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  Method for screening compounds inhibiting signal transduction through inflammatory cytokinesUSPTO Application #: 20060040316Title: Method for screening compounds inhibiting signal transduction through inflammatory cytokines Abstract: Revealed are that the actions of inflammatory cytokine and the production of inflammatory cytokines such as IL-1 and TNF induced by an inflammatory stimulus as well as the production of other inflammatory cytokines such as IL-6 induced by the former class of inflammatory cytokines are all suppressed by inhibiting the signal transduction through TAK1. (end of abstract) Agent: Fish & Richardson PC - Minneapolis, MN, US Inventors: Masayuki Tsuchiya, Toshihiko Ohtomo, Yasuhiro Sugamata, Kunihiro Matsumoto USPTO Applicaton #: 20060040316 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20060040316. Brief Patent Description - Full Patent Description - Patent Application Claims
TECHNICAL FIELD [0001] The present invention relates to a method for screening compounds inhibiting signal transduction through inflammatory cytokines. The present invention also relates to the compound that can be isolated by the screening method and the use thereof. The present invention further relates to an inhibitor of the signal transduction through inflammatory cytokines, for example, inhibitors of the action and production of inflammatory cytokines, and anti-inflammatory agent, which contain as an active ingredient a compound inhibiting the signal transduction through TAK1. BACKGROUND ART [0002] The system of mitogen-activated protein kinase (MAPK) is known as a system involved in intracellular signal transduction. [0003] MAPK system is a conserved eukaryotic signal transduction system, by which the receptor-mediated signals are converted to a variety of actions. MAPK system contains three types of protein kinases, namely, mitogen-activated protein kinase kinase kinase (MAPKKK), mitogen-activated protein kinase kinase (MAPKK), and mitogen-activated protein kinase (MAPK). MAPKK phosphorylates and activates MAPK, and MAPKKK phosphorylates and activates MAPKK (Nishida, E. et al., Trends Biochem. Sci. (1993) 18, 128; Blumer, K. J. et al., Trends Biochem. Sci. (1993) 19, 236; David R. J. et al., Trends Biochem. Sci. (1993) 19, 470; Marchall, C. J. et al., Cell (1995) 80, 179). [0004] TGF-.beta.-activated kinase 1 (TAK1), a member of the MAPKKK family, functions in the intracellular signal transduction system. The TAK1 protein was identified by Yamaguchi, K. et al. (Yamaguchi, K. et al., Science (1995) 270, 2008). The protein has been revealed to be involved in the signal transduction of TGF-.beta. and to be activated by TGF-.beta.. [0005] TAK1 binding protein 1 (TAB1) binds to TAK1 and participates in the signal transduction system of TGF-.beta. that activates TAK1. The TAB1 protein was identified by Shibuya, H. et al. (Shibuya, H. et al., Science (1996) 272, 1179-1182). TAB1 binds to TAK1 and activates the kinase activity of TAK1, thereby transducing the TGF-signal. [0006] A very recent report describes that TAK1 is also activated by tumor necrosis factor (TNF) and interleukin-1 (IL-1) (Shirakabe, K. et al., J. Biol. Chem. (1997) 272, 8141). It has also been reported that the activation of transcription factor NF-.kappa.B is induced after TAK1 activation (Moriguchi, T., et al., J. Biol. Chem. (1996) 271, 13675; Ponton, A., et al., J. Biol. Chem. (1996) 271, 8991; Sakurai S., et al., Biochem. Biophys. Res. Commun. (1998) 243, 545). [0007] However, it is not at all known that the inhibition of signal transduction through TAK1 actually results in the inhibition of cellular responses to the inflammatory stimulation such as LPS or cytokine stimulation, for example, the inhibition of signal transduction through an inflammatory cytokine as an inflammatory mediator as well as results in the inhibition of inflammatory cytokine actions and further the inhibition of inflammatory cytokine production. DISCLOSURE OF THE INVENTION [0008] The present invention provides a method for screening compounds inhibiting TAK1-associated signal transduction of inflammatory cytokines. The present invention also provides compounds which can be isolated by the screening method and the uses thereof. The present invention further provides inhibitors of the signal transduction through inflammatory cytokines, which contain as an active ingredient