Isoform-selective inhibitors and activators of PDE3 cyclic nucleotide phosphodiesterases

• US 8,722,866 B2
• Filed: 01/18/2007
• Issued: 05/13/2014
• Est. Priority Date: 08/01/2001
• Status: Expired due to Fees

• Current Assignee: University of Utah Research Foundation (State of Utah)
• Original Assignee: US Department of Veterans Affairs (Government of the United States of America)
• Inventors: Movsesian, Matthew A.
• Primary Examiner(s): ZARA, JANE J
• Application Number:
  US11/654,858
• Publication Number:
  US 20100267046A1
• Time in Patent Office: 2,672 Days
• Field of Search: 435/6.1, 435/7.1, 435/7.6, 435/91.1, 435/196, 435/375, 514/1, 514/2, 514/44, 536/23.1, 536/23.2, 536/24.31, 425/9.1, 425/9.2
• US Class Current: 536/23.2
• 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. A method of identifying an isoform-selective regulator of PDE3, said method comprising:

(a) obtaining a first isolated polypeptide (PDE3A1), wherein said first isolated polypeptide (PDE3A1) has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO;

1;

(b) identifying at least one test compound that binds to said first isolated polypeptide, (PDE3A1);

(c) assaying the at least one test compound for its ability to interfere with binding of said first isolated polypeptide (PDE3A1), to cAMP, cGMP, or another polypeptide;

(d) assaying the at least one test compound for its ability to interfere with binding of a second isolated polypeptide (PDE3A2), to cAMP, cGMP, or a another polypeptide; and

(e) identifying said at least one test compound as an isoform-selective regulator of PDE3 when said ability to interfere with said binding of cAMP, cGMP, or another polypeptide to said first isolated polypeptide (PDE3A1) is greater than said ability to interfere with binding of cAMP, cGMP or another polypeptide to said second isolated polypeptide (PDE3A2).

2. The method according to claim 1, wherein the first isolated (PDE3A1) polypeptide is identical in sequence to SEQ ID NO:

1.

3. The method according to claim 1, wherein the second isolated polypeptide (PDE3A2) is identical in sequence to SEQ ID NO:

2.

4. The method of claim 1, wherein the second isolated polypeptide (PDE3A2) is identical in sequence to SEQ ID NO:

3.

5. The method according to claim 1, wherein the first isolated polypeptide (PDE3A1) has the sequence of SEQ ID NO:

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

6. The method according to claim 5, wherein the substitution mutation substitutes an alanine or an aspartate residue for the serine residue.

7. The method according to claim 1, wherein the second isolated polypeptide (PDE3A2) has the sequence of SEQ ID NO:

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

8. The method according to claim 7, wherein the substitution mutation substitutes an alanine or an aspartate residue for the serine residue.

9. The method of claim 1, wherein the another polypeptide is a protein kinase, a protein phosphatase, PDE3A-binding proteins or a protein phosphorylase.

10. The method of claim 1 further including a step of using said at least one test compound to regulate at least one of the following of phosphorylation, dephosphorylation, catalytic activity, intracellular localization or protein-protein interactions of PDE3A2.