Method of inhibiting pathogenicity of infectious agents
View Patent ↗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.
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.
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.
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.
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.