Methods for identification and uses of anti-inflammatory receptors for eicosapentaenoic acid analogs

• US 20060024758A1
• Filed: 09/01/2005
• Published: 02/02/2006
• Est. Priority Date: 03/05/2003
• Status: Active Grant

• Current Assignee: Brigham and Women's Hospital Incorporated
• Original Assignee: Brigham and Women's Hospital Incorporated
• Inventors: Serhan, Charles N., Arita, Makoto
• Granted Patent: US 7,341,840 B2
• Time in Patent Office: Days
• US Class Current: 435/7.200
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: G01N 2333/4703 Regulators; Modulating activity
• CPC Class Codes: G01N 2333/715 for cytokines; for lymphokines; for interferons
• CPC Class Codes: G01N 2333/726 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• CPC Class Codes: G01N 2500/04 Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
• CPC Class Codes: G01N 33/5023 on expression patterns
• CPC Class Codes: G01N 33/5032 on intercellular interactions
• CPC Class Codes: G01N 33/5041 involving analysis of members of signalling pathways
• CPC Class Codes: G01N 33/5047 Cells of the immune system
• CPC Class Codes: G01N 33/505 involving T-cells
• 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

The present invention is directed to methods for the identification and uses of a receptors that interact with anti-inflammatory compounds derived from eicosapentaenoic acid (EPA). The receptors are of the G-protein coupled receptor (GPCR) family, and are useful to screen candidate substances for anti-inflammatory activity, especially substances that are analogs of EPA. Such analogs are termed “resolvins”; and are typically di- and tri-hydroxy EPA analogs. One analog herein denoted Resolvin E1 was identified in humans and prepared by total synthesis. In nanomolar range Resolvin E1 reduces dermal inflammation, peritonitis, dendritic cells (DCs) migration and IL-12 production. Also described herein is a receptor denoted Reso ER1 that interacts with Resolvin E1 to attenuate cytokine induced activation of inflammatory pathways mediated by transcription factor (NF)-kB. Treatment of DCs with small-interfering RNA specific for ResoE1 eliminated the ligand'"'"'s ability to regulate IL-12. Assays of anti-inflammatory activity based on these discoveries are also described.

Claims

1. A method for screening a candidate substance for anti-inflammatory activity, comprising

contacting a cell that expresses a Reso E receptor with the candidate substance;

and

detecting a biological activity mediated by the Reso E receptor.

2. The method of claim 1 wherein the biological activity is reduced cytokine induced activation of a NF-kB transcription factor relative to a cell not contacted with the candidate substance.

3. The method of claim 2 wherein the cytokine is selected from the group consisting of TNF-α

and IFN-γ

.

4. The method of claim 1 wherein the biological activity includes reduced cytokine induced COX-2 expression relative to a cell not contacted with the candidate substance.

5. The method of claim 1 comprising introducing an isolated nucleic acid into the cell prior to contacting the cell with the substance, the isolated nucleic acid being operably configured to express the Reso E receptor in the cell.

6. The method of claim 5 wherein the cell is from a cultured cell line.

7. The method of claim 6 wherein the cultured cell line does not endogenously express the Reso E receptor.

8. The method of claim 6 wherein the cultured cell line is a HEK293 cell line.

9. The method of claim 1 wherein the cell is from a cultured cell line that endogenously expresses the Reso E receptor.

10. The method of claim 9 wherein the cultured cell line is a THP-1 cell line.

11. The method of claim 1 wherein the biological activity is reduced expression of a reporter protein encoded by a nucleic acid sequence operably linked to a control sequence that is transcriptionally regulated by the NF-kB transcription factor.

12. The method of claim 11 wherein the reporter protein is luciferase.

13. The method of claim 1 wherein the cell is a leucocyte.

14. The method of claim 9 wherein the leucocyte is selected from the group consisting of monocyte, neutrophil, and T-lymphocyte.

15. The method of claim 14 wherein the leukocyte is a monocyte that is an APC selected from the group consisting of a macrophage and a dendritic cell.

16. The method of claim 1 wherein the cell is selected from the group consisting of a brain cell and a glioma cell.

17. The method of claim 1 wherein the cell is contained within a dorsal air pouch and wherein the method further includes contacting the dorsal air pouch with a cytokine that induces NF-kB expression.

