranoside) is used, and the degree of color development is measured using a spectrophotometer or the like. In the case of the chloramphene-cotyl acetyltransferase gene, kuram ramphenicol is used as a substrate, and the activity is measured by the degree of conversion to acetylated form. The timing of the measurement varies depending on conditions such as the expression time and half-life of the protein encoded by the reporter gene to be used, as well as the measurement method including the color development method. Usually, the test compound is used in the assay system. Measure 24 to 72 hours after addition. The means for measuring the activity of the reporter gene can also be appropriately changed depending on the type of the reporter gene, substrate, etc. used. Specifically, it is measured as follows. For example, an HNF4 expression vector, a reporter vector containing a reporter gene under the control of a promoter of a gene whose expression level is regulated by HNF4 expression, and a control reporter vector containing a reporter gene serving as an internal standard If the reporter vector and control reporter vector are to be introduced into preselected HNF4-expressing cells in the Atsushi system, use the test compound for 24 to 72 hours after adding the test compound to the Atsushi system. Similarly, when HNF4ο; an expression vector, a reporter vector and a control reporter vector are simultaneously introduced, 24 to 72 hours after the test compound has been added to the ATSY system, Measure reporter activity.

 Conveniently, the step of measuring the expression of the reporter gene can be performed using commercially available reagents, kits and the like.

 Further, the present invention provides a new HNF4α agonist obtained by the above-described screening method of the present invention. Such HNF4α-agonists (including salts, if present) may be solid, semi-solid or semi-solid, as a mixture with an organic or inorganic carrier or excipient suitable for oral or parenteral administration. It can be used in the form of a pharmaceutical preparation in liquid form. The agonists may be used, for example, for powders, tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, aerosols, sprays, and other forms suitable for use. It can be mixed with conventional, non-toxic, pharmaceutically acceptable carriers. If necessary, auxiliaries, stabilizers, thickeners and the like may be used. These carriers and excipients may be used after sterilization if necessary, or may be subjected to sterilization after formulation.

Therapeutically effective dose of HN F 4 _alpha Agonisu bets, the active ingredient may differ depending on the age Contact Yopi state of the individual patient to be treated, also determined in dependence on them. The pharmaceutical preparation containing the HNF 4 agonist is useful for treating diabetes, particularly for increasing the number of knee cells and improving the function of insulin-dependent diabetes and insulin-independent diabetes due to its HNF 4 agonist activity. Of these, it is useful for patients with abnormal insulin secretion.

The HNF 4 agonist obtained by the screening method of the present invention can be used for various applications in which the operation is useful. For example, for functional analysis of various HNFs that contribute to the HNF cascade, and for various types of HNF such as diabetes It is a useful tool for studying diseases.

Example

 Hereinafter, the present invention will be described specifically and in detail with reference to examples, but the present invention is not limited thereto. In this example, activation of HNF4 was performed by adding serum.

Example 1

HeLa cells were seeded on a 6-well plate in 10% serum-supplemented culture medium (D-MEM) at a cell count of 4 x 10 ⁵ cells / cell, and the HNF4a expression vector (Yang, Q. et al., R127 -HNF-4 is a loss of function mutation but not a rare polymorphism and causes Type II diabetes in a Japanese family with MODYl. Diabetologia 43: 520-524 (2000): pc DNA 3.1 (invitrogen ) BamHI site with HNF4o; (SEQ ID NO: 2) inserted therein) (HNFaZpcDNA3.1), HNF1α promoter Hotanorenoluciferase vector (ΗΝF1hi promoterno pGL3 basic; Promega Co., Ltd., and a Mycobacterium luciferase expression vector (internal standard) (pRL-SV40; Promega) were transfatated by the lipofection method (test cells).

 Similarly, the control vector (a vector similar to the above HNF4 expression vector except that it does not contain the HNF4 expression cassette) (pcDNA3.1), the reporter vector and the control vector reporter vector were subjected to lipofection method. Was transfected (control cells).

 The lipofection method was carried out by transfection using Cytolipofectamine Plus (Lifetech Oriental Co., Ltd .; hereinafter, plus reagent).

 The transfection was carried out using 0.5 μg of each vector and 18 μL of plus reagent and 8 μL of lipofectamine per 1 μL of a 6-well plate.

After treatment for 3 hours, triaxin C (BI0M0L Research Laboratories) 10% serum-containing culture medium (D-MEM) containing 10 μL (final concentration 10 μM) was added. Luciferase activity was measured 24 hours after the addition of triaxin C. The activity of HNF4α was shown as the ratio between the firefly luciferase activity and the Pseudomonas luciferase activity. The luciferase activity was measured using a Wallac 1420 ARVOsx multilabel counter. The results are shown in Figure 1.