a compound inhibiting the signal transduction through TAK1, for example, inhibitors of the action and production of inflammatory cytokines, and anti-inflammatory agents. [0009] The present invention has revealed that the inhibition of signal transduction through TAK1 leads to the inhibition of inflammatory cytokine actions, further the inhibition of production of inflammatory cytokines such as IL-1 and TNF, of which production is induced by inflammatory stimulation, and also the inhibition of production of other inflammatory cytokines such as IL-6, of which production is induced by the inflammatory cytokines. The present invention was based on this finding. [0010] Accordingly, the present invention provides: [0011] (1) a method for screening compounds inhibiting signal transduction through inflammatory cytokines, the method comprising the steps of: [0012] (a) contacting a test sample with TAK1 and TAB1; [0013] (b) detecting binding between the TAK1 and the TAB1; and [0014] (c) selecting a compound inhibiting the binding; [0015] (2) the method of (1), wherein the TAK1 and/or the TAB1 is fused with a peptide; [0016] (3) the method of (1) or (2), wherein the TAK1 or the TAB1 is linked to a support; [0017] (4) the method of any one of (1) to (3), wherein a label is attached to the TAK1 or the TAB1 and, wherein the binding is detected by detecting or measuring the label; [0018] (5) the method of any one of (1) to (3), wherein the binding is detected by detecting or measuring the TAB1 bound to TAK1 with a primary antibody against TAB1 or a primary antibody against the peptide fused with TAB1; [0019] (6) the method of any one of (1) to (3), wherein the binding is detected by detecting or measuring the TAK1 bound to the TAB1 with a primary antibody against TAK1 or a primary antibody against the peptide fused with the TAK1; [0020] (7) the method of any one of (1) to (3), wherein the binding is detected by detecting or measuring the TAB1 bound to the TAK1 with a primary antibody against TAB1 or a primary antibody against the peptide fused with TAB1, and a secondary antibody against the primary antibody; [0021] (8) the method of any one of (1) to (3), wherein the binding is detected by detecting or measuring the TAK1 bound to the TAB1 with a primary antibody against TAK1 or a primary antibody against the peptide fused with the TAK1, and a secondary antibody against the primary antibody; [0022] (9) the method of any one of (5) to (8), wherein the primary antibody or the secondary antibody is labeled with radioisotope, enzyme, or fluorescent substance; [0023] (10) the method of (2), wherein the binding is detected with, as an index, change in the expression level of a reporter gene which is activated in response to the binding; [0024] (11) the method of (10), wherein the reporter gene is luciferase, chloramphenicol acetyltransferase, green fluorescent protein, or .beta.-galactosidase; [0025] (12) a method for screening compounds inhibiting signal transduction through inflammatory cytokines, the method comprising the steps of: [0026] (a) contacting a test sample with TAK1; [0027] (b) detecting phosphorylation by the TAK1; and [0028] (c) selecting a compound inhibiting the phosphorylation; [0029] (13) a method for screening compounds inhibiting signal transduction through inflammatory cytokines, the method comprising the steps of: [0030] (a) contacting a test sample with TAK1 and TAB1; [0031] (b) detecting phosphorylation by the TAK1; and [0032] (c) selecting a compound inhibiting the phosphorylation; [0033] (14) the method of (12) or (13), wherein a substrate for the TAK1 is added and wherein the phosphorylation of the substrate by the TAK1 is detected; [0034] (15) the method of (14), wherein the substrate for the TAK1 is MKK6 and/or MKK3; [0035] (16) the method of any one of (12) to (15), wherein the TAK1 is fused with a peptide; [0036] (17) the method of any one of (12) to (16), wherein the TAK1 is linked to a support; [0037] (18) a method for screening compounds inhibiting signal transduction through inflammatory cytokines, the method comprising the steps of: [0038] (a) introducing a test sample into and/or contacting the sample with cells expressing TAK1; [0039] (b) detecting and/or measuring a biological activity transduced through the TAK1; and [0040] (c) selecting a compound reducing the biological activity; [0041] (19) the method of (18), wherein the biological activity