Compositions and methods for modulating c-Rel-dependent cytokine production

Abstract

The present invention is directed to compositions and methods for modulating c-Rel-dependent cytokine production without materially altering the level of expression of NFκB and/or the amount of IκB. The present invention is also directed to screening for modulators of c-Rel activity as determined by assaying for altered subcellular localization of c-Rel but where the level of expression of NFκB and/or the amount of IκB is materially unaltered.

1 . A method of identifying a molecule that alters the subcellular location of c-Rel in a cell comprising: 
 (a) contacting the cell with one or more candidate molecules; and    (b) detecting localization of c-Rel molecules in the cell,    wherein an increase or decrease in the amount of c-Rel localized to the nucleus without materially altering the level of expression of NFκB and/or amount of IκB relative to said amount in a cell not so contacted with the one or more candidate molecules indicates that the candidate molecules alter the subcellular localization of c-Rel.    
     
     
         2 . A method of identifying a molecule that alters the subcellular location of c-Rel in a cell comprising: 
 (a) recombinantly expressing within the cell one or more candidate molecules; and    (b) detecting localization of c-Rel molecules in the cell,    wherein an increase or decrease in the amount of c-Rel localized to the nucleus without materially altering the level of expression of NFκB and/or amount of IκB relative to said amount in a cell in which the one or more candidate molecules were not so expressed indicates that the candidate molecules alter the subcellular localization of c-Rel.    
     
     
         3 . The method according to  claim 1  or  2  wherein step (b) comprises contacting the cell with an antibody to c-Rel or a binding region of said antibody, and a fluorescently labeled binding partner of said antibody under conditions conducive to immunospecific binding.  
     
     
         4 . The method according to  claim 1  or  2  wherein step (b) comprises contacting the cell with a fluorescently labeled antibody to c-Rel or a binding region of said antibody under conditions conducive to immunospecific binding.  
     
     
         5 . The method according to  claim 1  or  2  wherein step (b) comprises sequencing by mass spectroscopy nuclear proteins isolated from the cell.  
     
     
         6 . The method according to  claim 1  or  2  wherein the cell is a cultured cell.  
     
     
         7 . A method of identifying a molecule that alters the subcellular location of c-Rel in a cell comprising: 
 (a) microinjecting into the cell one or more candidate molecules; and    (b) detecting localization of c-Rel molecules in the cell,    wherein an increase or decrease in the amount of c-Rel localized to the nucleus without materially altering the level of expression of NFκB and/or amount of IκB relative to said amount in a cell not so microinjected with the one or more candidate molecules indicates that the candidate molecules alter the subcellular localization of c-Rel.    
     
     
         8 . A method for screening for a molecule that modulates directly or indirectly c-Rel activity comprising: 
 (a) contacting a cell expressing c-Rel with one or more candidate molecules; and    (b) detecting the levels of c-Rel localized to the nucleus in said cell without materially altering the level of expression of NFκB and/or amount of IκB relative to said levels in a cell not contacted with said candidate molecules,    wherein a higher or lower level of c-Rel localization to the nucleus in the presence of said candidate molecules indicates that the molecules modulate the activity of c-Rel.    
     
     
         9 . A method for screening for a molecule that modulates directly or indirectly c-Rel activity comprising: 
 (a) recombinantly expressing one or more candidate molecules within a cell expressing c-Rel; and    (b) detecting the levels of c-Rel localized to the nucleus in said cell without materially altering the level of expression of NFκB and/or amount of IκB relative to said levels in a cell in which candidate molecules were not so expressed,    wherein a higher or lower level of c-Rel localization to the nucleus in the presence of said candidate molecules indicates that the molecules modulate the activity of c-Rel.    
     
     
         10 . The method according to  claim 8  or  9  wherein the candidate molecule decreases the amount of c-Rel localization to the nucleus, thereby being a candidate inhibitor of c-Rel activity.  
     
     
         11 . The method according to  claim 8  or  9  wherein the candidate molecule increases the amount of c-Rel localization to the nucleus, thereby being a candidate agonist of c-Rel activity.  
     
     
         12 . The method according to  claim 8  or  9  wherein the candidate molecules are derived from a constrained random peptide library.  
     
     
         13 . The method according to  claim 8  or  9  wherein c-Rel localization to the nucleus is detected by a method comprising contacting the cell with a molecule that binds to c-Rel and a molecule that binds to a nuclear-specific protein other than c-Rel under conditions conducive to binding, and detecting any binding of the molecules to the same subcellular location that occurs.  
     
     
         14 . The method according to  claim 8  or  9  wherein step (b) comprises contacting the cell with a fluorescently labeled antibody to c-Rel or a binding region of the antibody under conditions conducive to immunospecific binding.  
     
     
         15 . A compound that inhibits translocation of c-Rel to the nucleus but that does not materially alter expression of NFκB and/or amount of IκB.  
     
     
         16 . A pharmaceutical composition comprising the compound of  claim 15;  and a pharmaceutically acceptable carrier or excipient.  
     
     
         17 . The pharmaceutical composition of  claim 16 , further comprising a growth hormone or growth hormone secretagogue.  
     
     
         18 . The pharmaceutical composition of  claim 16 , further comprising a TNF antagonist.  
     
     
         19 . A method of treating or ameliorating an IL-12 production-related disease or disorder comprising administering the composition of  claim 17  to a subject in need there in an amount sufficient to treat or ameliorate said disease or disorder.  
     
     
         20 . The method of  claim 19 , further comprising co-administering a growth hormone, growth hormone secretagogue, a TNF antagonist, an anti-inflammatory agent, and/or an immunomodulatory agent.  
     
     
         21 . A method of treating or ameliorating an IL-12 production-related disease or disorder in a subject identified as in need thereof comprising administering the composition of  claim 17  to a subject in need there in an amount sufficient to treat or ameliorate said disease or disorder.  
     
     
         22 . A kit comprising, a compound of  claim 15  labeled for administration to a subject identified as in need thereof.