Method for screening compounds inhibiting signal transduction through inflammatory cytokines

• US 6,989,244 B1
• Filed: 10/21/1999
• Issued: 01/24/2006
• Est. Priority Date: 10/21/1998
• Status: Expired due to Fees

• Current Assignee: Chugai Seiyaku Kabushiki Kaisha (Roche Holding AG)
• Original Assignee: Chugai Seiyaku Kabushiki Kaisha (Roche Holding AG)
• Inventors: Tsuchiya, Masayuki, Ohtomo, Toshihiko, Matsumoto, Kunihiro, Sugamata, Yasuhiro
• Primary Examiner(s): Prouty, Rebecca E.
• Assistant Examiner(s): Swope, Sheridan
• Application Number:
  US09/830,144
• Time in Patent Office: 2,287 Days
• Field of Search: 435/15, 435/7, 435/1, 435/7.8, 435/194, 530/350, 536/23.2
• US Class Current: 435/15
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: G01N 2500/02 Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

The present invention relates to methods of screening for compounds that inhibit signal transduction by inflammatory cytokines. The methods include providing a sample that contains a TAK1 and a TAB1; contacting the sample with a compound; detecting binding between the TAK1 and the TAB1; and selecting the compound if binding between the TAK1 and TAB1 is inhibited in the sample compared to a control.

Claims

1. A method of screening for compounds that inhibit production of an inflammatory cytokine, comprising:

(a) providing a sample comprising a TAK1 and a TAB1;

(b) contacting a test compound with the TAK1 and the TAB1;

(c) detecting binding between the TAK1 and the TAB1; and

(d) selecting a compound that inhibits the binding;

wherein the TAK1 of (a) is selected from the group consisting of

(i) a protein comprising amino acids 76 to 303 of SEQ ID NO;

2; and

(ii) a protein that binds to amino acids 437 to 504 of SEQ ID NO;

4 and comprises an amino acid sequence encoded by a DNA sequence that hybridizes with the complement of nucleotides 408 to 1091 of SEQ ID NO;

1 under washing conditions of 42°

C., 5×

SSC, 0.1% sodium dodecyl sulfate, and 50% formamide;

and wherein the TAB1 of (a) is selected from the group consisting of

(iii) a protein comprising amino acids 437 to 504 of SEQ ID NO;

4; and

(iv) a protein that binds to amino acids 76 to 303 of SEQ ID NO;

2 and comprises an amino acid sequence encoded by a DNA that hybridizes with the complement of nucleotides 1338 to 1541 of SEQ ID NO;

3 under washing conditions of 42°

C., 5×

SSC, 0.1% sodium dodecyl sulfate, and 50% formamide, wherein the compound inhibits production of an inflammatory cytokine produced in response to lipopolysaccharide (LPS) or IL-1α

.

2. The method of claim 1, wherein the TAK1 and/or the TAB1 is fused with a peptide.

3. The method of claim 1, wherein the TAK1 or the TAB1 is linked to a support.

4. The method of claim 1, wherein a label is attached to the TAK1 or the TAB1 and the binding is detected by detecting or measuring the label.

5. The method of claim 1, 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 a peptide fused with the TAB1.

6. The method of claim 1, 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 a peptide fused with the TAK1.

7. The method of claim 1, wherein the binding is detected by detecting or measuring the TAB1 bound to the TAK1 with a primary antibody against the TAB1 or a primary antibody against a peptide fused with TAB1, and a secondary antibody against the primary antibody.

8. The method of claim 1, 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 a peptide fused with the TAK1, and a secondary antibody against the primary antibody.

9. The method of claim 7, wherein the primary antibody or the secondary antibody is labeled with a radioisotope, enzyme, or fluorescent substance.

10. The method of claim 1, wherein the TAK1 of (a) comprises amino acids 76 to 303 of SEQ ID NO:

2.

11. The method of claim 1, wherein the TAK1 of (a) is a protein that binds to amino acids 437 to 504 of SEQ ID NO:

4 and comprises an amino acid sequence encoded by a DNA that hybridizes with the complement of nucleotides 408 to 1091 of SEQ ID NO;

1 under washing conditions of 42°

C., 5×

SSC, 0.1% sodium dodecyl sulfate, and 50% formamide.

12. The method of claim 1, wherein the TAK1 of (a) is a protein that binds to amino acids 437 to 504 of SEQ ID NO:

4 and comprises an amino acid sequence that is encoded by a DNA that hybridizes with the complement of nucleotides 408 to 1091 of SEQ ID NO;

1 under washing conditions of 60°

C., 0.1×

SSC, and 0.1% sodium dodecyl sulfate.

13. The method of claim 1, wherein the TAB1 of (a) comprises amino acids 437 to 504 of SEQ ID NO:

4.

14. The method of claim 1, wherein the TAB1 of (a) is a protein that binds to amino acids 76-303 of SEQ ID NO:

2 and comprises an amino acid sequence encoded by a DNA that hybridizes with the complement of nucleotides 1338 to 1541 of SEQ ID NO;

3 under washing conditions of 42°

C., 5×

SSC, 0.1% sodium dodecyl sulfate, and 50% formamide.

15. The method of claim 1, wherein the TAB1 of (a) is a protein that binds to amino acids 76-303 of SEQ ID NO:

2 and comprises an amino acid sequence encoded by a DNA that hybridizes with the complement of nucleotides 1338 to 1541 of SEQ ID NO;

3 under washing conditions of 60°

C., 0.1×

SSC, and 0.1% sodium dodecyl sulfate.

16. The method of claim 1, wherein the inflammatory cytokine is IL-1, TNF, IL-10, or IL-6.

17. The method of claim 1, wherein the inflammatory cytokine is IL-1.

18. The method of claim 1, wherein the inflammatory cytokine is TNF.

19. The method of claim 1, wherein the inflammatory cytokine is IL-6.

20. The method of claim 1, wherein the inflammatory cytokine is IL-10.