is a biological activity of inflammatory cytokines; [0042] (20) the method of (18), wherein the biological activity is detected with, as an index, change in the expression level of a reporter gene which is activated in response to the activity; [0043] (21) a method for screening compounds inhibiting signal transduction through inflammatory cytokines, the method comprising the steps of: [0044] (a) introducing a test sample into and/or contacting the sample with cells expressing TAK1 and TAB1; [0045] (b) detecting and/or measuring a biological activity transduced through the TAK1 and the TAB1; and [0046] (c) selecting a compound reducing the biological activity; [0047] (22) the method of (21), wherein the biological activity is a biological activity of IL-1 or TNF; [0048] (23) the method of (21), wherein the biological activity is detected with, as an index, change in the expression level of a reporter gene which is activated in response to the activity; [0049] (24) the method of (20) or (23), wherein the reporter gene is luciferase, chloramphenicol acetyltransferase, green fluorescent protein, or .beta.-galactosidase; [0050] (25) the method of any one of (18) to (24), wherein an inflammatory stimulus is given to cells and wherein the biological activity transduced through TAK1 or through TAK1 and TAB1 is detected and/or measured; [0051] (26) the method of (25), wherein the inflammatory stimulus is IL-1, TNF, or LPS; [0052] (27) the method of any one of (1) to (26), wherein the inflammatory cytokine is IL-1, TNF, IL-10, or IL-6; [0053] (28) a compound for inhibiting signal transduction through inflammatory cytokines, the compound that can be isolated by the method of any one of (1) to (27); [0054] (29) a pharmaceutical composition comprising as an active ingredient the compound of (28); [0055] (30) an inhibitor of the signal transduction through inflammatory cytokines, the inhibitor having an activity of inhibiting signal transduction through TAK1; [0056] (31) an inhibitor of the activity of inflammatory cytokines, the inhibitor having an activity of inhibiting signal transduction through TAK1; [0057] (32) an inhibitor of the production of inflammatory cytokines, the inhibitor having an activity of inhibiting signal transduction through TAK1; [0058] (33) a pharmaceutical composition for inhibiting signal transduction through inflammatory cytokines, the pharmaceutical composition comprising as an active ingredient a compound inhibiting signal transduction through TAK1; [0059] (34) a pharmaceutical composition for inhibiting the activity of inflammatory cytokines, the pharmaceutical composition comprising as an active ingredient a compound inhibiting signal transduction through TAK1; [0060] (35) a pharmaceutical composition for inhibiting the production of inflammatory cytokines, the pharmaceutical composition comprising as an active ingredient a compound inhibiting signal transduction through TAK1; [0061] (36) the pharmaceutical composition of any one of (33) to (35) wherein the pharmaceutical composition is an anti-inflammatory agent; [0062] (37) the pharmaceutical composition of any one of (33) to (36), wherein the compound is a compound inhibiting binding between TAK1 and TAB1; [0063] (38) the pharmaceutical composition of any one of (33) to (36), wherein the compound is a compound inhibiting phosphorylation by TAK1; [0064] (39) the pharmaceutical composition of any one of (33) to (38), wherein the compound is a compound that can be isolated by the method of any one of (1) to (27); and [0065] (40) the pharmaceutical composition of any one of (33) to (39), wherein the inflammatory cytokine is IL-1, TNF, IL-10, or IL-6. [0066] In the present invention, the term "peptide" means a compound in which amino acids are bonded to each other by peptide bond. Accordingly, the inventive peptide includes long chains of amino acids, namely, polypeptides and proteins. [0067] In the screening of compounds based on inhibiting the binding between TAK1 and TAB1, there is no particular limitation on TAK1 used in the present invention, as far as the TAK1 has the activity of TAB1 binding. Such TAK1 includes not only the complete TAK1 having the amino acid sequence from Met at amino acid 1 to Ser at amino acid 579 in the amino acid sequence of SEQ ID NO: 2 but also a TAK1 species without the kinase activity of TAK1. [0068] TAB1 binds to a region containing TAK1 catalytic domain having the amino