Methods of treatment and uses for camkii and its interaction with hdacs and calpain

Abstract

The present invention provides for methods of treating and cardiac hypertrophy, heart failure, dilated cardiomyopathy or hypertension comprising the use of CaMKII-HDAC binding domains. The present invention discloses not only the fact that CaMKII binds to HDAC4 at a specific site, but that HDAC4 may dimerize with other HDACs. Both events can lead to export of HDACs from the nucleus to the cytoplasm, an event associated with the development of heart disease. Thus the methods of treatment and the screening methods of the present invention are novel attempts to prevent, treat or identify therapies for cardiac hypertrophy, heart failure, dilated cardiomyopathy or hypertension.

1 . A method of treating or preventing cardiac hypertrophy, heart failure, dilated cardiomyopathy, arrhythmias or hypertension comprising:
 (a) identifying a subject suffering from or at risk of developing cardiac hypertrophy, heart failure, dilated cardiomyopathy, arrhythmias or hypertension; and   (b) administering to said patient an agent that specifically inhibits the interaction between s class II Histone Deacetylase (HDAC) and Calcium/Calmodulin Kinase II (CaMKII).   
     
     
         2 . The method of  claim 1 , wherein said agent comprises a peptide. 
     
     
         3 . The method of  claim 2 , wherein said peptide is a peptide of HDAC4 comprising the docking site to CaMKII. 
     
     
         4 . The method of  claim 3 , wherein said peptide comprises a peptide of between 5 and 25 consecutive residues of HDAC4. 
     
     
         5 . The method of  claim 3 , wherein said peptide consists of only the HDAC/CaMKII docking site. 
     
     
         6 . The method of  claim 2 , wherein said peptide is a peptide of CaMKII comprising the docking site to class II HDACs. 
     
     
         7 . The method of  claim 2 , where said peptide is a peptide of CaMKII-α, β, γ, or δ comprising the docking site to class II HDACs. 
     
     
         8 . The method of  claim 2 , wherein said peptide comprises a peptide of between 5 and 25 consecutive residues of CaMKII. 
     
     
         9 . The method of  claim 3 , wherein said peptide consists of only the HDAC4/CaMKII docking site. 
     
     
         10 . A method of treating or preventing cardiac hypertrophy, heart failure, dilated cardiomyopathy, arrhythmias or hypertension comprising:
 (a) identifying a subject suffering from or at risk of suffering from cardiac hypertrophy, heart failure, dilated cardiomyopathy, arrhythmias or hypertension comprising; and   (b) administering to said patient an agent that specifically inhibits the dimerization of class II HDACs.   
     
     
         11 . The method of  claim 10 , wherein said agent is a peptide. 
     
     
         12 . The method of  claim 11 , wherein said peptide is an class II HDAC peptide. 
     
     
         13 . The method of  claim 12 , wherein said class II HDAC peptide comprises a peptide of between 5 and 50 consecutive residues of the dimerization region of class II HDACs. 
     
     
         14 . The method of  claim 12 , wherein said class II HDAC peptide is an HDAC 4 peptide. 
     
     
         15 . The method of  claim 12 , wherein said class II HDAC peptide specifically interacts with HDAC4. 
     
     
         16 . A method for identifying a compound that inhibits calpain protease/peptidase activity comprising:
 (a) providing a peptide generated from CaMKII that contains the calpain cleavage site,   (b) labeling the peptide with an agent that allows for downstream measurement of calpain cleavage,   (c) mixing the peptide with calpain and a compound of interest; and   (d) comparing the cleavage of the peptide in the presence of compound as compared to calpain mediated cleavage without compound;   wherein decreased cleavage of the peptide by calpain in the presence of the compound as compared to cleavage without compound identifies the compound as a compound that calpain protease/peptidase activity.   
     
     
         17 . The method of  claim 16 , wherein said peptide is a CaMKII α, β, δ, or γ peptide. 
     
     
         18 . The method of  claim 17 , wherein said CaMKII is CaMKIIδb. 
     
     
         19 . The method of  claim 16 , wherein said calpain is m-calpain or μ-calpain. 
     
     
         20 . The method of  claim 16 , where said peptide is labeled with a phosphoryl group and a fluorescent dye. 
     
