WO1999040202A1 - INHIBITEURS DE L'ACTIVATION DU FACTEUR NUCLEAIRE NF λB CIBLES SUR LA KINASE 1 ACTIVEE PAR LE FACTEUR DE CROISSANCE TRANSFORMANT BETA (TAK1) ET PROCEDE D'IDENTIFICATION - Google Patents

INHIBITEURS DE L'ACTIVATION DU FACTEUR NUCLEAIRE NF λB CIBLES SUR LA KINASE 1 ACTIVEE PAR LE FACTEUR DE CROISSANCE TRANSFORMANT BETA (TAK1) ET PROCEDE D'IDENTIFICATION Download PDF

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WO1999040202A1
WO1999040202A1 PCT/JP1999/000422 JP9900422W WO9940202A1 WO 1999040202 A1 WO1999040202 A1 WO 1999040202A1 JP 9900422 W JP9900422 W JP 9900422W WO 9940202 A1 WO9940202 A1 WO 9940202A1
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tak1
activation
cells
tak
tab1
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Japanese (ja)
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Takahisa Sugita
Hiroaki Sakurai
Noriko Kageyama
Ko Hasegawa
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Tanabe Pharma Corp
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Tanabe Seiyaku Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to an NF- (Nuclear Factor kappa B) activation inhibitor, and an agent for treating or preventing an autoimmune disease or an intractable disease exhibiting inflammatory symptoms. Further, the present invention relates to these novel screening methods and identification methods.
  • NF- Nuclear Factor kappa B
  • NF- / B known as one of the transcription factors, plays an important role in the transcriptional regulation of various genes involved in inflammatory and immune responses.
  • NF— is a force that exists as an inactive complex that binds to the regulatory protein I ⁇ ⁇ .
  • I / c ⁇ modifies and degrades. It is activated by detaching from the receiving complex.
  • the NF-II thus activated is translocated into the nucleus, and the specific nucleotide sequence (NF-/-consisting of about 10 bases) present in the upstream region (enhansa-one region) of various genes on the genome DN- cB binding sequence) to activate gene transcription.
  • the NF- / cB binding sequence is present not only in the immunoglobulin gene but also in the upstream region of genes such as IL-1 and inflammatory cytokines such as tumor necrosis factor, interferon, and cell adhesion factor.
  • A: B is involved in inflammation and immune response through induction of expression of these genes.
  • NF- is also involved in the pathogenesis of autoimmune diseases and inflammatory diseases.
  • Drugs that inhibit the activation of NF_ / cB are used in autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, systemic scleroderma) Disease, Behcet's disease, periarteritis nodosa, ulcerative colitis, glomerulonephritis, etc., intractable diseases with inflammatory symptoms (osteoarthritis, atherosclerosis, psoriasis, atopic dermatitis, etc.) It is known to be effective in treating and preventing various diseases such as viral diseases, endotoxin shock, and sepsis.
  • B activation inhibitors involve screening or identifying cells in vitro in the presence (or absence) of a stimulus, in the presence of a test drug, or in the presence of a test drug.
  • a method of detecting the activity of NF-AB by culturing in the absence of NF-AB is generally used.
  • the signal transduction pathway from the time when a cell receives a certain stimulus (signal) to the activation of NF- / cB is the existence of many steps involving various transduction molecules such as protein kinases. it is conceivable that. Therefore, for more efficient drug discovery research, it is desirable to clarify the key molecules that play a key role and to establish a new drug screening method that focuses on them.
  • NF- / B NF-a receptor associated factor 2
  • MAP KKK mitogen-activated protein kinase kinase kinase
  • NIK NF- ⁇ -inducing kinase
  • IKK I / cB kinase
  • ubiquitin-conjugating enzyme 26S proteosome, etc.
  • TGF-activated kinase 1 Transforming growth factor- ⁇ -activated kinase 1; also referred to as “TAK 1” was discovered as one of mammalian MAP KKKs (Yamaguchi et al. Science, vol. 270, pp. 2008-2011, 1995; JP-A-9-163990). TAK 1 is TGF— /?
  • TGF- transforming growth factor- ⁇
  • TAK1 becomes active by binding (interacting) with TAK1 binding protein 1 (TAB1), and it can function as MAPKKK in the signal transduction pathway. It is known (Shibuya et al., Science, Vol. 272, Vol. 1179-: 1182, 1996). However, nothing was known about the association between TAK1 and NF— / cB activation.
  • the present inventors isolated three allelic variants of human TAK1 cDNA, and furthermore, in a study using these, increased expression of human TAK1 together with TAB1 (over expression).
  • TAK1 interacts with TAB1 and interacts with IKK (I / cB kinase) complex to activate it.
  • IKK I / cB kinase
  • TAK1 force s an important signaling molecule in the signal transduction pathway (NF- / B activation pathway) leading to NF- ⁇ B activation, and drugs that suppress the function of TAK1 DISCLOSURE OF THE INVENTION that led to completion of the present invention, which was found to be a NF- / B activation inhibitor
  • the present invention provides a method for identifying or screening for a NF—; cB activation inhibitor, which comprises a step of assaying the modulatory effect of a test substance on the function of TAK 1 (TGF—; reactive kinase 1 :). Is the way.
  • the present invention provides a therapeutic and / or prophylactic agent for an autoimmune disease or an intractable disease exhibiting inflammatory symptoms, which comprises a step of assaying a modulatory effect of a test substance on the function of TAK1 in the NF- ⁇ activation pathway.