acid sequence from Met at amino acid 1 to Glu at amino acid 303 shown in SEQ ID NO: 2, and thereby activating TAK1. It has been disclosed herein that TAB1 binds to the amino acid sequence from Val at amino acid 76 to Gln at amino acid 303 of TAK1 sequence shown in SEQ ID NO: 2. A TAK1 species, which has the amino acid sequence from Val at amino acid 76 to Gln at amino acid 303 of TAK1 sequence shown in SEQ ID NO: 2, does not exhibit the kinase activity, but has the activity of binding to TAB1, and thus the species can be used in the present invention. [0069] Accordingly, TAK1 to be used in the present invention can be a TAK1 species which has the amino acid sequence from Val at amino acid 76 to Gln at amino acid 303 in SEQ ID NO: 2 and which has a amino acid sequence modified by one or more amino acid substitutions, deletions, and/or additions within the amino acid sequence from Met at amino acid 1 to Ile at amino acid 75 and within the amino acid sequence from Tyr at amino acid 304 to Ser at amino acid 579. [0070] As far as a TAK1 species has the TAB1-binding activity, TAK1 can be the TAK1 species of which amino acid sequence is modified by one or more amino acid substitutions, deletions, and/or additions in the amino acid sequence from Val at amino acid 76 to Gln at amino acid 303 in SEQ ID NO: 2. [0071] On the other hand, in the screening of compounds based on inhibiting TAK1 kinase activity, there is no particular limitation on TAK1 as far as the TAK1 species has the kinase activity. [0072] TAK1 to be used includes, for example, the complete TAK1 having the amino acid sequence from Met at amino acid 1 to Ser at amino acid 579 in the amino acid sequence shown in SEQ ID NO: 2 and having the biological activity of TAK1. It has been found that the biological activities of TAK1 are the binding activity to TAB1 as well as the kinase activity to MAPKK in its activated state. Namely, TAK1 is activated upon the binding with TAB1 and then exhibits the kinase activity. [0073] In more detail, it has been revealed that TAK1 exhibits the kinase activity (phosphorylation) in its activated state and the kinase activity is responsible for the phosphorylation of MAPKK, for example, MKK3 (Moriguchi, T. et al., J. Biol. Chem. (1996) 271, 13675-13679), MKK6 (Moriguchi, T. et al., J. Biol. Chem. (1996) 271, 13675-13679), or XMEK2/SEKI (Shibuya, H. et al., Science (1996) 272, 1179-1182); and the phosphorylation activates the kinase activity of MAPKK. [0074] TAK1 to be used in the present invention can also be a TAK1 species having the biological activity of TAK1 and having the amino acid sequence which is modified by one or more amino acid substitutions, deletions, and/or additions in the amino acid sequence shown in SEQ ID NO: 2. More specifically, TAK1 to be used in the present invention may have the amino acid sequence shown in SEQ ID NO: 2, in which one, two or more amino acid residues, preferably 1 to 20 amino acid residues, more preferably 1 to 10 amino acid residues are substituted, as far as the TAK1 species has the biological activity of TAK1. [0075] TAK1 to be used in the present invention may also have the amino acid sequence shown in SEQ ID NO: 2, in which one, two or more amino acid residues, preferably 1 to 276 amino acid residues, more preferably 1 to 10 amino acid residues are deleted. TAK1 to be used in the present invention may also have the amino acid sequence shown in SEQ ID NO: 2, in which one, two or more amino acid residues, preferably 1 to 30 amino acid residues, more preferably 1 to 20 amino acid residues are added. [0076] One example of TAK1 of which amino acid sequence is modified by one or more amino acid substitutions, deletions, and/or additions in the amino acid sequence of human TAK1 shown in SEQ ID NO: 2 is a mouse-derived TAK1 in which Ser is substituted for Gly at amino acid 16, Arg is substituted for His at amino acid 372, Val is substituted for Ala at amino acid 400, Ala is substituted for Thr at amino acid 403, and Ala is substituted for Thr at amino acid 449. Continue reading... Full patent description for Method for screening compounds inhibiting signal transduction through inflammatory cytokines Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Method for screening compounds inhibiting signal transduction through inflammatory cytokines patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. 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