Compositions and methods for modulating connexin hemichannels

Abstract

Disclosed are compositions and methods for modulating hemichannel function in a cell, tissue or organ. The invention also relates to useful screens for detecting such compounds, particularly those capable of modulating connexin phosphorylation. Further provided are therapeutic methods for preventing or treating conditions impacted by undesired hemichannel function in a mammal such as heart arrhythmia.

1 . A method of modulating a hemichannel in a cell, tissue or organ exposed to stress, the method comprising contacting the stressed cell, tissue or organ with a therapeutically effective amount of Compound 1, wherein the contact is sufficient to close the hemichannel in the stressed cell, tissue or organ, and wherein Compound 1 is defined as Ac- D -Tyr- D -Pro- D -4Hyp-Gly- D -Ala-Gly-NH 2 .  
     
     
         2 . The method of  claim 1 , wherein the method further comprises phosphorylating a tyrosine residue of connexin 43 (Cx 43).  
     
     
         3 . A method of preventing or treating tissue or organ stress in a mammal, the method comprising administering a therapeutically effective amount Compound 1, wherein the contact is sufficient to prevent or treat the stress in tissue or organ, and wherein Compound 1 is defined as Ac- D -Tyr- D -Pro- D -4Hyp-Gly- D -Ala-Gly-NH 2 .  
     
     
         4 . A method of increasing gap junction intracellular communication (GJIC) in a cell, tissue or organ, the method comprising administering a therapeutically effective amount of Compound 1, wherein the contact is sufficient to increase the GJIC in the cell, tissue or organ, and wherein Compound 1 is defined as Ac- D -Tyr- D -Pro- D -4Hyp-Gly- D -Ala-Gly-NH 2 .  
     
     
         5 . A method of treatment of burns comprising administering to a patient in need of such treatment a therapeutically effective amount of Compound 1, wherein Compound 1 is defined as Ac- D -Tyr- D -Pro- D -4Hyp-Gly- D -Ala-Gly-NH 2 .  
     
     
         6 . A method of treatment of thromboses comprising administering to a patient in need of such treatment a therapeutically effective amount of Compound 1, wherein Compound 1 is defined as Ac- D -Tyr- D -Pro- D -4Hyp-Gly- D -Ala-Gly-NH 2 .  
     
     
         7 . A method of treatment of respiratory and metabolic acidosis comprising administering to a patient in need of such treatment a therapeutically effective amount of Compound 1, wherein Compound 1 is defined as Ac- D -Tyr- D -Pro- D -4Hyp-Gly- D -Ala-Gly-NH 2 .  
     
     
         8 . A method of treatment of focal arrhythmia comprising administering to a patient in need of such treatment a therapeutically effective amount of Compound 1, wherein Compound 1 is defined as Ac- D -Tyr- D -Pro- D -4Hyp-Gly- D -Ala-Gly-NH 2 .  
     
     
         9 . A method of treating and preventing cell and tissue damage resulting from elevated levels of blood glucose comprising administering to a patient in need of such treatment a therapeutically effective amount of Compound 1, wherein Compound 1 is defined as Ac- D -Tyr-D-Pro- D -4Hyp-Gly- D -Ala-Gly-NH 2 .  
     
     
         10 . A method of treatment of chronic atrial fibrillation comprising administering to a patient in need of such treatment a therapeutically effective amount of Compound 1, wherein Compound 1 is defined as Ac- D -Tyr- D -Pro- D -4Hyp-Gly- D -Ala-Gly-NH 2 .  
     
     
         11 . A method of cytoprotecting tissue or an organ of a mammal in need of such treatment, the method comprising administering a therapeutically effective amount of Compound 1, wherein Compound 1 is defined as Ac- D -Tyr- D -Pro- D -4Hyp-Gly- D -Ala-Gly-NH 2 .  
     
     
         12 . A method of preventing or treating reperfusion injury in a mammal, the method comprising administering a therapeutically effective amount of Compound 1, wherein Compound 1 is defined as Ac- D -Tyr- D -Pro- D -4Hyp-Gly- D -Ala-Gly-NH 2 .  
     
     
         13 . The method of  claim 3  or  claim 14 , wherein the method further comprises phosphorylating a tyrosine residue of connexin 43 (Cx 43) and closing the hemichannel.  
     