  • the present invention is a novel NF-activation inhibitor selected or identified by the above-mentioned method, and a therapeutic or prophylactic agent for an autoimmune disease, an intractable disease exhibiting inflammatory symptoms, or the like.
  • FIG. 1 shows a comparison of the amino acid sequences of mouse TAK1 (mTAK1) and three kinds of human ⁇ 1 (hTAKla, hTAK1b and hTAK1c);
  • C A diagram showing the results of electrophoresis showing NF- / cB activation (nuclear translocation of NF-KB in gel shift assay) in cells in which TAK1 and TAB1 have been enhanced in expression;
  • Fig. 3 shows NF- / cB activation (luciferase activity in reporter attase) of cells in which expression of human TAK1 and TAB1 was enhanced;
  • Fig. 4 shows mutant humans. Diagram showing suppression of NF-KB activation in cells expressing TAK1 (results from gel shift to (A) and reporter to (B));
  • Figure 5 shows the results of immunoblotting of immunoprecipitated fractions containing TAK1 obtained from cells with enhanced expression of human TAK1 (intracellular interaction between TAK1 and TAB1).
  • Figure 6 shows the results of kinase assay of the immunoprecipitated fraction containing TAK1 obtained from cells with enhanced expression of human TAK1 (autophosphorylation of TAB1 by TAK1). ;
  • Fig. 7 shows the results of immunoplating of immunoprecipitated fractions containing TAK1 and cell lysates obtained from cells with enhanced expression of human TAK1 (interaction between TAK1 and IKK in cells).
  • Fig. 8 shows the results of IKK kinase assay (activation of the IKK complex by TAK1) of immunoprecipitated fractions containing IKK obtained from cells with enhanced expression of human TAK1.
  • FIG. 9 is a schematic diagram showing the function of TAK 1 in the NF- ⁇ B activation pathway (in the figure, TRAF2 indicates TNF — ⁇ receptor 1 * association factor-12, IKK indicates I / B Kinase, NIK is NF- / B-inducing kinase, NEMO is NF- ⁇ ⁇ Essential. 11:? 11 111: Complex 'associated' protein, Respectively).
  • TAK1 used in the present invention may be derived from any species, for example, derived from mammals such as human, mouse, rat, peacock, bush, dog, monkey, guinea pig and the like. No. Of these, the use of human-derived drugs is preferred for use in research and development of human therapeutics.
  • TAK 1 The cDNA and amino acid sequences of TAK 1 have already been reported
  • SEQ ID NOs: 3, 4, and 5 in the sequence listing below include the DNA sequences of three allelic variants of human TAK1 cDNA newly discovered by the inventors and the TAK encoded by them. The amino acid sequence of 1 was shown.
  • TAK1 functions as a main transmitter molecule in the NF- / cB activation pathway.
  • TAK 1 is activated by interacting (binding) with TAB 1 (TAK 1 binding protein 1) in cells, and becomes an active form showing protein kinase activity (MAPKKK activity). And phosphorylation of TAB1. TAK 1 also interacts functionally with the IKK complex. Activated TAK1 is thought to activate the IKK complex, function as a transfer molecule in the NF- activation pathway, and induce NF- / cB activation. Replacement form (Rule 26) 4/1
  • FIG. 9 shows a schematic diagram of the function of TAK1 in the NF-KB activation pathway.
  • the function of the test substance is assayed by focusing on the function of TAK1 as described above (particularly, the function in the activation pathway of NF-KB).
  • TAK 1 and TAB 1 When detecting the binding between TAK 1 and TAB 1, TAK 1 and TAB 1 may be used in their entirety, or at least a partial polypeptide containing a region involved in the binding of both. Good. Alternatively, use an appropriate tag label (glutathione-S-transferase, 6XHis, protein A, galactosidase, maltose-binding protein, flag antigen, Xpress antigen, HA antigen, Myc antigen, etc.) A fusion protein to which a partial polypeptide or the like has been added may be used.
  • an appropriate tag label glutase, 6XHis, protein A, galactosidase, maltose-binding protein, flag antigen, Xpress antigen, HA antigen, Myc antigen, etc.
  • a fusion protein to which a partial polypeptide or the like has been added may be used.
  • TAK 1 or TAB 1 labeled with RI, etc.
  • TAK 1 or TAB 1 labeled with RI, etc.
  • TAK 1 or TAK 1 labeled with RI, etc.
  • an antibody that recognizes TAK1, TAB1, or a tag label added thereto is used for detection.
  • a cell lysate is prepared from cells expressing TAK1 and TAB1, and the protein in the cell lysate is immunoprecipitated using an antibody that recognizes one of the proteins.
  • the interaction (coupling) of both proteins in the cell can be detected.
  • the two-hybrid system is a method using marker expression of a reporter as a marker (US Pat. No. 5,283,173 and roc.Natl.Acad.Sci. USA, Vol. 88, 9578-9958). P. 1991).
  • the genes encoding the first and second fusion proteins can be designed and constructed using conventional gene recombination techniques.
  • Host cells include, for example, yeast cells, insect cells, mammalian cells, and the like.
  • yeast cells are advantageous in that they can be cultured easily and quickly, and that it is easy to apply genetic recombination techniques such as introduction of a foreign gene.
  • the transcription factor may be any as long as it functions in the host cell.
  • yeast GAL4 protein (Keegan et al., Science, 231: 699-704, 1986, Ma et al., Cell, 48) Pp. 847-853, 1987)
  • GCN4 protein Hope et al., Cell, Vol. 46, 885-894, 1986
  • ADR1 protein Thukral et al., Molecular and
  • the response element may be a response element corresponding to a transcription factor.
