Methods and reagents for treating autoimmune disorders

Abstract

The present invention provides methods and reagents for identifying compounds to treat autoimmune diseases.

1 . A method for identifying candidate compounds to treat an autoimmune condition, comprising identifying compounds that: 
 a) reduce phosphorylation of a first target protein selected from the group consisting of GPBP, an α3 type IV collagen NC1 domain polypeptide comprising the amino acid sequence of SEQ ID NO:26, and a polypeptide comprising the amino acid sequence of SEQ ID NO:64; and    b) reduce formation of conformational isomers of a second target protein selected from the group consisting of an α3 type IV collagen NC1 domain polypeptide and myelin basic protein;    wherein such compounds are candidates for treating an autoimmune condition.    
     
     
         2 . The method of  claim 1  wherein identifying compounds that reduce phosphorylation of the target protein comprises: 
 i) incubating the first target protein and ATP in vitro in the presence or absence of one or more test compounds under conditions that promote phosphorylation of the target protein in the absence of the one or more test compounds;    ii) detecting phosphorylation of the first target protein; and    iii) identifying test compounds that reduce phosphorylation of the first target protein relative to phosphorylation of the first target protein in the absence of the one or more test compounds.    
     
     
         3 . The method of  claim 2  wherein the first target protein is GPBP and wherein the phosphorylation is autophosphorylation.  
     
     
         4 . The method of  claim 2  wherein the first target protein is the α3 type IV collagen NC 1 domain polypeptide comprising the amino acid sequence of SEQ ID NO:26, and wherein the method further comprises incubating in vitro the first target protein and ATP with GPBP, and wherein the phosphorylation is phosphorylation of the first target protein by GPBP.  
     
     
         5 . The method of  claim 2 , wherein the first target protein is an α3 type IV collagen NC1 domain polypeptide, and wherein the method further comprises determining an effect of the one or more test compounds on phosphorylation of individual conformational isomers of the first target protein.  
     
     
         6 . The method of  claim 2 , wherein the first target protein is an α3 type IV collagen NC1 domain polypeptide, and wherein the method further comprises determining an effect of the one or more test compounds on phosphorylation of an α3 type IV collagen NC1 domain polypeptide selected from the group consisting of α3(IV)NC1Ser9, α3(IV)NC1Asp9, and α3(IV)NC1Ala9.  
     
     
         7 . The method of  claim 1  wherein identifying compounds that reduce formation of conformational isomers of the target protein comprises: 
 i) providing cells expressing the second target protein;    ii) culturing the cells in the presence or absence of one or more test compounds, under conditions that promote conformational isomerization of the second target protein in the absence of the one or more test compounds;    iii) detecting conformational isomerization of the second target protein; and    iv) identifying test compounds that reduce conformational isomerization of the second target protein relative to conformational isomerization of the second target protein in the absence of the one or more test compounds.    
     
     
         8 . The method of  claim 7  wherein the second target protein is an α3 type IV collagen NC1 domain polypeptide.  
     
     
         9 . The method of  claim 1 , wherein identifying compounds that reduce formation of conformational isomers of the second target protein comprises: 
 i) contacting in vitro the second target protein with GPBP in the presence or absence of one or more test compounds under conditions that promote GPBP-induced conformational isomerization of the second target protein in the absence of the one or more test compounds;    ii) detecting GPBP-induced conformational isomerization of the second target protein; and    iii) identifying test compounds that reduce GPBP-induced conformational isomerization of the second target protein relative to GPBP-induced conformational isomerization of the second target protein in the absence of the one or more test compounds.    
     
     
         10 . The method of  claim 9  wherein the second target protein is an α3 type IV collagen NC1 domain polypeptide.  
     
     
         11 . The method of  claim 1 , wherein the method further comprises identifying compounds that reduce oligomerization of the second target protein.  
     
     
         12 . The method of  claim 11 , wherein the second target protein is an α3 type IV collagen NC1 domain polypeptide.  
     
     
         13 . The method of  claim 12  wherein identifying compounds that reduce oligomerization of the second target protein comprises: 
 i) incubating in vitro the second target protein, GPBP, and a redox system, in the presence or absence of one or more test compounds, under conditions to promote GPBP-induced-oligomerization of the second target protein in the absence of the one or more test compounds; and    ii) identifying test compounds that reduce GPBP-induced oligomerization of the second target protein relative to GPBP induced oligomerization of the second target protein in the absence of the one or more test compounds.    
     
     
         14 . The method of  claim 1 , wherein the autoimmune condition is selected from the group consisting of Goodpasture Syndrome, multiple sclerosis, systemic and cutaneous lupus erythematosus, pemphigus, pemphigoid and lichen planus.  
     
     
         15 . An isolated type IV collagen α3 NC1 domain conformational isomer, wherein the isolated conformational isomer has an amino acid sequence identical to that of wild type α3 type IV collagen NC1 domain, wherein the conformational isomer is stabilized by disulfide bonds, wherein the isolated conformational isomer has a molecular weight in a non-reducing sodium dodecyl sulfate gel selected from the group consisting of 22 kD, 23, kD, 25 kD, 27 kD, and 28 kD, and wherein the conformational isomer has a molecular weight of 29 kDa in a reducing sodium dodecyl sulfate gel.  
     
     
         16 . The conformational isomer of  claim 15 , wherein the isolated conformational isomer has a molecular weight in a non-reducing sodium dodecyl sulfate gel of 22 kD.  
     
     
         17 . The conformational isomer of  claim 15 , wherein the isolated conformational isomer has a molecular weight in a non-reducing sodium dodecyl sulfate gel of 23 kD.  
     
     
         18 . The conformational isomer of  claim 15 , wherein the isolated conformational isomer has a molecular weight in a non-reducing sodium dodecyl sulfate gel of 25 kD.  
     
     
         19 . The conformational isomer of  claim 15 , wherein the isolated conformational isomer has a molecular weight in a non-reducing sodium dodecyl sulfate gel of 27 kD.  
     
     
         20 . The conformational isomer of  claim 15 , wherein the isolated conformational isomer has a molecular weight in a non-reducing sodium dodecyl sulfate gel of 28 kD.  
     
     
         21 . An isolated type IV collagen α3 NC1 domain polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 66 and SEQ ID NO: 68.