Isoform-selective inhibitors and activators of PDE3 cyclic nucleotide phosphodiesterases

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.

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