Isoform-selective inhibitors and activators of PDE3 cyclic nucleotide phosphodiesterases

• US 20030158133A1
• Filed: 06/19/2002
• Published: 08/21/2003
• Est. Priority Date: 08/01/2001
• Status: Abandoned Application

• Current Assignee: University of Utah Research Foundation (State of Utah)
• Original Assignee: University of Utah Research Foundation (State of Utah)
• Inventors: Movsesian, Matthew A.
• Application Number:
  US10/175,161
• Publication Number:
  US 20030158133A1
• Time in Patent Office: Days
• US Class Current: 514/44
• CPC Class Codes: A61K 31/444 containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
• CPC Class Codes: A61K 31/4709 Non-condensed quinolines and containing further heterocyclic rings
• 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: A61P 7/02 Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: A61P 9/04 Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: A61P 9/06 Antiarrhythmics
• CPC Class Codes: A61P 9/12 Antihypertensives
• CPC Class Codes: C12N 9/18 Carboxylic ester hydrolases (3.1.1)
• CPC Class Codes: G01N 2333/916 acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
• 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 2500/04 Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

The present invention concerns methods and compositions related to type 3 phosphodiesterases (PDE3). Certain embodiments concern isolated peptides corresponding to various PDE3A isoforms and/or site-specific mutants of PDE3A isoforms, along with expression vectors encoding such isoforms or mutants. In specific embodiments, methods for identifying isoform selective inhibitors or activators of PDE3 are provided, along with methods of use of such inhibitors or activators in the treatment of dilated cardiomyopathy, pulmonary hypertension and/or other medical conditions related to PDE3 effects on cAMP levels in different intracellular compartments.

Claims

1. An isolated polypeptide consisting of an amino acid sequence that is at least 95% homologous to SEQ ID NO:

1, SEQ ID NO;

2 or SEQ ID NO;

3.

2. The isolated polypeptide of claim 1, wherein the isolated polypeptide is identical in sequence to SEQ ID NO:

1.

3. The isolated polypeptide of claim 1, wherein the isolated polypeptide is identical in sequence to SEQ ID NO:

2.

4. The isolated polypeptide of claim 1, wherein the isolated polypeptide is identical in sequence to SEQ ID NO:

3.

5. The isolated polypeptide of claim 1, wherein the isolated polypeptide has the sequence of SEQ ID NO:

1, with at least one substitution mutation at serine residues 292, 293, 312 or 438.

6. The isolated polypeptide of claim 5, wherein the substitution mutation substitutes an alanine or an aspartate residue for the serine residue.

7. The isolated polypeptide of claim 1, wherein the isolated polypeptide has the sequence of SEQ ID NO:

2, with at least one substitution mutation at serine residues 312 or 438.

8. The isolated polypeptide of claim 5, wherein the substitution mutation substitutes an alanine or an aspartate residue for the serine residue.

10. The expression vector of claim 9, wherein the isoform is the isolated polypeptide of claim 1.

11. The expression vector of claim 9, wherein the isoform is the isolated polypeptide of claim 2.

12. The expression vector of claim 9, wherein the isoform is the isolated polypeptide of claim 3.

13. The expression vector of claim 9, wherein the isoform is the isolated polypeptide of claim 4.

14. The expression vector of claim 9, wherein the isoform is the isolated polypeptide of claim 5.

15. The expression vector of claim 9, wherein the isoform is the isolated polypeptide of claim 7.

16. A method of identifying an isoform selective inhibitor or activator of type 3 phosphodiesterase (PDE3) comprising:

(a) obtaining an isolated polypeptide according to any of claims 1 to 8;

(b) identifying at least one test compound that binds to the isolated polypeptide; and

(c) assaying the at least one test compound for inhibition or activation of PDE3 catalytic activity.

17. The method of claim 16, wherein the at least one test compound is part of a phage display library.

18. The method of claim 17, wherein identifying at least one test compound comprises biopanning.

19. The method of claim 18, wherein the phage display library comprises random peptide sequences.

20. The method of claim 19, wherein the random peptide sequences are 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in length.

21. A method of identifying an isoform selective regulator of PDE3 comprising:

(a) obtaining an isolated polypeptide according to any of claims 1 to 8;

(b) identifying at least one test compound that binds to the isolated polypeptide; and

(c) assaying the at least one test compound for its ability to interfere with binding of a second polypeptide to PDE3.

22. The method of claim 21, wherein the second polypeptide is a protein kinase, a phosphatase or a phosphorylase.

9. The expression vector comprising a nucleic acid encoding an isoform of type 3 phosphodiesterase.

23. A method of modulating cardiac contractility comprising administering to an individual a pharmacologically effective amount of an isoform selective inhibitor, activator or regulator of PDE3.

25. The method of claims 23 or 24, wherein the inhibitor is an antisense inhibitor.

26. The method of claim 25, wherein the antisense inhibitor is transcribed from a vector.

24. A method of treating an individual with dilated cardiomyopathy and/or pulmonary hypertension comprising administering to the individual a pharmacologically effective amount of an isoform selective inhibitor, activator or regulator of PDE3.