High throughput assay systems and methods for identifying agents that alter surface expression of integral membrane proteins

Abstract

Disclosed are high throughput assay systems and methods for identifying agents that alter the level of surface expression of integral membrane proteins, such as cardiac ion channels, in mammalian cells. Also disclosed are therapeutic methods of using agents identified using such methods.

1 - 23 . (canceled)  
   
   
       24 . A method for preventing or treating cardiac arrhythmia comprising administering to a mammal in need thereof an effective amount of an active agent, wherein said active agent increases the level of surface expression of a first mutant form of hERG in a mammalian cell and does not increase the level of surface of a second mutant form of hERG in a mammalian cell as determined by the method comprising: 
 a) preparing a first medium containing mammalian cells that express said first mutant form of hERG, wherein said first mutant form is expressed on the surface of said mammalian cells at a level lower than that of a wild-type form of hERG;    b) adding to said first medium an effective amount of said active agent;    c) incubating said cells in the presence of said active agent for a sufficient period of time; and    d) adding to said first medium containing mammalian cells an effective amount of at least one antibody which binds to at least one extracellular epitope of said mutant form of hERG;    e) determining the level of binding of said at least one antibody to said extracellular epitope;    f) preparing a second medium containing mammalian cells that express a second mutant form of hERG, wherein said second mutant form is different from said first mutant form and is expressed on the surface of said mammalian cells at a level lower than that of a wild-type form of hERG;    g) adding to said second medium containing mammalian cells an effective amount of said active agent;    h) incubating said cells in the presence of said active agent for a sufficient period of time;    i) adding to said second medium containing mammalian cells an effective amount of at least one antibody which binds to at least one extracellular epitope of said second mutant form of hERG; and    j) determining the level of binding of said at least one antibody to said extracellular epitope of said second mutant form of hERG.    
   
   
       25 . The method according to  claim 24 , wherein said active agent is vanoxerine or a pharmaceutically acceptable salt thereof.  
   
   
       26 . The method according to  claim 24 , wherein step d) comprises adding an effective amount of at least one primary antibody and an effective amount of at least one secondary antibody, wherein said primary antibody binds to at least one extracellular epitope of said first mutant form of hERG and said secondary antibody binds to said first antibody.  
   
   
       27 . The method according to  claim 24 , wherein said level of binding is measured by fluorescence, luminescence, radioactivity, absorbance or a combination of two or more thereof.  
   
   
       28 . The method according to  claim 24 , wherein said at least one extracellular epitope contains a tag.  
   
   
       29 . The method according to  claim 28 , wherein said extracellular tag replaces at least a portion of an extracellular domain of hERG.  
   
   
       30 . The method according to  claim 28 , wherein said extracellular tag is inserted in an extracellular domain of hERG.  
   
   
       31 . The method according to  claim 28 , wherein said extracellular tag comprises a hemagglutinin (HA) tag.  
   
   
       32 . The method according to  claim 26 , wherein said primary antibody and/or said secondary antibody is coupled to an enzyme.  
   
   
       33 . The method according to  claim 32 , wherein said enzyme is selected from the group consisting of peroxidases, luciferases, alkaline phosphatases, glucose oxidases, beta-galactosidases and mixtures of two or more thereof.  
   
   
       34 . The method according to  claim 24 , wherein said first mutant form is a trafficking-deficient mutant.  
   
   
       35 . The method according to  claim 24 , wherein said first mutant form of hERG is G601S.  
   
   
       36 . The method according to  claim 24 , wherein said first mutant form of hERG is N470D.  
   
   
       37 . The method according to  claim 24 , wherein said second mutant form of hERG is G601S/F656C.  
   
   
       38 . The method according to  claim 24 , wherein said second mutant form of hERG is N470D/F656C.  
   
   
       39 . The method according to  claim 24 , wherein step i) comprises adding an effective amount of at least one primary antibody and an effective amount of at least one secondary antibody, wherein said primary antibody binds to at least one extracellular epitope of said second mutant form of hERG and said secondary antibody binds to said first antibody.  
   
   
       40 . The method according to claim  1 , wherein said membrane protein is a mutant form that is expressed on the surface of said cells at a level less than a wild-type form of said protein.  
   
   
       41 . (canceled)