     
         21 . The method of  claim 16 , wherein comparing the cleavage of said peptide by calpain further comprises measurement using fluorescence resonance energy transfer (FRET). 
     
     
         22 . The method of  claim 16 , wherein said peptide is coupled to amino-4-methylcoumarin (AMC). 
     
     
         23 . A method for identifying a compound capable of disrupting the interaction between CaMKIIδb and HDAC comprising:
 (a) providing a CaMKIIδb protein labeled with a first agent and a HDAC protein labeled with a second agent, wherein each protein is either directly or indirectly labeled, and further wherein said first agent emits a measurable signal when in proximity of said second agent, 
 (b) mixing said proteins in a solution, 
 (c) adding a compound of interest to the mixture and comparing the signal; 
 wherein a decrease in the measured signal as compared to the signal without compound identifies the compound as a compound capable of disrupting the interaction between CaMKIIδb and HDAC. 
 
     
     
         24 . The method of  claim 23 , wherein said CaMKIIδb and HDAC are purified. 
     
     
         25 . The method of  claim 23 , wherein said CaMKIIδb and HDAC are human. 
     
     
         26 . The method of  claim 23 , wherein said CaMKIIδb and HDAC are rodent. 
     
     
         27 . The method of  claim 23 , wherein said HDAC is HDAC4. 
     
     
         28 . The method of  claim 23 , wherein said first and second agents are labeled antibodies wherein the antibody to CaMKIIδb is labeled with a different agent than the antibody to HDAC. 
     
     
         29 . The method of  claim 23 , comprising the use of a labeled secondary antibody that recognizes the primary antibody to CaMKIIδb and another labeled secondary antibody that recognizes the primary antibody to HDAC, wherein each secondary antibody is labeled with a different agent. 
     
     
         30 . The method of  claim 28 , wherein said antibody is labeled with a fluorescent molecule. 
     
     
         31 . The method of  claim 29 , wherein said secondary antibody is labeled with a fluorescent molecule. 
     
     
         32 . The method of  claim 23 , wherein said first or second agent is a fluorescent molecule. 
     
     
         33 . The method of  claim 23 , wherein said first agent is a scintillation proximity (SPA) bead and said second agent is a radionuclide. 
     
     
         34 . The method of  claim 23 , wherein said second agent is a SPA bead and said first agent is a radionuclide. 
     
     
         35 . The method of  claim 23 , wherein said indirect labeling comprises FRET. 
     
     
         36 . The method of  claim 23 , wherein only one protein is labeled and wherein fluorescent polarization (FP) is used to measure the signal. 
     
     
         37 . The method of  claim 23 , further comprising a secondary assay to measure the signal. 
     
     
         38 . The method of  claim 37 , wherein said secondary assay is an enzyme linked immunosorbent assay (ELISA). 
     
     
         39 . The method of  claim 38 , wherein said ELISA utilizes a lanthanide chelate conjugated secondary antibody. 
     
     
         40 . A method of screening for a compound capable of inhibiting the nuclear export of HDAC as mediated by CaMKIIδb comprising:
 (a) transfecting or infecting cells with a vector that expresses a tagged and activated HDAC protein and with a vector that expresses an activated CaMKIIdb; 
 (b) adding a compound of interest to the cells; 
 (c) measuring the amount of HDAC located in the nucleus and cytoplasm of cells exposed to the agent as opposed to cells left untreated; 
 wherein an increase in the amount of HDAC found in the nucleus as compared to the amount found in the cytoplasm of untreated cells identifies the compound as a compound capable of inhibiting the nuclear export of HDAC as mediated by CaMKIIδb. 
 
     
     
         41 . The method of  claim 40 , wherein said HDAC is HDAC 4. 
     
     
         42 . The method of  claim 40 , wherein said tagged HDAC protein is tagged at the carboxyterminus with GFP, myc, HA, flag, or 6Xhistdine. 
     
     
         43 . The method of  claim 40 , wherein said activated CaMKIIδb comprises a mutation at position 287 from threonine to aspartic acid. 
     
     
         44 . The method of  claim 40 , wherein said measuring comprises fluorescent microscopy.