  • the response element may be UASg (upper region of galactose metabolism).
  • Site A GAL4-specific DNA sequence called upstream activation site of galactose genes) can be used.
  • the reporter gene is also not particularly limited.
  • stable and active genes such as the E. coli-derived /? Galactosidase gene (1 ac Z), the bacterial transposon-derived chloramphenicol acetyltransferase gene (CAT), and the firefly-derived luciferase gene (Luc).
  • An enzyme gene or the like which can be easily measured quantitatively can be suitably used.
  • a solution containing TAK 1 and TAB 1 and a solution containing ATP (labeled with RI or the like as necessary) are added to the solution containing the substrate protein, and the solution is added in the presence or absence of the test substance.
  • An enzymatic reaction is performed, and protein kinase activity is measured using the incorporation of phosphoric acid into the substrate protein as an index to test the effect of the test substance.
  • Chohachi 1 1 and Choha81, those expressed in a suitable host cell (eg, a yeast cell, an insect cell, or a mammalian cell) by a gene recombination technique can be used. 7
  • TAK1 the N-terminal region of TAK1 is involved in binding to TAB1, and TAK1 in which the N-terminus (22 amino acids on the N-terminal side) has been deleted has an active signal even when it does not bind to TAB1. Since it is known to act as a transfer molecule (Yamaguchi et al., And Shibuya et al.), Instead of using both TAK1 and TAB1, an active mutation that deletes the N-terminus and shows TAB1-independent activity You can use type TAK 1.
  • TAK1 itself, TAB1, or a partial peptide thereof can be used.
  • a molecule functionally interacting with IKK and the IKK complex or a partial peptide thereof can also be used as a substrate protein.
  • XMEK 2 SEK 1 of African Megafrog (Shibuya et al., Science, Vol. 272, No. 1179-: 1182, 1996), human MKK3 (Derijard et al., Science, Vol. 267, No. 682) ⁇ 685, 1995), Human MKK6 (MAPKK6)
  • Protein kinase kinase and their partial peptides can also be used as substrates.
  • TAK 1 protein kinase activity can be measured using MAP KK activation (increase in phosphorylation activity against MAPK (mitogen-activated protein kinase)) as an index.
  • test cells use cells overexpressing TAK1 (more specifically, active TAK1) as test cells .
  • TAK1 more specifically, active TAK1
  • test cells include cells having enhanced expression of both TAK1 and TAB1, and can be obtained by introducing a vector for expression of TAK1 and TAB1 into an appropriate host cell.
  • cells in which the N-terminus is deleted and the expression of an active mutant TAK1 exhibiting TAB1-independent activity is enhanced may be used.
  • test cells are cultured, for example, in the presence or absence of a test substance.
  • a fraction containing the IKK complex is obtained from the cultured cells by immunoprecipitation or the like, and is used to perform an IKK kinase reaction, measure the activity of the IKK complex, and determine the effect of the test substance. Is tested.
  • cells that enhance the expression of active TAK1 are used as test cells, and cultured in the presence or absence of a test substance.
  • NF- / cB activation is detected by gel shift assay and the effect of the test substance is assayed.
  • Activated TAK1 expression-enhancing cells are control cells.
  • TAK1 which acts as a signal transduction molecule
  • TAK1 acts as a signal transduction molecule
  • cell lines derived from mammals such as humans can be suitably used.
  • human HeLa cells, human Jurkat cells, and human cells examples include THP-1 cells, monkey COS-7 cells, Chinese Hamster CHO cells, and the like. Of these, human HeLa cells, human Jurkat cells, and human THP-1 cells are preferred.
  • TAK1, TAB1, or a fusion protein thereof when expression of TAK1, TAB1, or a fusion protein thereof is enhanced, it can be carried out using known sequence information and ordinary gene recombination techniques.
  • TAK1 The sequence information of TAK1 is as described above, and the cDNA sequence and amino acid sequence of TAB1 have also been reported (Genbank / EMBL database Accession No. U49928; Shibuya et al., Science, Vol. 1179-; 1182, 1996).
  • TAB1 may be derived from any species, and includes, for example, those derived from mammals such as human, mouse, rat, porcupine, pig, dog, monkey, and guinea pig. Of these, the use of human-derived drugs is preferred for use in research and development of human therapeutics.
  • CDNAs or genes such as TAK 1 and TAB 1 can be synthesized using primers and probes designed and synthesized based on information on known amino acid sequences and base sequences, using ordinary PCR (Polymerase Chain Reaction) and RT-PCR. It can be isolated by a method or screening from a DNA library. These can be incorporated into an appropriate vector to construct an expression vector.
  • the vector of all a suitable promoter (eg, CMV promoter, SV 40 promoter, LTR promoter, Eronge one Chillon 1 alpha promoter one, etc.) vector for animal cells (e.g., retroviral-based vectors, Papi port - Mavirus vector, vaccinia virus vector, SV40 vector, etc.) can be used.
  • a suitable promoter eg, CMV promoter, SV 40 promoter, LTR promoter, Eronge one Chillon 1 alpha promoter one, etc.
  • retroviral-based vectors e.g., Papi port - Mavirus vector, vaccinia virus vector, SV40 vector, etc.
  • NF- «B activation is performed by the known gel shift assay (Sakurai et al., Journal of Neurochemistry Vol. 59, pp. 2067-2075, 1992; Sakurai et al., Biochimica Biophysica Acta, Vol. 1316, pp. 132-138) , 1996), reporter method (Tanaka et al., Journal of Veterinary Medical Science, Vol. 59, pp. 575-579, 1997; EP-6252920-A; JP-A-7-2) (Japanese Patent Application Laid-Open No. 9-158559; Japanese Patent Application Laid-Open No. Hei 9-2217561).