18. The method of claim 17 wherein the cytokine is selected from the group consisting of TNF-α

and IFN-γ

.

19. The method of claim 17 wherein the biological activity is reduced recruitment of leukocytes into the dorsal pouch relative to cells not contacted with the candidate substance.

20. The method of claim 1 wherein the biological activity is binding of a hydrolysis resistant GTP analog to a G-protein that interacts with the Reso E receptor.

21. The method of claim 1 wherein the cell is a splenic cell and the biological activity is reduced expression of a cytokine that induces monocyte migration to splenic cells relative to a cell not contacted with the candidate substance.

22. The method of claim 21 wherein the splenic cell comprises an activated splenic dendric cell, and the biological activity is reduced expression of IL-12 from the splenic dendritic cell.

23. The method of claim 22 wherein the splenic dendritic cell is a CD11c⁺ cell.

24. The method of claim 1 wherein contacting the cell comprises administering the candidate substance to an animal in vivo, and the biological activity is reduced expression of 11-12 and reduced migration of dendritic cells into T-cell areas of a spleen relative to a an animal not administered the candidate substance.

25. The method of claim 1 wherein the Reso E receptor is selected from the group consisting of the murine Reso E receptor according to SEQ.ID NO:

______, a rat Reso E receptor according to SEQ.ID NO;

______ and human Reso ER1 according to SEQ.ID NO;

______.

26. The method of claim 1 wherein the Reso E receptor is human Reso ER1.

27. The method of claim 1 wherein the candidate substance comprises a resolvin molecule.

28. The method of claim 27 wherein the resolvin molecule is a poly-hydroxylated eicosapentaenoic acid. (pH-EPA)

29. The method of claim 27 wherein the resolvin molecule is Reso-E1.

33. The method of claim 1, wherein the receptor is a G-protein coupled receptor (GPCR).

34. The method of claim 33, wherein the GPCR is selected from the group consisting of FPR (P21462), FPRL2(P25089), GPR1(A55733), GPR32(075388), Dez(Q99788), CRTH2(Q9Y5Y4), C3AR(Q16581) and C5AR(P21730).

35. The method of claim 34, wherein the GPCR is Dez (Q99788) according to SEQ.ID NO:

______.

36. The method of claim 1, wherein the receptor is introduced into a HEK293 cell by introducing an isolated nucleic acid configured to express the receptor in the HEK293 cell.

30. A method of identifying a receptor that mediates an anti-inflammatory activity of a resolvin substance, comprising

introducing a nucleic acid configured to express a G-protein coupled receptor (GPCR) into a cell that does not endogenously express the GPCR;

contacting the cell with a substance comprising a resolvin; and

detecting that the cell has a reduced cytokine induced activation of a NF-kB transcription factor relative to a cell not contacted by the substance.

31. A method for screening a candidate substance for anti-inflammatory activity, comprising,

detecting that the candidate substance alters an activity mediated by a GPCR receptor.

32. A method for identifying a substance that interacts with a receptor that mediates an anti-inflammatory activity, comprising interacting a candidate substance with a receptor that reduces cytokine induced (NF)-kB activation.

37. An isolated nucleic acid operably configured to express a nucleic acid that encodes a sufficient amount of a resolvin receptor polypeptide to mediate a biological response of the polypeptide when introduced into a cell.

38. The isolated nucleic acid of claim 37 wherein the resolvin receptor polypeptide is according to SEQ.ID NO:

______.

39. The isolated nucleic acid of claim 37 wherein the nuclidic acid encoding the polypeptide comprises the Reso-R1/Dezaccording to SEQ.ID NO:

______.

40. The isolated nucleic acid of claim 37 wherein the polypeptide sequence is a human sequence.

41. The isolated nucleic acid of claim 37 wherein the polypeptide sequence is a murine sequence.

42. A method of identifying a receptor that mediates an anti-inflammatory activity comprising;

providing a siRNA against a nucleic acid that encodes a candidate receptor,

expressing the siRNA in a cell that expresses the candidate receptor;

subjecting the cell to a condition that induces pro-inflammatory response; and

detecting whether expressing the siRNA in the cell increases the pro-inflammatory response relative to a cell subjected to the condition but not expressing the siRNA.

43. The method of claim 42 wherein the pro-inflammatory response is increased production of IL-12.