Axin domain-like polypeptide inhibitors of glycogen synthase kinase 3 beta activity and activators of wnt signaling

• US 20010052137A1
• Filed: 03/01/2001
• Published: 12/13/2001
• Est. Priority Date: 03/01/2000
• Status: Abandoned Application

• Current Assignee: Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Original Assignee: Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Inventors: Klein, Peter S.
• Application Number:
  US09/798,831
• Publication Number:
  US 20010052137A1
• Time in Patent Office: Days
• US Class Current: 800/18
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• 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 14/4703 Inhibitors; Suppressors

Abstract

The invention relates to polypeptides which inhibit the activity of glycogen synthase kinase-3 beta (GSK-3 beta) in vivo and which also activate wnt signaling. The polypeptides have an amino acid sequence which includes one or both of an axin/GSK-3 beta interaction domain (GID) and an axin/axin interaction domain (AID). These polypeptides are useful for treating a number of disorders (e.g. bipolar disorder, mania, depression, Alzheimer'"'"'s disease, diabetes, and leukopenia) which are presently treated by administration of lithium. The invention also includes antibodies (including fragments of antibodies) which bind specifically with the polypeptides described in the disclosure, and to transgenic mice which comprise a transgene encoding such polypeptides.

Claims

1. A composition that inhibits glycogen synthase kinase 3 beta activity, the composition comprising a polypeptide of not more than about 60 amino acid residues, the polypeptide having an amino acid sequence which comprises the sequence

Val-Xaa₅-Pro-Xaa₇-Xaa₈-Phe-Ala-Xaa₁₁-Glu-Leu-Ile-Xaa₁₅-Arg-Leu-Glu-Xaa₁₉-Xaa₂₀-Xaa₂₁-Xaa₂₂-Xaa₂₃-Xaa₂₄-Glu,

wherein

each of Xaa₇, Xaa₈, Xaa₁₁, and Xaa₁₉ is independently any amino acid residue,

Xaa₅ is a negatively-charged amino acid residue,

Xaa₁₅ is a polar amino acid residue,

Xaa₂₀ is a non-polar aliphatic amino acid residue, and

at least two of Xaa₂₁, Xaa₂₂, Xaa₂₃, Xaa₂₄ are polar amino acid residues, the balance of Xaa₂₁, Xaa₂₂, Xaa₂₃, Xaa₂₄ being any amino acid residue.

2. The composition of

claim 1

, wherein at least two of Xaa₂₁, Xaa₂₂, Xaa₂₃, Xaa₂₄ are charged amino acid residues.

3. The composition of

claim 2

, wherein Xaa₇ is a polar amino acid residue, Xaa₈ is Lys, Xaa₂₀ is Val, Xaa₂₁ is any amino acid residue, Xaa₂₂ is a positively-charged amino acid residue, Xaa₂₃ is a polar amino acid residue, and Xaa₂₄ is Arg.

4. The composition of

claim 3

, wherein the amino acid sequence comprises the sequence

Xaa₁-Xaa₂-Xaa₃-Val-Xaa₅-Pro-Xaa₇-Xaa₈-Phe-Ala-Xaa₁₁-Glu-Leu-Ile-Xaa₁₅-Arg-Leu-Glu-Xaa₁₉-Xaa₂₀-Xaa₂₁-Xaa₂₂-Xaa₂₃-Xaa₂₄-Glu,

wherein

each of Xaa₁, Xaa₂, and Xaa₃ is independently any amino acid residue.

5. The composition of

claim 4

, wherein Xaa₁ is a negatively-charged amino acid residue, Xaa₂ is a non-polar amino acid residue, and Xaa₃ is a positively-charged amino acid residue.