  • a PHA-induced IL-12 production model using a human T cell line (Jurkat cells) (Wacholtz et al., Cell Immunology 135, pp. 285-298, 1991), LP S + IFN-y-induced iNOs production model using human macrophage cell line RAW2 64.7 (Xie et al., Science, vol. 256, vol. 225-228, 1992) and in vitro models such as TNF-induced IL-6 production model using human HeLa cells, rat adjuvant arthritis model (Connor et al., European Journal of Pharmacology, vol. 273). Pp.
  • RNA was prepared from human cervical carcinoma-derived cell line HeLa (ATCC CCL2). This was designated as ⁇ , and a single-stranded cDNA was prepared using an oligo dT primer.
  • the single-stranded cDNA obtained above was designated as type III, and a human TAK1 cDNA fragment was obtained by PCR (polymerase chain reaction). Used for PCR Ten
  • the primer was the cDNA sequence of mouse TAK1 (Genbank / EMBL database Accession No. D76446; Yamaguchi, Science Vol. 270, pp. 2008-2011,
  • a 30-mer synthetic primer consisting of a sequence containing a recognition site for restriction enzyme cleavage (10 bases) and a translation initiation codon of mouse TAK1 cDNA and a downstream sequence (20 bases) (described later)
  • SEQ ID NO: 1 in the sequence listing
  • SEQ ID NO: 2 in the sequence listing below
  • HeLa mRNA prepared in the same manner as described above was converted into type III, and cDNA (h) containing the entire coding region of human TAK1 was separately separated by RT-PCR (Reverse transcript-polymerase chain reaction). TAK1c—cDNA) was obtained.
  • a primer the same synthetic primer as described above was used.
  • the DNA sequences of the three obtained cDNAs were determined by the dideoxy method.
  • the DNA sequence of the region including the coding region and the human TAK1 hTAK1
  • SEQ ID NO: 3 SEQ ID NO: 4
  • SEQ ID NO: 5 SEQ ID NO: 5
  • cDNA sequences of hTAK la, hTAK lb and hTAK lc show homology in the coding regions of 91.7%, 87.6% and 86.8%, respectively, as compared to the cDNA sequence of mouse TAK1. Atsuta.
  • hTAK1a consists of 579 amino acid residues. Compared to mouse TAK1, 4 amino acid substitutions were observed, and the homology in the amino acid sequence was 99.3%.
  • hTAKlb is composed of 606 amino acid residues, and has an insertion of 27 amino acids which seems to be caused by a splicing mutation at the C-terminal side as compared with hTAK1a.
  • HTAK1c is composed of 567 amino acid residues. Compared to hTAK1a, hTAK1b has a 27-amino acid insertion at the C-terminal, similar to hTAK1b, and 3 A 9 amino acid deletion was found.
  • TAK1 derived from human T cell line Jurkat described in SEQ ID NO: 5 of JP-A-9-1163990 has one amino acid substitution (the 372nd A amino acid) when compared with the amino acid sequence of hTAK1a. rg ⁇ His), which is considered to be an allelic variant.
  • RNA prepared from HeLa in the same manner as in (1) above was used as type III, and cDNA of human TAB1 was obtained by RT-PCR.
  • the primer was designed with reference to the reported cDNA sequence of human TAB1 (Genbank / EMBL database Accession No. U49928; Shibuya et al., Science. Vol. 272, pp. 1179-1182, 1996). And synthesized on a DNA synthesizer.
  • a sense primer a 30-mer synthetic primer consisting of a sequence containing a recognition site for restriction enzyme cleavage (10 bases) and a translation initiation codon of TAB1 cDNA and a sequence downstream thereof (20 bases) (later).
  • the antisense primer includes a sequence containing a recognition site for restriction enzyme cleavage (10 bases) and the termination codon of TAB1 cDNA and its upstream complementary sequence (20 bases).
  • a 30-mer synthetic primer consisting of bases) was used.
  • Example 2 Detection of NF-A: B Activity in Cells with Enhanced TAK1 Expression (1) Obtaining Cells with Enhanced Human TAK1 Expression
  • the recombinant plasmid for TAK1 expression was transfected (transient transfection) into HeLa cells together with the recombinant plasmid for TAB1 expression or alone. At this time, the transfection was performed using a cationic ribosome for transfection (trade name: LipofectAMINE, manufactured by Life Technologies). 12
  • cells with enhanced TAK1 expression or cells with enhanced TAK1-TAB1 expression were obtained. These cells were cultured in Dulbecco's Eagle's medium (Gibco) containing high glucose supplemented with 10% fetal serum, penicillin (100 units Zml) and streptomycin (100 gZml). .
  • This nuclear extract (5 / g) and a detection probe labeled with RI were combined with a binding buffer (20 mM HEPES (pH 7.9), 0.3 mM EDTA, 0.2 mM EGTA, 80 mM NaCl, 10% glycerol, 2 g
  • a binding buffer (20 mM HEPES (pH 7.9), 0.3 mM EDTA, 0.2 mM EGTA, 80 mM NaCl, 10% glycerol, 2 g
  • the reaction solution was subjected to polyacrylamide gel electrophoresis. After the gel was dried under reduced pressure, NF- / cB bound to the probe was detected by autoradiography.
  • 0ct-] (Octamer-1) is a constitutively expressed transcription factor.
  • a detection probe a double-stranded synthetic DNA labeled with 32 P was used.