     
         14 . A method of preventing or treating tissue or organ stress in a mammal, the method comprising administering a therapeutically effective amount of at least one compound selected from the group consisting of compounds represented as Formula I or II as described above, wherein the contact is sufficient to prevent or treat the stress in tissue or organ, and wherein Formula I is defined as follows:  
       
         
           
           
               
               
           
         
       
       wherein, 
 R1 represents H or acetyl (Ac)  
 R2 represents a sidechain of one of the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe,  
 R3 represents any amino acid sidechain,  
 R4 represents a sidechain of one of the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe,  
 R5 represents OH or NH2,  
 and a, S, T, P and Q are integers and independently=0 or 1;  
 and salts thereof,  
 and wherein Formula II is defined as follows:  
   R1-X1-X2-X3-R2  
 wherein,  
 X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp,  
 X2 is 0, Ala-Gly-T4c-Pro, Ala-Sar-Hyp-Pro, Ala-Asn, D-Asn-D-Ala, D-Asn, Gly, Ala, D-Ala,  
 β-Ala, Asn or,  
 X3 is Tyr, D-Tyr, Gly or Phe, and  
 R1 is H or Ac,  
 R2 is OH or NH2,  
 with the proviso that X1 and X2 are not both 0;  
 and salts thereof.  
 
     
     
         15 . A method of increasing gap junction intracellular communication (GJIC) in a cell, tissue or organ, the method comprising administering a therapeutically effective amount of at least one compound selected from the group consisting of compounds represented as Formula I or II, wherein the contact is sufficient to increase the GJIC in the cell, tissue or organ, and wherein Formula I is defined as follows:  
       
         
           
           
               
               
           
         
       
       wherein, 
 R1 represents H or acetyl (Ac)  
 R2 represents a sidechain of one of the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe,  
 R3 represents any amino acid sidechain,  
 R4 represents a sidechain of one of the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe,  
 R5 represents OH or NH2,  
 and a, S, T, P and Q are integers and independently=0 or 1;  
 and salts thereof,  
 and wherein Formula II is defined as follows:  
   R1-X1-X2-X3-R2  
 wherein,  
 X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp,  
 X2 is 0, Ala-Gly-T4c-Pro, Ala-Sar-Hyp-Pro, Ala-Asn, D-Asn-D-Ala, D-Asn, Gly, Ala, D-Ala,  
 β-Ala, Asn or,  
 X3 is Tyr, D-Tyr, Gly or Phe, and  
 R1 is H or Ac,  
 R2 is OH or NH2,  
 with the proviso that X1 and X2 are not both 0;  
 and salts thereof.  
 
     
     
         16 . A method of treatment of burns comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound according to Formula I or II that blocks connexin hemichannel opening, wherein Formula I is defined as follows:  
       
         
           
           
               
               
           
         
       
       wherein, 
 R1 represents H or acetyl (Ac)  
 R2 represents a sidechain of one of the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe,  
 R3 represents any amino acid sidechain,  
 R4 represents a sidechain of one of the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe,  
 R5 represents OH or NH2,  
 and a, S, T, P and Q are integers and independently=0 or 1;  
 and salts thereof,  
 and wherein Formula II is defined as follows:  
   R1-X1-X2-X3-R2  
 wherein,  
 X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp,  
 X2 is 0, Ala-Gly-T4c-Pro, Ala-Sar-Hyp-Pro, Ala-Asn, D-Asn-D-Ala, D-Asn, Gly, Ala, D-Ala,  
 β-Ala, Asn or,  
 X3 is Tyr, D-Tyr, Gly or Phe, and  
 R1 is H or Ac,  
 R2 is OH or NH2,  
 with the proviso that X1 and X2 are not both 0;  
 and salts thereof.  
 
     
     
         17 . A method of treatment of thromboses comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound according to Formula I or II that blocks connexin hemichannel opening, and wherein Formula I is defined as follows:  
       
         
           
           
               
               
           
         
       
       wherein, 
 R1 represents H or acetyl (Ac)  
 R2 represents a sidechain of one of the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe,  
 R3 represents any amino acid sidechain,  
 R4 represents a sidechain of one of the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe,  
 R5 represents OH or NH2,  
 and a, S, T, P and Q are integers and independently=0 or 1;  
 and salts thereof,  
 and wherein Formula II is defined as follows:  
   R1-X1-X2-X3-R2  
 wherein,  
 X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp,  
 X2 is 0, Ala-Gly-T4c-Pro, Ala-Sar-Hyp-Pro, Ala-Asn, D-Asn-D-Ala, D-Asn, Gly, Ala, D-Ala,  
 β-Ala, Asn or,  
 X3 is Tyr, D-Tyr, Gly or Phe, and  
 R1 is H or Ac,  
 R2 is OH or NH2,  
 with the proviso that X1 and X2 are not both 0;  
 and salts thereof.  
 
