Toll-like receptor assays

• US 20050208470A1
• Filed: 12/16/2004
• Published: 09/22/2005
• Est. Priority Date: 12/16/2003
• Status: Abandoned Application

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: Latz, Eicke, Golenbock, Douglas T., Visintin, Alberto
• Application Number:
  US11/014,351
• Publication Number:
  US 20050208470A1
• Time in Patent Office: Days
• US Class Current: 435/4
• CPC Class Codes: A61K 49/0008 Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
• CPC Class Codes: G01N 2500/02 Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• CPC Class Codes: G01N 33/5035 on sub-cellular localization
• CPC Class Codes: G01N 33/5041 involving analysis of members of signalling pathways
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

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.

Claims

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.

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.

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.