  • sequence of the NF- ⁇ NF detection probe the same sequence as the NF-A: B binding sequence present in the LTR (Long Terminal Repeat) of HIV was used.
  • oligo nucleotides containing the consensus sequence AGCTAAAT were used as the sequence of the Oct-1 detection probe.
  • TAK1 As described above, activation of NF- / cB was observed in association with enhancement of the action of human TAK1, and thus TAK1 was expressed in the signal transduction pathway leading to the activation of NF- ⁇ B. It turned out to play a major role as a transfer molecule.
  • a reporter plasmid (p (kB) 4-Luc) was prepared by incorporating an oligonucleotide linked to four NF-KB binding sequences (GGGGACTTTCC) upstream of the firefly luciferase gene (Luc).
  • the recombinant plasmid for TAK1 expression together with the recombinant plasmid for TAB1 expression, if necessary, is transfected into HeLa cells (transient) according to the method described in (1) above. Transfection (transient transfection). However, the reporter plasmid (p (kB) 4-Luc) obtained above was used for the transfusion.
  • transfectants containing reporter plasmid and recombinant plasmid for TAK1 expression were obtained.
  • the luciferase activity was measured for the extract prepared by lysing the cells.
  • the luciferase activity was measured using a luciferase zea kit, Pitka Gene (trade name, manufactured by Toyo Ink Co., Ltd.) and a chemiluminescence measuring device (trade name: MicroLumant LB96P, manufactured by Berthold Japan KK).
  • cells expressing only human TAK1 had enhanced luciferase activity compared to cells containing only the vector. Little increase (ie, activation of NF- / cB) was observed. However, cells with enhanced expression of human TAK1 together with TAB1 showed a marked increase in luciferase activity (ie, the ability of NF- ⁇ to activate) as compared to cells containing only vector-1. s accepted.
  • the reporter assay (Lucifera assay) also increases the activity of ⁇ F— ⁇ with the enhanced action of human TAK1. observed, TAK 1 force? this and the force that is the major acts as a signaling molecule? has been confirmed.
  • the translation region of the human TAK1 cDNA obtained in (1) of Example 1 was cut out and ligated with the DNA binding region of the transcription factor GAL4 (amino acid residues 1 to 147 of GAL4).
  • GAL4 amino acid residues 1 to 147 of GAL4
  • pGBT9 a vector for the yeast two-hybrid system
  • the translation region of the human TAB cDNA obtained in (2) of Example 1 was cut out, and this was ligated to a DNA encoding the transcriptional activation region of GAL4 (amino acid residue at position 768 to 881 of GAL4).
  • pGAD424 (Clontech, vector for yeast two-hybrid system).
  • the fusion protein expression plasmids pGBT9-TAKl and pGAD424-TABl obtained above are introduced into a host yeast cell strain SFY526 (manufactured by Clontech).
  • the cell line SFY526 is a cell line in which the fusion gene of GAL1 and lacZ is integrated into the chromosome and has a deletion mutation in the GAL4 gene (Bartel et al., BioTechniques, Vol. 14, pp. 920-924). , 1993). Transformation is carried out by culturing in a synthetic medium lacking tryptophan and leucine, which are selective markers for each plasmid, to obtain a transformant in which both plasmids have been introduced.
  • the yeast transformant obtained above is cultured in a liquid medium. At the time of culturing, the test substance is added (or not added) to the medium. After culturing for 4 to 5 hours, the yeast cells are recovered by centrifugation, and the binding (interaction) between TAK1 and TAB1 is detected using the /?-Galactosidase activity as an index.
  • test substance decreases the concentration of -galactosidase activity in a concentration-dependent manner, the test substance is considered to have an effect of inhibiting the binding between TAK1 and TAB1.
  • Human TAK1 (or human TAK1 lacking the N-terminus (22 amino acids)) is expressed and purified in an insect cell system as follows. That is, an appropriate DNA sequence designed to add a tag peptide (6XHis or glutathione-S-transferase) using the translation region of human TAK1 cDNA obtained in (1) of Example 1 above.
  • LT multicloning site of LT (Pharmingen) to obtain human TAK1 expression plasmid.
  • the resulting plasmid is introduced into host insect cells SF21, and the resulting transformed cells are cultured to express human peptide-tagged human TAK1 (or N-terminal-deleted human TAK1). Then, the extract is purified from the cell extract by affinity chromatography using the added tag peptide.
  • human TAB1 is expressed and purified in an insect cell system.
  • human MKK3 and human MKK6 are expressed and purified as follows. First, according to the method of Moriguchi et al. (Journal of Biological Chemistry, Vol. 271, pp. 13675-13679, 1996), sequence information on human MKK3 (Genbank / EMBL database Accession No. L36719) Derijard et al., Science, 267, 682-685, 1995) and sequence information on human MKK6.
  • the resulting plasmid is introduced into host Escherichia coli (such as the strain JM109), and the resulting transformed cells are cultured to express human MKK3 and human MKK6 to which the tag peptide has been added, respectively.
  • the solution is purified by affinity chromatography using the added tag peptide.
  • human TAK 1 (or N-terminal-deleted human TAK 1) obtained above as an enzyme (MAPKKK) in combination with human TAB 1 as necessary, using human MKK3 or human MKK6 as a substrate, Perform the enzymatic reaction in the presence or absence of the test substance.
  • Substrate protein is used immobilized by the pre-plate, the reaction is Tris buffer containing 32 P or 33 P-labeled ATP100 M (20m Tris - HC1, pH7.5, 2mM EGTA, lOmM MgCl 2) in 30 ° C Do it.