     
     
         18 . A method of treatment of respiratory and metabolic acidosis comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound according to Formula I or II that blocks connexin hemichannel opening, wherein Formula I is defined as follows:  
       
         
           
           
               
               
           
         
       
       wherein, 
 R1 represents H or acetyl (Ac)  
 R2 represents a sidechain of one of the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe,  
 R3 represents any amino acid sidechain,  
 R4 represents a sidechain of one of the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe,  
 R5 represents OH or NH2,  
 and a, S, T, P and Q are integers and independently=0 or 1;  
 and salts thereof,  
 and wherein Formula II is defined as follows:  
   R1-X1-X2-X3-R2  
 wherein,  
 X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp,  
 X2 is 0, Ala-Gly-T4c-Pro, Ala-Sar-Hyp-Pro, Ala-Asn, D-Asn-D-Ala, D-Asn, Gly, Ala, D-Ala,  
 β-Ala, Asn or,  
 X3 is Tyr, D-Tyr, Gly or Phe, and  
 R1 is H or Ac,  
 R2 is OH or NH2,  
 with the proviso that X1 and X2 are not both 0;  
 and salts thereof.  
 
     
     
         19 . A method of treatment of focal arrhythmia comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound according to Formula I or II that blocks connexin hemichannel opening, wherein Formula I is defined as follows:  
       
         
           
           
               
               
           
         
       
       wherein, 
 R1 represents H or acetyl (Ac)  
 R2 represents a sidechain of one of the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe,  
 R3 represents any amino acid sidechain,  
 R4 represents a sidechain of one of the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe,  
 R5 represents OH or NH2,  
 and a, S, T, P and Q are integers and independently=0 or 1;  
 and salts thereof,  
 and wherein Formula II is defined as follows:  
   R1-X1-X2-X3-R2  
 wherein,  
 X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp,  
 X2 is 0, Ala-Gly-T4c-Pro, Ala-Sar-Hyp-Pro, Ala-Asn, D-Asn-D-Ala, D-Asn, Gly, Ala, D-Ala,  
 β-Ala, Asn or,  
 X3 is Tyr, D-Tyr, Gly or Phe, and  
 R1 is H or Ac,  
 R2 is OH or NH2,  
 with the proviso that X1 and X2 are not both 0;  
 and salts thereof.  
 
     
     
         20 . A method of treating and preventing cell and tissue damage resulting from elevated levels of blood glucose comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound according to Formula I or II that blocks connexin hemichannel opening, wherein Formula I is defined as follows:  
       
         
           
           
               
               
           
         
       
       wherein, 
 R1 represents H or acetyl (Ac)  
 R2 represents a sidechain of one of the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe,  
 R3 represents any amino acid sidechain,  
 R4 represents a sidechain of one of the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe,  
 R5 represents OH or NH2,  
 and a, S, T, P and Q are integers and independently=0 or 1;  
 and salts thereof,  
 and wherein Formula II is defined as follows:  
   R1-X1-X2-X3-R2  
 wherein,  
 X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp,  
 X2 is 0, Ala-Gly-T4c-Pro, Ala-Sar-Hyp-Pro, Ala-Asn, D-Asn-D-Ala, D-Asn, Gly, Ala, D-Ala,  
 β-Ala, Asn or,  
 X3 is Tyr, D-Tyr, Gly or Phe, and  
 R1 is H or Ac,  
 R2 is OH or NH2,  
 with the proviso that X1 and X2 are not both 0;  
 and salts thereof.  
 
     
     
         21 . A method of treatment of chronic atrial fibrillation comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound according to Formula I or II that blocks connexin hemichannel opening, and wherein Formula I is defined as follows:  
       
         
           
           
               
               
           
         
       
       wherein, 
 R1 represents H or acetyl (Ac)  
 R2 represents a sidechain of one of the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe,  
 R3 represents any amino acid sidechain,  
 R4 represents a sidechain of one of the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe,  
 R5 represents OH or NH2,  
 and a, S, T, P and Q are integers and independently=0 or 1;  
 and salts thereof,  
 and wherein Formula II is defined as follows:  
   R1-X1-X2-X3-R2  
 wherein,  
 X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp,  
 X2 is 0, Ala-Gly-T4c-Pro, Ala-Sar-Hyp-Pro, Ala-Asn, D-Asn-D-Ala, D-Asn, Gly, Ala, D-Ala,  
 β-Ala, Asn or,  
 X3 is Tyr, D-Tyr, Gly or Phe, and  
 R1 is H or Ac,  
 R2 is OH or NH2,  
 with the proviso that X1 and X2 are not both 0;  
 and salts thereof.  
 