6. The composition of

claim 4

, wherein

Xaa₁ is selected from the group consisting of Asp, Glu, and Met;

Xaa₂ is selected from the group consisting of Ile, Val, and Thr;

Xaa₃ is selected from the group consisting of His, Arg, and Pro;

Xaa₅ is selected from the group consisting of Asp and Glu;

Xaa₇ is selected from the group consisting of Glu, Gln, and Ala;

Xaa₈ is selected from the group consisting of Lys, Thr, and Ala;

Xaa₁₁ is selected from the group consisting of Ala and Glu;

Xaa₁₅ is selected from the group consisting of Ser, Asn, and His;

Xaa₁₉ is selected from the group consisting of Gly, Glu, Ala, and Lys;

Xaa₂₀ is selected from the group consisting of Val and Leu;

Xaa21 is selected from the group consisting of Leu, Gln, and Lys;

Xaa₂₂ is selected from the group consisting of Arg, Lys, and Leu;

Xaa₂₃ is selected from the group consisting of Asp, Glu, and Thr; and

Xaa₂₄ is selected from the group consisting of Arg and Leu.

7. The composition of

claim 6

, wherein the sequence is selected from the group consisting of SEQ ID NOs:

1-7.

8. The composition of

claim 6

, wherein

Xaa₁ is selected from the group consisting of Asp and Glun;

Xaa₂ is selected from the group consisting of Ile and Val;

Xaa₃ is selected from the group consisting of His and Arg;

Xaa₇ is selected from the group consisting of Glu and Gln;

Xaa₈ is Lys;

Xaa₁₉ is selected from the group consisting of Gly, Glu, and Ala;

Xaa₂₀ is Val;

Xaa₂₁ is selected from the group consisting of Leu and Gln;

Xaa₂₂ is selected from the group consisting of Arg and Lys; and

Xaa₂₄ is Arg.

9. The composition of

claim 8

, wherein the sequence is selected from the group consisting of SEQ ID NOs:

1-4.

10. The composition of

claim 1

, wherein the polypeptide comprises not more than about 30 amino acid residues.

11. The composition of

claim 1

, further comprising a pharmaceutically acceptable carrier.

12. A kit for inhibiting glycogen synthase kinase 3 beta activity, the kit comprising the composition of

claim 1

and an instructional material selected from the group consisting of an instructional material that describes administration of the composition to an animal in order to inhibit glycogen synthase kinase 3 beta activity, an instructional material that describes administration of the composition to an animal in order to activate wnt signaling, an instructional material that describes administration of the composition to an animal in order to alleviate a disorder known to be alleviated by administration of lithium, and an instructional material that describes administration of the composition to a mammal in order to inhibit spermatozoal motility.

13. A method of inhibiting glycogen synthase kinase 3 beta activity in an animal, the method comprising administering the composition of

claim 1

to the animal.

14. A method of activating wnt signaling in an animal, the method comprising administering the composition of

claim 1

to the animal.

15. A method of alleviating, in an animal, a disorder known to be alleviated by administration of lithium, the method comprising administering the composition of

claim 1

to the animal.

16. The method of

claim 15

, wherein the disorder is selected from the group consisting of bipolar disorder, mania, depression, Alzheimer'"'"'s disease, diabetes, and leukopenia.

17. A method of inhibiting motility of mammalian spermatozoa, the method comprising contacting the composition of

claim 1

and the spermatozoa, whereby spermatozoal motility is inhibited.

18. A method of inhibiting phosphorylation of a protein in a cell, wherein the protein is selected from the group consisting of beta-catenin, glycogen synthase, phosphatase inhibitor I-2, the type-II subunit of cAMP-dependent protein kinase, the G-subunit of phosphatase-1, ATP-citrate lyase, acetyl coenzyme A carboxylase, myelin basic protein, a microtubule-associated protein, a neurofilament protein, an N-CAM cell adhesion molecule, nerve growth factor receptor, c-Jun transcription factor, JunD transcription factor, c-Myb transcription factor, c-Myc transcription factor, L-myc transcription factor, adenomatous polyposis coli tumor suppressor protein, and tau protein, the method comprising providing the composition of

claim 1

to the cell.

19. A composition that inhibits glycogen synthase kinase 3 beta activity in vivo, the composition comprising a polypeptide having the amino acid sequence of at least a portion of the region between the GID domain and the DIX domain of an axin.