  • TAK1 and TAB1 expression vectors and transfection vector plasmid pFLAG—CMV2 is a vector for expressing flag antigen-tagged proteins in mammalian cells. is there.
  • human TAK1 human TAK1a
  • pFLAG-CMV2 EcoRI-XbaI restriction enzyme cleavage site of pFLAG-CMV2
  • mutagenesis was performed into the TAK1 translation region of the F1ag-TAK1 expression vector to perform various mutations.
  • the mutant expression vector was obtained and the nucleotide sequence was determined.
  • an expression vector of the flagged mutant TAK1 (F1ag-TAK1K63W) was obtained. Is the mutant TAK1 expressed by this expression vector the lysine residue at position 63 of wild-type TAK1? It has been replaced by a tributofan residue, and has lost the kinase activity of TAK 1.
  • the flag-added wild-type or mutant TAK1 (Flag-TAK1 or F1ag-TAK1K63W) expression vector is transfected into HeLa cells alone or together with the TAB1 expression vector. It was clarified and transiently expressed. As a control, only the vector was used instead of the TAK1 expression vector. The transfection was carried out using a ribofectamine reagent (manufactured by Life Technologies), and the same TAB1 expression vector as that in Example 2 (1) was used.
  • the expression vector of the mutant TAK1 (F1ag-TAK1K63W) obtained in (1) above was transfected into HeLa cells.
  • the amount of F 1 ag—TAK 1 K63W expression vector used for transfection is 0 g, 0.03 ⁇ g, and 0.1 / g, and the total DNA amount is the same (0. 1 / g) 17
  • the reporter plasmid (NF-A: p (kB) 4-Luc containing the NF-A: B binding sequence and the firefly luciferase gene) obtained in (3) of Example 2 was used. At the same time.
  • TNF-— was added to the medium to a final concentration of 20 ng / ml (control was without TNF- ⁇ ). Further, after culturing for 5 hours, the cells were lysed and the luciferase activity was measured in the same manner as in Example 2, (3).
  • Fig. 4 ( ⁇ ) The results are shown in Fig. 4 ( ⁇ ).
  • the unmarked, 10, and ++ of TAK1 K63W indicate the amounts of F1ag-TAK1163W expression vector added at 0 g, 0.03 / g and 0.1 / g.)
  • the increase in luciferase activity (activation of NF-) induced by the TNF- "stimulation was dependent on the dose of the mutant TAK1-expressing vector used in the transfectants. Dependent and suppressed.
  • mutant TAK1 lacking kinase activity suppresses NF_ / cB activation when expressed in cells.
  • TAK1 plays a major role in the NF- / cB activation pathway, as well as the result of (2) above, and also inhibits TAK1 kinase activity and TAK1 activation. This strongly supports that the inhibiting drug suppresses the activation of NF- ⁇ B.
  • TAK1 and TAB1 in cells were detected by immunoprecipitation using cells in which TAK1 expression was enhanced together with TAB1.
  • the expression vector of the wild-type TAK1 (Flag-TAK1) or the mutant TAK1 (F1ag-TAK1K63W) to which the flag was added was used alone. Alternatively, it was transfected into HeLa cells together with the TAB1 expression vector.
  • a cell lysate was prepared as follows. That is, cells were dissolved in a cell lysis buffer (25 mM HEPES (pH 7.7), 0.3 M NaCl, 1.5 mM gCl 2 , 0.2 mM EDTA, 0.1% Triton X-100, 20 mM ⁇ -glycerophosphate, O.lmM sodium orthovanadate, 0.5 mM 18
  • a cell lysis buffer 25 mM HEPES (pH 7.7), 0.3 M NaCl, 1.5 mM gCl 2 , 0.2 mM EDTA, 0.1% Triton X-100, 20 mM ⁇ -glycerophosphate, O.lmM sodium orthovanadate, 0.5 mM 18
  • the cell lysate obtained above was incubated with an anti-flag antibody (M5, manufactured by Kodak) for 1.5 hours on ice and further added with Protein G Sepharose (manufactured by Pharmacia) at 4 ° C for 1.5 hours. With gentle mixing, the immune complexes were adsorbed to the Protein G Sepharose beads. After collecting the beads by centrifugation, the beads are washed five times with a washing buffer (20 mM HEPES (pH 7.7), 50 mM NaCl, 2.5 mM MgCl 2 , O.lmM EDTA, 0.05% Triton X-100). Was used in the following procedure as an immunoprecipitated fraction.
  • a washing buffer (20 mM HEPES (pH 7.7), 50 mM NaCl, 2.5 mM MgCl 2 , O.lmM EDTA, 0.05% Triton X-100.
  • TAK 1 was detected.
  • Antibodies for detecting TAK 1 and TAB 1 include anti-TAK 1 antibody (M-17)
  • FIG. 5 shows the results of immunoplating of the anti-flag immunoprecipitated fraction.
  • the upper row shows the detection results with the anti-TAB1 antibody, and the lower row shows the detection results with the anti-TAK1 antibody.
  • TAB1 coexisted in the anti-flag immunoprecipitated fraction of cells in which wild-type TAK1 (F1ag-TAKl) was enhanced.
  • TAB1 was also co-present in the immunoprecipitated fraction in cells in which the expression of mutant TAK1 (Flag-TAKlK63W) was enhanced in place of the wild type.
  • TAB1 was co-immunoprecipitated with TAK1 (wild type and mutant type), indicating that TAK1 and TAB1 interact in cells.
