Bacterial small-molecule three-hybrid system

• US 20030203471A1
• Filed: 04/24/2002
• Published: 10/30/2003
• Est. Priority Date: 04/24/2002
• Status: Active Grant

• 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., Althoff, Eric A.
• Granted Patent: US 7,083,918 B2
• Time in Patent Office: Days
• US Class Current: 435/252.3
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/4705 stimulating, promoting or activating activity
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

A transgenic bacterial cell comprising

(a) a dimeric small molecule which comprises a first moiety known to bind a first receptor domain covalently linked to a second moiety known to bind a second receptor domain;

(b) nucleotide sequences which upon transcription encode

i) a first fusion protein comprising the first receptor domain, and

ii) a second fusion protein comprising the second receptor domain; and

(c) a reporter gene wherein expression of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein. The cell is also adapted for use in a method for identifying a molecule that binds to a known target in a bacterial cell from a pool of candidate molecules, and a method for identifying an unknown target receptor to which a molecule is capable of binding in a bacterial cell. Also described are compounds and kits for carrying out the methods.

Claims

1. A transgenic bacterial cell comprising

(a) a dimeric small molecule which comprises a first moiety known to bind a first receptor domain covalently linked to a second moiety capable of binding a second receptor domain, wherein the first and second moieties are different;

(b) nucleotide sequences which upon transcription encode

i) a first fusion protein comprising the first receptor domain, and

ii) a second fusion protein comprising the second receptor domain; and

(c) a reporter gene wherein expression of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein.

2. The bacterial cell of claim 1, wherein the dimeric small molecule has the formula:

H1-Y—

H2

wherein each of H1 and H2 may be the same or different and capable of binding to a receptor which is the same or different with a IC₅₀ of less than 100 μ

M; and

wherein Y is a linker which may be present or absent.

3. The bacterial cell of claim 2, wherein H1 or H2 is capable of binding to a receptor with a IC₅₀ of less than 10 μ

M.

4. The bacterial cell of claim 2, wherein H1 or H2 is capable of binding to a receptor with a IC₅₀ of less than 1 μ

M.

5. The bacterial cell of claim 2, wherein H1 or H2 is capable of binding to a receptor with a IC₅₀ of less than 100 nM.

6. The bacterial cell of claim 2, wherein H1 or H2 is capable of binding to a receptor with a IC₅₀ of less than 10 nM.

7. The bacterial cell of claim 2, wherein H1 or H2 is capable of binding to a receptor with a IC₅₀ of less than 1 nM.

8. The bacterial cell of claim 2, wherein H1 or H2 is a methotrexate moiety, FK506 moiety, an FK506 analog, a tetracycline moiety, or a cephem moiety.

9. The bacterial cell of claim 8, wherein H1 or H2 is a methotrexate moiety.

10. The bacterial cell of claim 8, wherein H1 or H2 is an FK506 analog.

11. The bacterial cell of claim 10, wherein the FK506 analog has the structure:

12. The bacterial cell of claim 2, wherein the dimeric small molecule has the structure:

13. The bacterial cell of claim 12, wherein n is an integer from 2 to 12.

14. The bacterial cell of claim 12, wherein n is an integer from 3 to 9.

15. The bacterial cell of claim 12, wherein n is 8.

16. The bacterial cell of claim 1, wherein the first fusion protein further comprises a DNA binding domain, and the second fusion protein further comprises a transcription activation domain.

17. The bacterial cell of claim 1, wherein the first fusion protein further comprises a transcription activation domain, and the second fusion protein further comprises a DNA binding domain.

18. The bacterial cell of claim 16 or 17, wherein the transcription activation domain is α

NTD.

19. The bacterial cell of claim 16 or 17, wherein the DNA-binding domain is λ

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

20. The bacterial cell of claim 1, wherein the first or the second receptor domain is that of dihydrofolate reductase (“

DHFR”

), glucocorticoid receptor, FKBP12, FKBP mutants, tetracycline repressor, or a penicillin binding protein.

21. The bacterial cell of claim 20, wherein the DHFR is the E. coli DHFR (“

eDHFR”

).

22. The bacterial cell of claim 1, wherein the first fusion protein is DHFR-λ

cI or FKBP12-λ

cI.

23. The bacterial cell of claim 1, wherein the second fusion protein is DHFR-α

NTD or FKBP12-α

NTD.

24. The bacterial cell of claim 1, wherein the reporter gene is Lac Z, araBAD, aadA (spectinomycin resistance), his3, β

-lactamase, GFP, luciferase, TetR (tetracyclin resistance), KanR (kanamycin resistance), Cm (chloramphenicol resistance).