20. The composition of

claim 19

, wherein the polypeptide has the amino acid sequence of at least a portion of residues 489-777 of SEQ ID NO:

8.

21. The composition of

claim 20

, wherein the polypeptide has the amino acid sequence of residues 489-777 of SEQ ID NO:

8.

22. A method of alleviating, in an animal, a disorder known to be alleviated by administration of lithium, the method comprising administering the composition of

claim 19

to the animal.

23. A method of identifying an inhibitor of glycogen synthase kinase 3 beta activity, the method comprising assessing glycogen synthase kinase 3 beta activity in an assay system in the presence and absence a polypeptide having an amino acid sequence that consists of less than all of the sequence between the GID domain and the DIX domain of an axin, whereby the polypeptide is an inhibitor of glycogen synthase kinase 3 beta activity if the activity in the assay system is greater in the absence of the polypeptide than in the presence of the polypeptide.

24. The method of

claim 23

, wherein the polypeptide has an amino acid sequence that consists of less than all of residues 489-777 of SEQ ID NO:

8.

25. An antibody that binds specifically with a polypeptide having an amino acid sequence which comprises the sequence

Val-Xaa₅-Pro-Xaa₇-Xaag-Phe-Ala-Xaa₁₁-Glu-Leu-Ile-Xaa₁₅-Arg-Leu-Glu-Xaa₁₉-Xaa₂₀-Xaa₂₁-Xaa₂₂-Xaa₂₃-Xaa₂₄-Glu,

wherein

each of Xaa₇, Xaa₈, Xaa₁₁, and Xaa₁₉ is independently any amino acid residue,

Xaa₅ is a negatively-charged amino acid residue,

Xaa₁₅ is a polar amino acid residue,

Xaa₂₀ is a non-polar aliphatic amino acid residue, and

at least two of Xaa₂₁, Xaa₂₂, Xaa₂₃, Xaa₂₄ are polar amino acid residues, the balance of Xaa₂₁, Xaa₂₂, Xaa₂₃, Xaa₂₄ being any amino acid residue.

26. The antibody of

claim 25

, wherein the sequence is selected from the group consisting of SEQ ID NOs:

1-7.

27. A method of alleviating, in an animal, a disorder known to be alleviated by administration of lithium, the method comprising administering to the animal the antibody of

claim 25

.

28. A transgenic animal comprising an expressible transgene, wherein the transgene encodes a polypeptide of not more than about 60 amino acid residues, the polypeptide having an amino acid sequence which comprises the sequence

Val-Xaa₅-Pro-Xaa₇-Xaa₈-Phe-Ala-Xaa₁₁-Glu-Leu-Ile-Xaa₁₅-Arg-Leu-Glu-Xaa₁₉-Xaa₂₀-Xaa₂₁-Xaa₂₂-Xaa₂₃-Xaa₂₄-Glu,

wherein

each of Xaa₇, Xaa₈, Xaa₁₁ and Xaa₁₉ is independently any amino acid residue,

Xaa₅ is a negatively-charged amino acid residue,

Xaa₁₅ is a polar amino acid residue,

Xaa20 is a non-polar aliphatic amino acid residue, and

at least two of Xaa₂₁, Xaa₂₂, Xaa₂₃, Xaa₂₄ are polar amino acid residues, the balance of Xaa₂₁, Xaa₂₂, Xaa₂₃, Xaa₂₄ being any amino acid residue.

29. The transgenic animal of

claim 28

, wherein the sequence is selected from the group consisting of SEQ ID NOs:

1-7.

30. The transgenic animal of

claim 28

, wherein the polypeptide binds with glycogen synthase 3 beta.

31. The transgenic animal of

claim 28

, wherein the polypeptide inhibits glycogen synthase 3 beta activity.

32. The transgenic animal of

claim 28

, wherein the animal is a mouse.

33. The transgenic animal of

claim 28

, wherein the transgene comprises an inducible promoter operably linked with the portion of the transgene encoding the polypeptide.