Specific inhibitors of NFAT activation by calcineurin and their use in treating immune-related diseases

• US 7,084,241 B2
• Filed: 01/31/2002
• Issued: 08/01/2006
• Est. Priority Date: 02/12/1998
• Status: Expired due to Term

• Current Assignee: CBR Institute for Biomedical Research Inc.
• Original Assignee: CBR Institute for Biomedical Research Inc.
• Inventors: Hogan, Patrick G., Rao, Anjana, Aramburu, Jose
• Primary Examiner(s): Saoud, Christine J.
• Application Number:
  US10/066,151
• Publication Number:
  US 20020132300A1
• Time in Patent Office: 1,643 Days
• Field of Search: 530/300, 530/330, 530/328, 530/326, 530/324
• US Class Current: 530/300
• 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 5/101 the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu

Abstract

Isolated peptide fragments of the conserved regulatory domain of NFAT protein capable of inhibiting protein—protein interaction between calcineurin and NFAT, or a biologically active analog thereof are described. Isolated polynucleotides and gene therapy vectors encoding such peptide fragments are also described. In addition, methods for treating immune-related diseases or conditions and methods for high throughput screening of candidate agents are described. Pharmaceutical compositions are also provided.

Claims

1. An isolated polypeptide comprising a sequence of at least 6 amino acids but no more than 150 amino acids of the conserved regulatory domain of Nuclear Factor of Activated T-cell (NFAT) protein, wherein said at least six amino acids consists of SEQ ID NO:

77, wherein X₁ is S or R, X₂ is E, R, or Q, and X₃ is I or F; and

wherein said polypeptide inhibits protein—

protein interaction between calcineurin and NFAT.

2. The isolated polypeptide of claim 1, wherein said sequence of at least six amino acids is selected from the group consisting of SEQ ID NO:

5, SEQ ID NO;

6, SEQ ID NO;

7, and SEQ ID NO;

71.

3. The polypeptide of claim 1, wherein said polypeptide comprises a sequence of at least 6 amino acid residues and less than 100 amino acid residues of the conserved regulatory domain of NFAT protein.

4. The polypeptide of claim 1, wherein said polypeptide comprises a sequence of at least 6 amino acid residues and less than 50 amino acid residues of the conserved regulatory domain of NFAT protein.

5. The polypeptide of claim 1, wherein said polypeptide comprises a sequence of at least 6 amino acid residues and less than 30 amino acid residues of the conserved regulatory domain of NFAT protein.

6. The polypeptide of claim 1, wherein said polypeptide comprises a sequence of at least 6 amino acid residues and less than 20 amino acid residues of the conserved regulatory domain of NFAT protein.

7. The polypeptide of claim 1, wherein said polypeptide comprises a sequence of at least 6 amino acid residues and less than 10 amino acid residues of the conserved regulatory domain of NFAT protein.

20. The isolated polypeptide of claim 1, wherein said at least six amino acids consists of SEQ ID NO:

5.

8. An isolated polypeptide comprising a sequence of 6 amino acid residues, and only 6 amino acid residues, of the conserved regulatory domain of NFAT protein, wherein said 6 amino acids consists of any one of SEQ ID NO:

5, SEQ ID NO;

6, SEQ ID NO;

7, and SEQ ID NO;

71, and wherein said polypeptide inhibits protein—

protein interaction between calcineurin and NFAT.

9. An isolated polypeptide consisting of any one of SEQ ID NO:

5, SEQ ID NO;

6, SEQ ID NO;

7, and SEQ ID NO;

71.

10. The isolated polypeptide of claim 9, wherein said polypeptide inhibits protein—

protein interaction between calcineurin and NFAT.

11. A fusion protein comprising the isolated polypeptide of claim 9 fused to at least one protein, wherein said at least one protein is other than an NFAT protein.

12. The fusion protein of claim 11, wherein said at least one protein comprises a maltose binding protein.

13. The fusion protein of claim 11, wherein said at least one protein comprises a glutathione S-transferase (GST) protein.

14. The fusion protein of claim 11 wherein said at least one protein comprises a green fluorescent protein, or a variant thereof.

15. The fusion protein of claim 11 wherein said at least one protein comprises a peptide tag.

16. The fusion protein of claim 11 wherein said at least one protein comprises thioredoxin.

17. The fusion protein of claim 11, wherein said at least one protein is fused to said isolated polypeptide at the N-terminus of said isolated polypeptide.

18. The fusion protein of claim 11, wherein said at least one protein is fused to said isolated polypeptide at the C-terminus of said isolated polypeptide.

19. The fusion protein of claim 17, further comprising a protein other than an NFAT protein fused to said isolated polypeptide at the C-terminus of said isolated polypeptide.