Method of inhibiting pathogenicity of infectious agents

• US 7,001,718 B2
• Filed: 12/16/2002
• Issued: 02/21/2006
• Est. Priority Date: 12/20/2001
• Status: Expired due to Fees

• Current Assignee: Massachusetts Institute of Technology, Arizona Board of Regents (University of Arizona)
• Original Assignee: Massachusetts Institute of Technology, Arizona Board of Regents (University of Arizona)
• Inventors: Rich, Alexander, Jacobs, Bertram L.
• Primary Examiner(s): MOSHER, MARY
• Application Number:
  US10/321,785
• Publication Number:
  US 20030211964A1
• Time in Patent Office: 1,163 Days
• Field of Search: 435/5, 435/32, 435/7.1, 436/501
• US Class Current: 435/5
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/06 for tuberculosis
• CPC Class Codes: A61P 31/08 for leprosy
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: A61P 31/16 for influenza or rhinoviruses
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 31/22 for herpes viruses
• CPC Class Codes: A61P 33/00 Antiparasitic agents
• CPC Class Codes: A61P 33/02 Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
• CPC Class Codes: A61P 33/04 Amoebicides
• CPC Class Codes: A61P 33/06 Antimalarials
• CPC Class Codes: A61P 33/10 Anthelmintics
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Abstract

The present invention relates to methods of detecting or identifying inhibitors of a Z-DNA binding ligand to Z-DNA, methods of inhibiting the pathogenicity of an infectious agent, antiviral therapies, and compounds that inhibit complex formation between a Z-DNA binding ligand and Z-DNA.

Claims

1. A method of detecting and/or identifying an inhibitor of binding of a Z-DNA binding ligand to Z-DNA, comprising the steps of:

a) combining an agent to be tested, Z-DNA, and a composition comprising a Z-DNA binding ligand, under conditions suitable for binding of said Z-DNA binding ligand to said Z-DNA; and

b) detecting the formation of a complex between said Z-DNA binding ligand and said Z-DNA, wherein inhibition of formation of said complex by said agent relative to a suitable control is indicative that the agent is an inhibitor,

wherein said Z-DNA binding ligand is a fusion protein comprising a Z-DNA binding ligand and a detectable label, and wherein formation of said complex is detected by detecting said detectable label.

2. The method of claim 1, wherein said Z-DNA binding ligand is a fusion protein comprising a Z-DNA binding ligand selected from the group consisting of E3L, ADAR1 and DLM-1.

3. The method of claim 1, wherein said fusion protein comprises a Z-DNA binding ligand and a green fluorescent protein.

4. The method of claim 1, wherein said fusion protein comprises a Z-DNA binding ligand and a glutathione S-transferase protein.

5. The method of claim 1, wherein said detectable label is selected from the group consisting of an epitope tag, an affinity label, an enzyme, and a fluorescent moiety.

6. The method of claim 1, wherein said method is a competition assay in which binding is determined in the presence of one or more Z-DNA binding ligand.

7. The method of claim 1, wherein the agent to be tested is selected from the group consisting of small organic molecules, peptides, proteins and peptidomimetics.

8. The method of claim 1, wherein the agent to be tested is an antibody or antigen-binding fragment thereof.

9. The method of claim 8, wherein said antibody or antigen-binding fragment binds to said ligand.

10. The method of claim 8, wherein said antibody or antigen-binding fragment binds to Z-DNA.

11. The method of claim 1, wherein said Z-DNA consists essentially of an oligonucleotide.

12. The method of claim 1, wherein said Z-DNA consists essentially of a plasmid.

13. A method of detecting and/or identifying an anti-infective agent, comprising the steps of:

a) combining an agent to be tested, Z-DNA and a composition comprising a Z-DNA binding ligand under conditions suitable for binding of said Z-DNA binding ligand to said Z-DNA;

b) detecting the formation of a complex between said Z-DNA binding ligand and said Z-DNA, wherein inhibition of formation of said complex by said agent relative to a suitable control is indicative that the agent is an anti-infective agent; and

c) assessing the anti-infective activity of said agent in a suitable assay.

24. The method of claim 13, wherein said Z-DNA binding ligand is selected from the group consisting of E3L, ADAR1, DLM-1, and a fusion protein comprising any one of the foregoing.

25. The method of claim 13, wherein said Z-DNA binding ligand is a fusion protein comprising a Z-DNA binding ligand and a detectable label, and wherein formation of said complex is detected by detecting said detectable label.

26. The method of claim 25, wherein said detectable label is a green fluorescent protein or a glutathione S-transferase protein.

27. The method of claim 13, wherein said Z-DNA binding ligand is labeled with a detectable label.

28. The method of claim 27, wherein said detectable label is selected from the group consisting of a radioisotope, an epitope tag, an affinity label, an enzyme, a fluorescent moiety and a chemiluminescent group.

29. The method of claim 13, wherein said method is a competition assay in which binding is determined in the presence of more than one Z-DNA binding ligand.

30. The method of claim 13, wherein the agent to be tested is selected from the group consisting of small organic molecules, peptides, proteins and peptidomimetics.

