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WO2002072874A1 - Procede de criblage d'un agoniste hnf4$g(a) - Google Patents

Procede de criblage d'un agoniste hnf4$g(a) Download PDF

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Publication number
WO2002072874A1
WO2002072874A1 PCT/JP2002/002325 JP0202325W WO02072874A1 WO 2002072874 A1 WO2002072874 A1 WO 2002072874A1 JP 0202325 W JP0202325 W JP 0202325W WO 02072874 A1 WO02072874 A1 WO 02072874A1
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Prior art keywords
hnf4α
gene
cells
promoter
vector
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English (en)
Japanese (ja)
Inventor
Junichiro Miyagawa
Kazuya Yamagata
Yuji Matsuzawa
Tadashi Yamamoto
Chiaki Kimura
Ikuo Kawamura
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Fujisawa Pharmaceutical Co Ltd
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Fujisawa Pharmaceutical Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • the present invention relates to a staring method for HNF 4 agoquest.
  • HNF 4a hepatocyte nuclear factor 4 ⁇
  • SHN F4 ⁇ a genetic abnormality in SHN F4 ⁇
  • MODY Maturity-onset type diabetes of the young
  • 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.
  • 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.
  • 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 ⁇ is involved in pancreatic ⁇ -cell-specific transcription of the pdx-1 gene.Mol.Cell Biol.
  • the HNF4agonist is useful not only for analyzing the function of HNF4 ⁇ 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.
  • it aims to provide HNF4 ⁇ agonists useful as therapeutic agents for diabetes.
  • 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 ⁇ under serum-added culture conditions
  • HNF4 ⁇ 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 ⁇ synthase inhibitor.
  • 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 HNF4 ⁇ 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.
  • FIG. 1 is a graph showing the effect of triaxin C on HNF4a activity in HNF4 ⁇ -expressing cells transfected with an HNF4 ⁇ expression vector and in control cells not expressing HNF4a.
  • FIG. 2 is a graph showing the effect of triaxin C on HNF4 ⁇ activity in previously cloned HNF4 ⁇ -expressing cells.
  • 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 4 ⁇ .
  • a substance having an action of enhancing the binding ability of ⁇ is intended.
  • An example is a substance that increases the ability of HNF4 ⁇ to bind to the HNF1 ⁇ promoter.
  • 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.
  • the action of HNF4a which binds to the HNF1 ⁇ promoter and subsequently promotes transcription and expression of genes downstream of the HNFla motor.
  • 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,
  • HNF4a expresses HNF4a and expresses a reporter protein under the control of a promoter of a gene whose expression level is regulated by HNF4.
  • HNF4a 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.
  • 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 HNF4 ⁇ -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.
  • HNF1 promoter 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.
  • PLL pyruvate kinase-L
  • PEPCK PEPCK promoter
  • an HNF1a promoter which is usually present downstream of the HNF4 gene and whose expression is regulated by HNF4 ⁇ is used.
  • HNF4 ⁇ 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 HNF4 ⁇ activity is determined. Is measured to obtain cells with high HNF4 ⁇ activity, that is, cells with high expression of HNF4 ⁇ .
  • 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.
  • 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.
  • a selectable marker gene a gene conferring resistance to a drug such as neomycin, zeocin, hygromycin, Auxotrophic mutation, etc.
  • a selectable marker gene a gene conferring resistance to a drug such as neomycin, zeocin, hygromycin, Auxotrophic mutation, etc.
  • the HNF4 ⁇ -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.
  • 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 HNF4 ⁇ ; contained in the expression vector may be a DNA known per se, for example, the sequence described in Gene (1994), 147: 269-272.
  • 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
  • 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.
  • 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.
  • 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)).
  • 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 HNF4 ⁇ ) 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 HNF1 ⁇ promoter, and such HNF1 ⁇ 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.
  • a reporter vector containing a luciferase gene as a reporter gene under the control of the HNF1 ⁇ promoter is commercially available (eg, Promega).
  • HNF4 ⁇ expression cassette and the reporter protein expression cassette so that they are on the same vector. Can be.
  • 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, ⁇ -galata tosidase (/ 3-Gal) gene, and the like.
  • CAT chloramphenicol acetyl acetyltransferase
  • GFP green 'fluorescein' protein
  • ⁇ -galata tosidase ⁇ -galata tosidase (/ 3-Gal) gene, and the like.
  • a second 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.
  • 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.
  • 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.
  • HNF4 ⁇ expression vector 0.2 to 2.5 / ig per 1 to 4 ⁇ 10 4 cells repo ⁇ "ter vector 0.2 to 2.5. 5 ⁇ g, 0.25 to 2.5 ng of the control reporter vector are introduced, and the control vector without the HNF4 ⁇ expression cassette used as a control is used for the assay. It is used in an amount according to the HNF4 ⁇ expression vector.
  • control vector is equivalent to the HNF4 ⁇ expression vector except that it does not have the HNF4 ⁇ expression cassette.
  • the HNF4 ⁇ 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.
  • 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 HNF4 ⁇ 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).
  • control cells that do not express HNF4 ⁇ ie, cells that are equivalent to the test cells except that they do not express ⁇ F4 ⁇
  • 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 ⁇ synthase inhibitor.
  • 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).
  • CoA coenzyme A
  • No particular limitation is imposed as long as it inhibits the activity of the enzyme.
  • 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.
  • 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 ⁇ / M or more, preferably about 10 or more.
  • test cell and / or the test compound 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.
  • the following method is exemplified. ⁇
  • the cells After transfection of the reporter vector into the HNF4 ⁇ 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.
  • 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 ⁇ progenitor cells differentiate into ⁇ 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.
  • the reporter gene to be used is a firefly luciferase gene, beetle luciferin or the like is used as a substrate
  • the reporter gene used is a mushroom luciferase gene, coelenterazine is used as a substrate
  • 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.
  • the activity of the reporter gene 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.
  • 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.
  • an HNF4 expression vector 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
  • 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.
  • HNF4 ⁇ 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.
  • the step of measuring the expression of the reporter gene can be performed using commercially available reagents, kits and the like.
  • HNF4 ⁇ -agonists 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.
  • 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.
  • 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.
  • D-MEM serum-supplemented culture medium
  • control vector a vector similar to the above HNF4 expression vector except that it does not contain the HNF4 expression cassette
  • reporter vector a vector similar to the above HNF4 expression vector except that it does not contain the HNF4 expression cassette
  • control vector reporter vector were subjected to lipofection method.
  • 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 ⁇ g of each vector and 18 ⁇ L of plus reagent and 8 ⁇ L of lipofectamine per 1 ⁇ L of a 6-well plate.
  • HNF4 ⁇ expression vector Seed 4 x 10 5 HeLa cells per 6 cm culture dish and transfected with 1.5 ⁇ g of HNF4 ⁇ expression vector, 18 AtL plus reagent, and 8 L of lipofectamine. went.
  • HNF4 ⁇ expression vector the HNF4 ⁇ expression vector
  • a colony was selected and seeded on a 24-well plate. For each colony selected, one reporter vector was transfected, and one having strong HNF4 ⁇ activity was selected.
  • HeLa cells (1 ⁇ 10 6 ) stably expressing HNF4 ⁇ were seeded on a 10-cm culture dish, and the HNF1 ⁇ promoter firefly luciferase vector (Promega) was prepared using the lipofection method. Mistake luciferase expression vector (Internal standard; Promega) was transferred.
  • the HNF1 promoter / firefly luciferase vector (4 ⁇ g) and ⁇ were added using a plus reagent (20 ⁇ L) and ribophenetoamine (30 ⁇ L).
  • the M. luciferase expression vector (2 ng) was transfected.
  • 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 ⁇ L of triaxin C (BI0M0L Research Laboratories) (final concentration: 0.1 ⁇ , 1 ⁇ M 10 ⁇ ). (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.