  • wild-type TAK 1 and TAB 1 both showed a slight decrease in mobility in SDS-polyacrylamide gel electrophoresis when co-expressed, but mutants without kinase activity In the case of type TAK 1, no such decrease in mobility was observed. This decrease in mobility was thought to reflect that both proteins were phosphorylated by functional interaction.
  • TAK1 immunoprecipitated from cells with enhanced expression of TAK1 together with TAB1 was subjected to kinase assay to detect autophosphorylation by TAK1 and phosphorylation of TAB1 as follows. .
  • the expression vector of the flagged wild-type TAK1 (Flag-TAK1) or mutant TAK1 (F1ag-TAK1K63W) was used alone or in TAB.
  • the cells were transfected into HeLa cells together with one expression vector.
  • a cell lysate was prepared from the cells 24 hours after the transfusion in the same manner as in Example 6, and immunoprecipitated with an anti-flag antibody.
  • the immunoprecipitated fraction was treated with a 30/1 kinase buffer (20 mM HEPES (pH).
  • TAK1 was activated by coexisting with TAB1, and that autophosphorylation of TAK1 and phosphorylation of TAB1 by TAK1 occurred.
  • Example 8 Interaction between TAK 1 and IKK in cells
  • the interaction (binding) between TAK1 and IKK in the cells was detected by the epidemicipitation method.
  • the expression vector of IKK was integrated by incorporating the cDNAs of human I ⁇ "and human I ⁇ /? Into vector-plasmid pcDNA3.1 (+) HisB (Invitrogen).
  • Human IKKa (Genbank / EMBL accession No. AF 012890; Cell, Vol. 90, pp. 373-383, 1997), and human I ⁇ /? (Genbank / EMBL accession No. AF029684; Science) , 278, 866-869, 1997) was obtained from human monocyte-derived cell line (THP-1) mRNA by reverse transcription PCR (Reverse transcriptase-polymerase chain reaction). Was used.
  • I ⁇ expression vectors I ⁇ expression vector and I I /? Expression vector
  • IKK Xpress-IKK ⁇ or Xpress-I ⁇ / ?) Can be expressed.
  • the expression vector of the flagged wild-type TAK1 (Flag-TAK1) was transfected into HeLa cells alone or together with the TAB1 expression vector.
  • the expression vector of IKK (Xpress- ⁇ or Xpress-IKK / 3) obtained above was simultaneously added (or not added) and transfection was performed.
  • a cell lysate was prepared from the cells 24 hours after the transfusion and immunoprecipitated with an anti-flag antibody in the same manner as in Example 6. After immunoprecipitation fraction and cell lysate were subjected to SDS-polyacrylamide electrophoresis, immunoblotting was performed to detect IKK and TAK1.
  • Antibodies for detecting IKK include anti-Xpress antibody (M—21) (manufactured by Santa Cruz Biotechnology) and anti-TAK1 antibody (M-17) (manufactured by Santa Cruz Biotechnology) was used.
  • FIG. 7 shows the results of immunoblotting of the anti-flag immunoprecipitated fraction.
  • the upper row shows the results of detection of the anti-flag immunoprecipitated fraction with anti-Xpress antibody
  • the middle row shows the results of detection of the cell lysate with anti-Xpress antibody
  • the lower row shows the detection of anti-flag immunoprecipitated fraction with anti-TAK1 antibody The result.
  • IKK IKK. And /?) was co-immunoprecipitated with TAK1, indicating that TAK1 and IKK (IKK "and /?) Interact intracellularly.
  • IKK was not detected in the anti-flag immunoprecipitated fraction in cells that had enhanced expression of TAB1 together with TAK1 and IKK, indicating that TAK1 was not activated. It was considered that in the state, the ability to generate a stable bond with IKK in the cell, and in the state activated by TAB 1, the stable bond with the bond to IKK in the cell was not observed.
  • TAK 1 is NIK (Regnier et al., 1997; Woronicz et al,
  • NF- / cB activation is promoted by phosphorylating IKK (or a molecule that interacts functionally with the IKK complex) to promote kinase activity of IKK. It is considered to induce.
  • IKK phosphorylating IKK
  • TAK1 TAK1
  • IKK kinase A kinase reaction using I / cB as a substrate (IKK kinase) was performed on the IKK complex immunoprecipitated from cells in which TAK1 and TAB1 were enhanced in the following manner. Activation of the complex was detected.
  • the expression vector of XKK-tagged IKK (Xpress- ⁇ or Xpress-IKK ⁇ ) was added at the same time as transfection. I made an action.
  • a cell lysate was prepared from the cells 24 hours after the transfusion in the same manner as in Example 6, and immunoprecipitation was performed.
  • the antibody used for immunoprecipitation uses an anti-I « « antibody (H-744) (manufactured by Santa Cruz Bio-technology) to immunoprecipitate the endogenous ⁇ ⁇ ⁇ complex.
  • the immunoprecipitation fraction obtained above was subjected to an in vitro kinase reaction in the same manner as in Example 7. However, as a substrate, recombinant I / cB (2.5 g) was added to the reaction system. After completion of the reaction, the reaction solution was subjected to SDS-polyacrylamide gel electrophoresis, and the gel after the electrophoresis was subjected to photoradiography.
  • Recombinant I / cB used as a reaction substrate includes a partial polysaccharide comprising the first to 54th amino acid residues of human IA: B at the C-terminus of GST (glutathione-S-transferase). Peptide-linked fusion peptides (hereinafter, GST-I / cB «1-54) were used.
  • Recombinant I was prepared from a culture of a transformant in which the expression vector for GST-I / cB «1-54 was introduced into an E. coli host.