     
     
         22 . A method of treatment of epilepsia comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound according to Formula I or II that promotes connexin hemichannel opening, and wherein Formula I is defined as follows:  
       
         
           
           
               
               
           
         
       
       wherein, 
 R1 represents H or acetyl (Ac)  
 R2 represents a sidechain of one of the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe,  
 R3 represents any amino acid sidechain,  
 R4 represents a sidechain of one of the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe,  
 R5 represents OH or NH2,  
 and a, S, T, P and Q are integers and independently=0 or 1;  
 and salts thereof,  
 and wherein Formula II is defined as follows:  
   R1-X1-X2-X3-R2  
 wherein,  
 X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp,  
 X2 is 0, Ala-Gly-T4c-Pro, Ala-Sar-Hyp-Pro, Ala-Asn, D-Asn-D-Ala, D-Asn, Gly, Ala, D-Ala,  
 β-Ala, Asn or,  
 X3 is Tyr, D-Tyr, Gly or Phe, and  
 R1 is H or Ac,  
 R2 is OH or NH2,  
 with the proviso that X1 and X2 are not both 0;  
 and salts thereof.  
 
     
     
         23 . A method of cytoprotecting tissue or an organ of a mammal in need of such treatment, the method comprising administering a therapeutically effective amount of at least one compound selected from the group consisting of the compounds represented by Formula I or II, and wherein Formula I is defined as follows:  
       
         
           
           
               
               
           
         
       
       wherein, 
 R1 represents H or acetyl (Ac)  
 R2 represents a sidechain of one of the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe,  
 R3 represents any amino acid sidechain,  
 R4 represents a sidechain of one of the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe,  
 R5 represents OH or NH2,  
 and a, S, T, P and Q are integers and independently=0 or 1;  
 and salts thereof,  
 and wherein Formula II is defined as follows:  
   R1-X1-X2-X3-R2  
 wherein,  
 X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp,  
 X2 is 0, Ala-Gly-T4c-Pro, Ala-Sar-Hyp-Pro, Ala-Asn, D-Asn-D-Ala, D-Asn, Gly, Ala, D-Ala,  
 β-Ala, Asn or,  
 X3 is Tyr, D-Tyr, Gly or Phe, and  
 R1 is H or Ac,  
 R2 is OH or NH2,  
 with the proviso that X1 and X2 are not both 0;  
 and salts thereof.  
 
     
     
         24 . The method of  claim 11  or  claim 23 , wherein the method further comprises exposing the tissue or organ of the mammal to ischemic conditions.  
     
     
         25 . The method of  claim 24 , wherein the organ to be cytoprotected is associated with a fibrous capsule or bone.  
     
     
         26 . The method of  claim 25 , wherein the organ is heart, kidney, brain, spinal cord or bone marrow.  
     
     
         27 . The method of  claim 26 , wherein the heart has been subjected to an infarction and the ischemia is associated with myocardial cell swelling.  
     
     
         28 . The method of  claim 27 , wherein the compound is Ac-Gly-Asn-Tyr-NH 2  (Compound 2).  
     
     
         29 . A method of preventing or treating reperfusion injury in a mammal, the method comprising administering a therapeutically effective amount of at least one compound selected from the group consisting of the compounds represented by Formula I or II, and wherein Formula I is defined as follows:  
       
         
           
           
               
               
           
         
       
       wherein, 
 R1 represents H or acetyl (Ac)  
 R2 represents a sidechain of one of the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe,  
 R3 represents any amino acid sidechain,  
 R4 represents a sidechain of one of the amino acids Gly, Tyr, D-Tyr, Phe and D-Phe,  
 R5 represents OH or NH2,  
 and a, S, T, P and Q are integers and independently=0 or 1;  
 and salts thereof,  
 and wherein Formula II is defined as follows:  
   R1-X1-X2-X3-R2  
 wherein,  
 X1 is 0, Ala, Gly, β-Ala, Tyr, D-Tyr, Asp,  
 X2 is 0, Ala-Gly-T4c-Pro, Ala-Sar-Hyp-Pro, Ala-Asn, D-Asn-D-Ala, D-Asn, Gly, Ala, D-Ala,  
 β-Ala, Asn or,  
 X3 is Tyr, D-Tyr, Gly or Phe, and  
 R1 is H or Ac,  
 R2 is OH or NH2,  
 with the proviso that X1 and X2 are not both 0;  
 and salts thereof.  
 
     
     
         30 . The method of  claim 12  or  claim 29 , wherein the method further comprises exposing the heart of the mammal to infarct conditions and establishing coronary perfusion.  
     
     
         31 . The method of  claim 30 , wherein the method further comprises administering a thrombolytic agent or providing coronary angioplasty to facilitate coronary perfusion into the infarcted heart.  
     
     
         32 . The method of  claim 31 , wherein the compound is Ac-Gly-Asn-Tyr-NH 2  (Compound 2).