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Abstract

L'invention porte sur un procédé de criblage d'un agoniste HNF4α qui consiste à (1) mettre un composé test en contact avec des cellules exprimant un gène HNFα et exprimant également un gène rapporteur sous la régulation d'un gène promoteur dont l'expression est contrôlée par HNF4α, en présence d'un inhibiteur de synthase acyle CoA ; et (2) mesurer le degré d'expression du gène rapporteur dans les cellules précitées. Selon ce procédé, il est possible d'inhiber l'activation de HNF4α issu d'une substance contenue dans le système de criblage et de pouvoir ainsi cribler un agoniste HNF4α de façon plus précise avec un échantillon de contrôle réduit.
PCT/JP2002/002325 2001-03-14 2002-03-13 Procede de criblage d'un agoniste hnf4$g(a) Ceased WO2002072874A1 (fr)

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Cited By (1)

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WO2006008008A3 (fr) * 2004-07-23 2006-06-22 Bayer Healthcare Ag Agents diagnostiques et therapeutiques pour pathologies associees au facteur nucleaire d'hepatocyte 4, alpha (hnf4a)

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WO1992011365A1 (fr) * 1990-12-21 1992-07-09 The Rockefeller University Facteur de transcription enrichi par extraits hepatiques
WO1998021363A1 (fr) * 1996-11-15 1998-05-22 Millennium Pharmaceuticals, Inc. Compositions et therapies utilisables contre le diabete de type ii lie au hnf-4
WO2000017334A2 (fr) * 1998-09-23 2000-03-30 Ludmila Solomin Analyse de recepteurs nucleaires actives par des ligands in vivo

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