  • the expression vector for GST-I « ⁇ 1-54 is the cDNA of human I ⁇ ⁇ « (Genbank / EMBL accession No. M69043; Cell, Vol. 65, 1281-: 1289, 1991). Created by inserting the cDNA portion encoding the 1st to 54th amino acid residues into the BamHI-Ec0RI cleavage site of vector-plasmid pGEX-2T (Pharmacia) did.
  • FIG. (A) shows the results of kinase assay of endogenous I KK complex (immunoprecipitated fraction with anti-I ⁇ antibody), and ( ⁇ ) shows the results of exogenous I ⁇ (immunoprecipitated fraction with anti-Xpress antibody). ) This is the result of the kinase assay.
  • TAK1 activated by TAB1 activates NF- / cB by activating ⁇ and IK ⁇ .
  • the effect of the test substance on IKK complex activity due to TAK1 can be assayed. That is, cells in which the expression of TAK1 and TAB1 are enhanced are obtained, and cultured in the presence or absence of the test substance. After culture twenty three
  • the method of the present invention provides a method for identifying and screening NF— ⁇ activation inhibitors that focuses on new transmitter molecules. According to the present invention, a new type of NF- ⁇ B activity inhibitor having an action point at T A ⁇ 1 can be obtained.
  • the method of the present invention is also useful as a method for identifying and screening Z or prophylactic drugs for diseases such as autoimmune diseases and intractable diseases exhibiting inflammatory symptoms.
  • the drug selected or identified by the method of the present invention has an obvious point of action, which is advantageous for development as a pharmaceutical.
  • drugs that have the effect of inhibiting or suppressing the function of TAK1 include new types of NF- ⁇ B activation inhibitors, autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, systemic scleroderma) , Pettiet's disease, periarteritis nodosa, ulcerative colitis, glomerulonephritis, etc., intractable diseases with inflammatory symptoms (osteoarthritis, atherosclerosis, psoriasis, atopic dermatitis, etc.) It is a therapeutic and / or prophylactic agent for diseases such as viral diseases, endotoxin shock, and sepsis.
  • autoimmune diseases rheumatoid arthritis, systemic lupus erythematosus, systemic scleroderma
  • Pettiet's disease rheumatoid arthritis, systemic lupus erythematosus, systemic sc

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Abstract

L'invention concerne des inhibiteurs de l'activation du NF λB ciblant une nouvelle molécule de transfert; des moyens de prévention/traitement pour les maladies auto-immunes, etc.; et des procédés nouveaux permettant d'identifier ou de cribler les moyens considérés. L'invention concerne également un procédé permettant d'identifier ou de cribler des inhibiteurs de l'activation du NF λB, qui consiste à examiner l'effet d'une substance test quant à la modulation de la fonction TAK1; un procédé permettant d'identifier ou de cribler des moyens de traitement et/ou de prévention pour les maladies auto-immunes ou les maladies incurables assorties d'une inflammation, qui consiste à examiner l'effet d'une substance test quant à la modulation de la fonction TAK1 dans le trajet d'activation du NF λB; et de nouveaux inhibiteurs de l'activation du NF λB, ainsi que des moyens de prévention/traitement pour les maladies auto-immunes, les maladies incurables assorties d'une inflammation, etc., ces moyens étant criblés ou identifiés par le biais des procédés décrits ci-dessus.
PCT/JP1999/000422 1998-02-06 1999-02-02 INHIBITEURS DE L'ACTIVATION DU FACTEUR NUCLEAIRE NF λB CIBLES SUR LA KINASE 1 ACTIVEE PAR LE FACTEUR DE CROISSANCE TRANSFORMANT BETA (TAK1) ET PROCEDE D'IDENTIFICATION Ceased WO1999040202A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000023610A1 (fr) * 1998-10-21 2000-04-27 Chugai Seiyaku Kabushiki Kaisha Methode de criblage de composes empechant la transduction de signal de cytokines inflammatoires
WO2004083854A1 (fr) * 2003-03-17 2004-09-30 Novartis Ag Identification de composes organiques capables de moduler l'activite de tak1
EP1608755A4 (fr) * 2003-04-01 2006-12-06 Yissum Res Dev Co Blocage de l'osteogenese par mediation du tak-1

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SAKURAI H., ET AL.: "TGF-BETA-ACTIVATED KINASE 1 STIMULATES NF-KB ACTIVATION BY AN NF-KB-INDUCING KINASE-INDEPENDENT MECHANISM.", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 243., 1 January 1998 (1998-01-01), US, pages 545 - 549., XP002921907, ISSN: 0006-291X, DOI: 10.1006/bbrc.1998.8124 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000023610A1 (fr) * 1998-10-21 2000-04-27 Chugai Seiyaku Kabushiki Kaisha Methode de criblage de composes empechant la transduction de signal de cytokines inflammatoires
US6989244B1 (en) 1998-10-21 2006-01-24 Chugai Seiyaku Kabushiki Kaisha Method for screening compounds inhibiting signal transduction through inflammatory cytokines
US8105799B2 (en) 1998-10-21 2012-01-31 Chugai Seiyaku Kabushiki Kaisha Methods of screening compounds inhibiting signal transduction through inflammatory cytokines
WO2004083854A1 (fr) * 2003-03-17 2004-09-30 Novartis Ag Identification de composes organiques capables de moduler l'activite de tak1
EP1608755A4 (fr) * 2003-04-01 2006-12-06 Yissum Res Dev Co Blocage de l'osteogenese par mediation du tak-1

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