 As shown in Fig. 1, the specific activity of luciferase was suppressed by 58% by adding triacsin C compared to the case of non-added calyx, and the addition of triaxin was able to suppress the activation of HNF4 based on serum stimulation. Indicated.

Example 2

(1) Preparation of HeLa cells with high expression of HNF4α

Seed 4 x 10 ⁵ HeLa cells per 6 cm culture dish and transfected with 1.5 μg of HNF4α expression vector, 18 AtL plus reagent, and 8 L of lipofectamine. went. Here, the HNF4α expression vector

The same one used in 1 was used.

 The next day, collect the cells after the transfection, dilute 100-fold, and

It was diluted 0-fold and re-inoculated in a 10-cm culture dish with 20 cells of each dilution. Two days later, 50 ^ gZmL of Geneticin (G418) (Lifetech Oriental) was added to select for expression cells.

 One month later, a colony was selected and seeded on a 24-well plate. For each colony selected, one reporter vector was transfected, and one having strong HNF4α activity was selected.

 By this step, six clones stably expressing HNF4a were obtained.

(2) Using one of the clones obtained in (1) above, HNF 4 activity in the presence of triaxin C was measured in the same manner as in Example 1.

HeLa cells (1 × 10 ⁶ ) stably expressing HNF4α were seeded on a 10-cm culture dish, and the HNF1α promoter firefly luciferase vector (Promega) was prepared using the lipofection method. Mistake luciferase expression vector (Internal standard; Promega) was transferred. In the same manner as in Example 1, in a 10-cm culture dish, the HNF1 promoter / firefly luciferase vector (4 μg) and ゥ were added using a plus reagent (20 μL) and ribophenetoamine (30 μL). The M. luciferase expression vector (2 ng) was transfected.

After treatment with transfection for 3 hours, the cells were detached by washing with PBS-EDTA and trypsin treatment, and were seeded again on a 96-well plate at 1 × 10 ⁴ cells. After confirming that the cells have adhered to the plate, add 10% serum-containing medium containing 10 μL of triaxin C (BI0M0L Research Laboratories) (final concentration: 0.1 μΜ, 1 μM 10 μΜ). (D-MEM) was added. Luciferase activity was measured 24 hours after the addition of triaxin C. The activity of HNF 4a was shown as the ratio of firefly luciferase activity to D. luciferase activity. The Luciferase activity was measured using a Wallac 1420 ARVOsx multilabel counter. The result is shown in figure 2.

 As shown in Fig. 2, when the specific activity in the medium without triaxin C was set to 100 ° Zo, the HNF4α activity was increased by adding 0.1 μΜ, 1 and 10 μ は of triaxin C, respectively. 89.9, 68.5 and 31.0%. This result suggests that the addition of triacsin to HNF4a-expressing cells obtained by previously cloned cells can suppress serum-stimulated activation of HNF4.

 Industrial applications

Screening of HNF4α agonists in the presence of an acyl C Α synthase inhibitor such as triacsin to suppress the activation of HNF4α caused by endogenous HNF4α agonists Becomes possible. Therefore, it is possible to more accurately detect the HNF4α agonist activity and obtain various types of agonists, thereby providing a useful tool for the development of a novel therapeutic agent for diabetes. This application is based on a patent application No. 2001-073167 filed in Japan, the contents of which are incorporated in full herein.

Claims

The scope of the claims 1. (l) A cell expressing the HNF4α gene and expressing the reporter gene under the control of the promoter of a gene whose expression level is regulated by HNF4α, A method of contacting a test compound in the presence of: and (2) a step of measuring the expression of a reporter gene in the cell.  2. The screening method according to claim 1, wherein the promoter of a gene whose expression level is regulated by HNF4 is HNF1 promoter. 3. The screening method according to claim 1, wherein the acyl CoA synthetase inhibitor is triaxin C.  4. The screening method according to claim 2, wherein the acyl CoA synthetase inhibitor is triaxin C. 5. The screening method according to claim 1, wherein the HNF4agonist is _a drug for treating diabetes.  6. The screening method according to claim 2, wherein the HNF 4 agonist is a drug for treating diabetes.  7. The screening method according to claim 3, wherein the HNF agonist is a drug for treating diabetes. 8. The screening method according to claim 4, wherein the HNF4α agonist is a drug for treating diabetes.  9. An HNF4α agonist obtained by the screening method according to claim 1.