Toll-like receptor assays

Abstract

Methods of identifying compounds that modulate the interaction between a TLR and a molecule that interacts with the TLR by direct binding or by inclusion in a complex that associates with the TLR are described. Methods of identifying molecules that interact with a TLR are also described.

1 . A method of identifying a compound that modulates Toll-like receptor (TLR) signaling, the method comprising: 
 (a) providing a sample comprising a TLR polypeptide;    (b) contacting the sample with a cognate TLR ligand and a test compound, thereby forming a test sample;    (c) incubating the test sample for a time and under conditions sufficient for the TLR ligand to bind to the TLR polypeptide in the absence of the test compound; and    (d) determining binding between the TLR polypeptide and the TLR ligand in the test sample,    wherein a difference in binding in the test sample compared to a control indicates that the test compound is a candidate compound for modulating TLR signaling.    
     
     
         2 . The method of  claim 1 , wherein the TLR ligand or the TLR polypeptide is biotinylated.  
     
     
         3 . The method of  claim 1 , wherein the TLR polypeptide is a chimeric polypeptide comprising a TLR protein or fragment thereof, and a second protein.  
     
     
         4 . The method of  claim 3 , wherein the second protein comprises a fluorescent protein.  
     
     
         5 . The method of  claim 4 , wherein the fluorescent protein is Green fluorescent protein (GFP) or a fluorescent variant thereof.  
     
     
         6 . The method of  claim 5 , wherein the second protein comprises an Fc fragment.  
     
     
         7 . The method of  claim 1 , wherein the TLR ligand is bound to a solid surface.  
     
     
         8 . The method of  claim 7 , wherein the TLR ligand is biotinylated and the solid surface comprises avidin, streptavidin, or a deglycosylated form of avidin.  
     
     
         9 . The method of  claim 1 , wherein the TLR polypeptide is bound to a solid surface.  
     
     
         10 . The method of  claim 9 , wherein the TLR polypeptide is biotinylated and the solid surface comprises avidin, streptavidin, or a deglycosylated form of avidin.  
     
     
         11 . The method of  claim 1 , wherein one or more of the TLR polypeptide and the TLR ligand is labeled.  
     
     
         12 . The method of  claim 11 , wherein the label comprises europium or allophycocyanin  
     
     
         13 . The method of  claim 1 , wherein the binding between the TLR polypeptide and TLR ligand is detected by time resolved fluorimetry.  
     
     
         14 . The method of  claim 1 , wherein the binding between the TLR polypeptide and the TLR ligand is detected with an antibody that specifically binds to the TLR polypeptide.  
     
     
         15 . The method of  claim 14 , wherein the antibody is labeled with a lanthanide chelate fluorophore and time-resolved fluorimetry is used to detect the binding.  
     
     
         16 . The method of  claim 14 , the TLR polypeptide is a chimeric TLR polypeptide, and the antibody binds to the second protein in the chimeric polypeptide.  
     
     
         17 . The method of  claim 1 , wherein the TLR polypeptide and TLR ligand are in solution.  
     
     
         18 . The method of  claim 1 , wherein the particle comprises a bead.  
     
     
         19 . The method of  claim 1 , wherein the TLR polypeptide is a TLR9 polypeptide or a TLR2 polypeptide.  
     
     
         20 . The method of claim, wherein the difference in binding is one or more of a difference in a rate of binding; a rate of dissociation; a difference in an amount of binding; or a difference in an affinity of binding.  
     
     
         21 . The method of  claim 1 , further comprising: 
 (a) administering a compound that modulates Toll-like receptor (TLR) signaling to an animal model of a disorder associated with TLR signalling; and    (b) evaluating an effect of the compound on a parameter of the disorder in the animal,    (c) wherein a positive effect of the compound indicates that the compound is a candidate therapeutic compound for the treatment of the disorder.    
     
     
         22 . The method of  claim 21 , further comprising optimizing the compound.  
     
     
         23 . The method of  claim 21 , further comprising formulating a therapeutic composition comprising a candidate therapeutic compound and a pharmaceutically acceptable carrier.