Ca2+/calmodulin-dependent protein kinase IV

• US 20030059854A1
• Filed: 04/11/2002
• Published: 03/27/2003
• Est. Priority Date: 04/11/2001
• Status: Active Grant

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Means, Anthony R.
• Granted Patent: US 7,105,312 B2
• Time in Patent Office: Days
• US Class Current: 435/7.9
• CPC Class Codes: A61K 49/0004 Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12Y 207/11017 Ca2+/Calmodulin-dependent protein kinase (2.7.11.17)

Abstract

The present invention relates to Ca²⁺/calmodulin-dependent protein kinase IV (CaMKIV) and, in particular, to methods of screening compounds for their ability to act as CaMKIV agonists or antagonists. The invention further relates to compounds identified using such methods and to therapeutic methodologies based on same.

Claims

1. A method of screening a test compound for its ability to act as a CaMKIV agonist comprising:

i) contacting CaMKIV and a substrate therefor, in the presence and absence of a test compound, under conditions such that CaMKIV-dependent phosphorylation of said substrate can be effected, and

ii) determining the level of phosphorylation of said substrate resulting from step (i) and comparing said level with a level of phosphorylation of said substrate in the absence of said test compound,

wherein an increase in phosphorylation of said substrate in the presence of said test compound indicates that said test compound is a CaMKIV agonist.

2. The method according to claim 1 wherein said substrate is a peptide substrate.

3. The method according to claim 1 wherein said CaMKIV and said substrate are present in a cell free system.

4. The method according to claim 1 wherein said CaMKIV and said substrate are present in a cell.

5. The method according to claim 1 wherein, in step (i), said CaMKIV and said substrate therefor are contacted with said test compound in the presence of CaMKIV kinase, calmodulin and calcium under conditions such that CaMKIV kinase dependent phosphorylation of CaMKIV can be effected.

6. A method of screening a test compound for its ability to inhibit differentiation of CD4+ T cells into Th2 cells comprising:

i) contacting CaMKIV and a substrate therefor in the presence of said test compound, under conditions such that CaMKIV-dependent phosphorylation of said substrate can be effected, and

ii) determining the level of phosphorylation of said substrate resulting from step (i) and comparing said level with a level of phosphorylation of said substrate obtained in the absence of said test compound,

wherein a reduction in the level of phosphorylation of said substrate in the presence of said test compound indicates that said test compound has said inhibitory activity.

7. The method according to claim 6 wherein said substrate is a peptide substrate.

8. The method according to claim 6 wherein said CaMKIV and said substrate are present in a cell free system.

9. The method according to claim 6 wherein said CaMKIV and said substrate are present in a cell.

10. The method according to claim 6 wherein, in step (i), said CaMKIV and said substrate therefor are contacted with said test compound in the presence of CaMKIV kinase, calmodulin and calcium under conditions such that CaMKIV kinase dependent phosphorylation of CaMKIV can be effected.

11. A method of preventing allergic asthma comprising administering to a patient in need of said prevention a compound identifiable by the method according to claim 6.

12. A method of screening a test compound for its ability to enhance hematopoietic cell differentiation comprising:

i) contacting CaMKIV and a substrate therefor in the presence of said test compound, under conditions such that CaMKIV-dependent phosphorylation of said substrate can be effected, and

ii) determining the level of phosphorylation of said substrate resulting from step (i) and comparing said level with a level of phosphorylation of said substrate obtained in the absence of said test compound,

wherein an increase in the level of phosphorylation of said substrate in the presence of said test compound indicates that said test compound has said enhancing activity.

13. The method according to claim 12 wherein said substrate is a peptide substrate.

14. The method according to claim 12 wherein said CaMKIV and said substrate are present in a cell free system.

15. The method according to claim 12 wherein said CaMKIV and said substrate are present in a cell.

16. The method according to claim 12 wherein, in step (i), said CaMKIV and said substrate therefor are contacted with said test compound in the presence of CaMKIV kinase, calmodulin and calcium under conditions such that CaMKIV kinase dependent phosphorylation of CaMKIV can be effected.

17. A method of treating a disease resulting from a defect in hematopoietic cell differentiation comprising administering to a patient in need of such treatment a compound identifiable by the method according to claim 12.

18. The method according to claim 17 wherein said disease is aplastic anemia.