Toll-Like Receptor 9 Modulators

Abstract

TLR9 is localized to endoplasmic reticulum and upon stimulation with a TLR9 ligand, is transported to a tubular lysosomal compartment as is CpG-DNA. Furthermore, it is shown that TLR9 and CpG-DNA directly bind. It was also found that the MyD88 translocates in response to activation of TLR9-mediated signaling. Methods of identifying compounds that affect translocation and activity of TLR9 and MyD88 are described.

1 . A method of identifying a compound that modulates Toll-Like Receptor 9 (TLR9) signalling, the method comprising:
 (a) providing a cell that expresses a TLR9 polypeptide;   (b) contacting the cell with a test compound, thereby providing a test sample;   (c) incubating the test sample under conditions and for a time sufficient for TLR9 polypeptide localization, translocation, and/or aggregation to occur in the absence of the test compound; and   (d) detecting one or more of TLR9 polypeptide localization, translocation, or aggregation,   wherein a difference in TLR9 polypeptide localization, translocation, or aggregation in the test sample compared to TLR9 localization, translocation, or aggregation in a reference indicates that the test compound is a compound that modulates TLR9 signalling.   
     
     
         2 . The method of  claim 1 , wherein the test sample further comprises a TLR9 ligand. 
     
     
         3 . The method of  claim 2 , wherein the TLR9 ligand is a CpG-DNA. 
     
     
         4 . The method of  claim 1 , wherein the TLR9 polypeptide is labeled. 
     
     
         5 . The method of  claim 1 , wherein the TLR9 polypeptide is a fusion protein. 
     
     
         6 . The method of  claim 1 , wherein the TLR9 polypeptide is a fluorescent fusion protein. 
     
     
         7 . The method of  claim 1 , wherein the TLR9 polypeptide is a fusion protein comprising a Yellow Fluorescent Protein (YFP), a Cyan Fluorescent Protein (CFP), Green Fluorescent Protein (GFP), Red Fluorescent Protein (RFP), or a fluorescent variant thereof. 
     
     
         8 . The method of  claim 1 , wherein TLR9 polypeptide localization is affected. 
     
     
         9 . The method of  claim 1 , wherein a rate of TLR9 polypeptide localization is affected. 
     
     
         10 . The method of  claim 1 , wherein localization of TLR9 polypeptide to endoplasmic reticulum (ER) is affected. 
     
     
         11 . The method of  claim 1 , wherein localization of TLR9 polypeptide to a tubular lysosomal compartment (TLC) is affected. 
     
     
         12 . The method of  claim 1 , wherein a rate of TLR9 polypeptide aggregation is affected. 
     
     
         13 . The method of  claim 1 , wherein aggregation of TLR9 polypeptide in a tubular lysosomal compartment (TLC) is affected. 
     
     
         14 . The method of  claim 1 , wherein aggregation of TLR9 polypeptide is detected using Fluorescence Resonance Energy Transfer (FRET). 
     
     
         15 . The method of  claim 1 , wherein translocation of TLR9 polypeptide is affected. 
     
     
         16 . The method of  claim 15 , wherein the translocation is from endoplasmic reticulum (ER) to a tubular lysosomal compartment (TLC). 
     
     
         17 . The method of  claim 1 , wherein a rate of TLR9 polypeptide translocation is affected. 
     
     
         18 . The method of  claim 1 , further comprising detecting Myeloid Differentiation Primary Response Gene 88 (MyD88) polypeptide localization. 
     
     
         19 . The method of  claim 18 , wherein the MyD88 polypeptide is a fusion protein. 
     
     
         20 . The method of  claim 18 , wherein the MyD88 polypeptide is a fluorescent fusion protein. 
     
     
         21 . The method of  claim 1 , wherein following step (c), the cell is fractionated and fractions are tested for the presence of TLR9 polypeptide. 
     
     
         22 . The method of  claim 1 , further comprising testing the test compound for the ability to modulate TLR9 polypeptide-mediated signaling. 
     
     
         23 . The method of  claim 1 , wherein the TLR9 polypeptide comprises a full-length TLR9 protein. 
     
     
         24 . The method of  claim 1 , wherein the TLR9 polypeptide comprises a fragment of TLR9 comprising one or more regions selected from the group consisting of LRR region 1, LRR region 2; transmembrane domain; Toll/IL-1 Resistance (TIR) domain; a ligand binding domain; and a localization signal domain. 
     
     
         25 . The method of  claim 24 , wherein the ligand binding domain comprises one or more CXXC motifs. 
     
     
         26 . The method of  claim 25 , wherein the CXXC motif is CRRC or CMEC. 
     
     
         27 . The method of  claim 1 , wherein the test compound is a modified CpG-DNA. 
     
     
         28 . The method of  claim 1 , wherein the sample comprises a cell. 
     
     
         29 . The method of  claim 1 , wherein the sample comprises a cell extract. 
     
     
         30 . The method of  claim 1 , wherein the sample comprises a cell-free preparation.