31. The method of claim 13, wherein the agent to be tested is an antibody or antigen-binding fragment thereof.

32. The method of claim 13, wherein said Z-DNA consists essentially of an oligonucleotide.

33. The method of claim 13, wherein said Z-DNA consists essentially of a plasmid.

14. A method of detecting and/or identifying an anti-infective agent, comprising the steps of:

a) contacting a cell comprising a Z-DNA binding ligand and Z-DNA with an agent to be tested, under conditions suitable for binding of said Z-DNA binding ligand to said Z-DNA; and

b) detecting the formation of a complex between said Z-DNA binding ligand and said Z-DNA, wherein inhibition of formation of said complex by said agent relative to a suitable control is indicative that the agent is an anti-infective agent; and

c) assessing the anti-infective activity of said agent in a suitable assay.

15. The method of claim 14, wherein binding of said Z-DNA binding ligand to Z-DNA can mediate cellular signaling and/or a cellular response, and said binding is detected by detecting a signaling activity or cellular response in response to said binding.

16. The method of claim 14, wherein the assay of step c) is an assay for anti-viral activity.

17. A method of detecting and/or identifying an inhibitor of binding of a Z-DNA binding ligand to Z-DNA, comprising the steps of:

a) combining an agent to be tested, a composition comprising a Z-DNA binding ligand and Z-DNA, under conditions suitable for binding of said Z-DNA binding ligand to said Z-DNA; and

b) detecting the formation of a complex between said Z-DNA binding ligand and said Z-DNA, wherein inhibition of formation of said complex by said agent relative to a suitable control is indicative that the agent is an inhibitor,

wherein said Z-DNA binding ligand is selected from the group consisting of E3L, ADAR1, DLM-1, and a fusion protein comprising any one of the foregoing.

18. The method of claim 17, wherein said Z-DNA binding ligand is a fusion protein comprising E3L, ADAR1 or DLM-1.

19. The method of claim 18, wherein said fusion protein comprises a green fluorescent protein or a glutathione S-transferase protein.

20. The method of claim 17, wherein said Z-DNA binding ligand is labeled with a detectable label.

21. The method of claim 20, wherein said detectable label is selected from the group consisting of a radioisotope, an epitope tag, an affinity label, an enzyme, a fluorescent moiety and a chemiluminescent group.

22. The method of claim 17, wherein said Z-DNA consists essentially of an oligonucleotide.

23. The method of claim 17, wherein said Z-DNA consists essentially of a plasmid.

34. A method of detecting and/or identifying an anti-viral agent, comprising the steps of:

a) combining an agent to be tested, Z-DNA and a composition comprising a Z-DNA binding ligand, under conditions suitable for binding of said Z-DNA binding ligand to said Z-DNA;

b) detecting the formation of a complex between said Z-DNA binding ligand and said Z-DNA, wherein inhibition of formation of said complex by said agent relative to a suitable control is indicative that the agent is an anti-viral agent; and

c) assessing the anti-viral activity of said agent in a suitable assay.

35. The method of claim 34, wherein said Z-DNA binding ligand is selected from the group consisting of E3L, ADAR1, DLM-1, and a fusion protein comprising any one of the foregoing.

36. The method of claim 34, wherein said Z-DNA binding ligand is a fusion protein comprising a Z-DNA binding ligand and a detectable label, and wherein formation of said complex is detected by detecting said detectable label.

37. The method of claim 36, wherein said detectable label is a green fluorescent protein or a glutathione S-transferase protein.

38. The method of claim 34, wherein said Z-DNA binding ligand is labeled with a detectable label.

39. The method of claim 38, wherein said detectable label is selected from the group consisting of a radioisotope, an epitope tag, an affinity label, an enzyme, a fluorescent moiety and a chemiluminescent group.

40. The method of claim 34, wherein said method is a competition assay in which binding is determined in the presence of more than one Z-DNA binding ligand.

41. The method of claim 34, wherein the agent to be tested is selected from the group consisting of small organic molecules, peptides, proteins and peptidomimetics.

42. The method of claim 34, wherein the agent to be tested is an antibody or antigen-binding fragment thereof.

43. The method of claim 42, wherein said antibody or antigen-binding fragment binds to said ligand.

44. The method of claim 42, wherein said antibody or antigen-binding fragment binds to Z-DNA.

45. The method of claim 34, wherein said Z-DNA consists essentially of an oligonucleotide.

46. The method of claim 34, wherein said Z-DNA consists essentially of a plasmid.

47. The method of claim 34, wherein step a) is performed by contacting a cell comprising a Z-DNA binding ligand and Z-DNA with the agent to be tested.

48. A method of detecting and/or identifying an anti-poxvirus agent, comprising the steps of:

a) combining an agent to be tested, Z-DNA and a composition comprising a protein, said protein comprising a poxvirus E3L or a Z-DNA binding domain thereof, under conditions suitable for binding of said poxvirus E3L or a Z-DNA binding domain thereof to said Z-DNA; and

b) detecting the formation of a complex between said poxvirus E3L or a Z-DNA binding domain thereof and said Z-DNA, wherein inhibition of formation of said complex by said agent relative to a suitable control is indicative that the agent is an anti-poxvirus agent.

49. The method of claim 48, wherein said poxvirus E3L is a vaccinia virus E3L or a variola virus E3L.

50. The method of claim 48, further comprising the step of assessing the anti-viral activity of the agent against poxvirus in a suitable assay.

51. The method of claim 48, wherein step a) is performed by contacting a cell comprising a protein, said protein comprising a poxvirus E3L or a Z-DNA binding domain thereof and Z-DNA with the agent to be tested.