Orthogonal chemical inducer of dimerization

• US 20090162858A1
• Filed: 09/18/2008
• Published: 06/25/2009
• Est. Priority Date: 09/18/2007
• Status: Abandoned Application

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Cornish, Virginia W.
• Application Number:
  US12/284,038
• Publication Number:
  US 20090162858A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection
• 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 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)

Abstract

A method for identifying a molecule as being able to bind a protein target in a cell, comprising (a) covalently bonding the molecule to trimethoprim (TMP) to form a screening molecule, (b) introducing the screening molecule into the cell which (A) expresses (i) a first fusion protein comprising a binding domain capable of binding TMP, and (ii) a second fusion protein comprising the protein target, and (B) comprises a reporter gene, wherein one of the fusion proteins comprises a DNA-binding domain and the other fusion protein comprises a transcription activation domain and wherein expression of the reporter gene is conditioned on the proximity of the DNA-binding domain to the transcription activation domain; and (c) determining whether the screening molecule binds to the first fusion protein and to the second fusion protein by determining whether the cell expresses the reporter gene, wherein expression of the reporter gene indicates that the molecule binds the protein target and wherein lack of expression of the reporter gene indicates that the molecule does not bind the protein target.

Claims

1. A method for identifying a molecule as being able to bind a protein target in a cell, comprising:

(a) covalently bonding the molecule to trimethoprim (TMP) to form a screening molecule;

(b) introducing the screening molecule into the cell which (A) expresses (i) a first fusion protein comprising a binding domain capable of binding TMP, and (ii) a second fusion protein comprising the protein target, and (B) comprises a reporter gene, wherein one of the fusion proteins comprises a DNA-binding domain and the other fusion protein comprises a transcription activation domain and wherein expression of the reporter gene is conditioned on the proximity of the DNA-binding domain to the transcription activation domain; and

(c) determining whether the screening molecule binds to the first fusion protein and to the second fusion protein by determining whether the cell expresses the reporter gene,

wherein expression of the reporter gene indicates that the molecule binds the protein target and wherein lack of expression of the reporter gene indicates that the molecule does not bind the protein target.

6. The method of claim 1 or 3, where the cell is a yeast cell, a bacteria cell, an insect cell or a mammalian cell.

8. The method of claim 1 or 3, wherein the first fusion protein or the second fusion protein is dihydrofolate reductase-LexA.

9. The method of claim 1 or 3, wherein the first fusion protein or the second fusion protein is dihydrofolate reductase-B42.

12. The method of claim 1 or 3 wherein the reporter gene is lacZ, leu2, ura3, his3, or trp, trp1, trp2, ade2, lys2, cyh1, can1, gfp, cat, araBAD, aaDa, KanR, or Cm.

13. The method of claim 1 or 3, wherein the transcription activation domain is α

NTD.

14. The method of claim 1 or 3, wherein the DNA-binding domain is λ

cI, AraC, LexA, Gal4, or zinc fingers.

15. The method of claim 1 or 3, wherein TMP has the following structure:

19. The method of claim 1 or 3, wherein TMP has the following structure:

2. (canceled)

3. A method for identifying a protein target as capable of binding a molecule in a cell, comprising:

(a) providing a screening molecule comprising trimethoprim (TMP) covalently bonded to the molecule;

(b) introducing the screening molecule into the cell which (A) expresses (i) a first fusion protein comprising a binding domain capable of binding TMP, and (ii) a second fusion protein comprising the target protein, and (B) comprises a reporter gene, wherein one of the fusion proteins comprises a DNA-binding domain and the other fusion protein comprises a transcription activation domain and wherein expression of the reporter gene is conditioned on the proximity of the DNA-binding domain to the transcription activation domain; and

(c) determining whether the protein target is capable of binding the molecule by determining whether the cell expresses the reporter gene,

wherein expression of the reporter gene indicates that the protein target is capable of binding the molecule and wherein lack of expression of the reporter gene indicates that the protein target is not capable of binding the molecule.

4. (canceled)

5. (canceled)

7. (canceled)

10. (canceled)

11. (canceled)

16. (canceled)

17. (canceled)

18. (canceled)

20. (canceled)

21. (canceled)

22. (canceled)

23. (canceled)

24. (canceled)

25. (canceled)

26. (canceled)

27. (canceled)

28. (canceled)

29. (canceled)

30. (canceled)

31. (canceled)

32. (canceled)

33. (canceled)

34. (canceled)

35. (canceled)

36. (canceled)

37. (canceled)

38. (canceled)

39. A method for identifying a first molecule as a competitive inhibitor of a second molecule'"'"'s binding to a protein target in a cell, comprising:

(a) covalently bonding the second molecule to trimethoprim (TMP) to form a screening molecule;

(b) introducing the screening molecule into the cell which (A) expresses (i) a first fusion protein comprising a binding domain capable of binding TMP, and (ii) a second fusion protein comprising the protein target, and (B) comprises a reporter gene, wherein one of the fusion proteins comprises a DNA-binding domain and the other fusion protein comprises a transcription activation domain and wherein expression of the reporter gene is conditioned on the proximity of the DNA-binding domain to the transcription activation domain;

(c) quantitating cellular expression of the reporter gene;

(d) introducing the first molecule into the cell repeating steps (b) and (c) in the presence of the first molecule;

wherein a decreased expression of the reporter gene in the presence of the first molecule indicates that the first molecule is a competitive inhibitor of the second molecule'"'"'s binding to the protein target.

50. The method of claim 39, wherein TMP has the following structure:

54. The method of claim 39, wherein TMP has the following structure:

40. (canceled)

41. (canceled)

42. (canceled)

43. (canceled)

44. (canceled)

45. (canceled)

46. (canceled)

47. (canceled)

48. (canceled)

49. (canceled)

51. (canceled)

52. (canceled)

53. (canceled)