25. The bacterial cell of claim 24, wherein the reporter gene is Lac Z.

26. A transgenic bacterial cell comprising

(a) a dimeric small molecule which comprises a methotrexate moiety covalently linked to a moiety capable of binding a receptor domain;

(b) nucleotide sequences which upon transcription encode

i) a first fusion protein comprising a DHFR domain, and

ii) a second fusion protein comprising the receptor domain; and

(c) a reporter gene wherein expression of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein.

27. The bacterial cell of claim 26 wherein the moiety known to bind a receptor domain is capable of binding to a receptor with a IC₅₀ of less than 100 nM.

28. The bacterial cell of claim 26, wherein the moiety known to bind a receptor domain is capable of binding to a receptor with a IC₅₀ of less than 10 nM.

29. The bacterial cell of claim 26, wherein the moiety known to bind a receptor domain is capable of binding to a receptor with a IC₅₀ of less than 1 nM.

30. A method for identifying a molecule that binds a known target receptor in a bacterial cell from a pool of candidate molecules, comprising:

(a) forming a dimeric molecule by covalently bonding each molecule in the pool of candidate molecules to a ligand capable of selectively binding to a receptor;

(b) introducing the dimeric molecule into a bacterial cell culture comprising bacterial cells that express

a first fusion protein which comprises the known target receptor domain against which the candidate molecule is screened,

a second fusion protein which comprises the receptor domain to which the ligand selectively binds, and

a reporter gene wherein expression of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein;

(c) permitting the dimeric molecule to bind to the first fusion protein and to the second fusion protein, bringing the two fusion proteins into proximity so as to activate the expression of the reporter gene;

(d) selecting the bacterial cell that expresses the reporter gene; and

(e) identifying the small molecule that binds the known target receptor.

31. The method of claim 30, wherein the steps (b)-(e) of the method are iteratively repeated in the presence of a preparation of random small molecules for competitive binding with the screening molecule so as to identify a molecule capable of competitively binding the known target receptor.

32. The method of claim 30, wherein the dimeric molecule is obtained from a combinatorial library.

33. The method of claim 30, wherein the dimeric molecule comprises a ligand capable of selectively binding to a receptor with a IC₅₀ of less than 100 μ

M.

34. The method of claim 33, wherein the dimeric molecule comprises a ligand capable of selectively binding to a receptor with a IC₅₀ of less than 100 nM.

35. The method of claim 34, wherein the dimeric molecule comprises a ligand capable of selectively binding to a receptor with a IC₅₀ of less than 1 nM.

36. The method of claim 30, wherein the dimeric molecule comprises a methotrexate moiety, FK506 moiety, an FK506 analog, a tetracycline moiety, or a cephem moiety.

37. The method of claim 30, wherein the dimeric molecule comprises a methotrexate moiety.

38. The method of claim 30, wherein the dimeric molecule comprises an FK506 analog.

39. The method of claim 38, wherein the FK506 analog has the structure:

40. The method of claim 30, wherein the first fusion protein further comprises a DNA binding domain, and the second fusion protein further comprises a transcription activation domain.

41. The method of claim 30, wherein the first fusion protein further comprises a transcription activation domain, and the second fusion protein further comprises a DNA binding domain.

42. The method of claim 40 or 41, wherein the transcription activation domain is α

NTD.

43. The method of claim 40 or 41, wherein the DNA-binding domain is λ

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

44. The method of claim 30, wherein the first or the second fusion protein comprises a receptor domain of dihydrofolate reductase (“

DHFR”

), glucocorticoid receptor, FKBP12, FKBP mutants, tetracycline repressor, or a penicillin binding protein.

45. The method of claim 44, wherein the DHFR is the E. coli DHFR (“

eDHFR”

).

46. The method of claim 30, wherein the first fusion protein is DHFR-λ

cI or FKBP12-λ

cI.

47. The method of claim 30, wherein the second fusion protein is DHFR-α

NTD or FKBP12-α

NTD.

48. The method of claim 30, wherein the reporter gene is Lac Z, araBAD, aadA, his3, β

-lactamase, GFP, luciferase, TetR, KanR, Cm.

49. The method of claim 48, wherein the reporter gene is Lac Z.

72. A process for screening a chemical library for a molecule that binds a known target receptor, comprising providing a chemical library, providing a bacterial cell that expresses the known target receptor as a part of a fusion protein, and identifying the molecule that binds the known target receptor in the bacterial cell by the method of claim 30.

73. A molecule identified by the method of claim 72, wherein the molecule was not previously known to bind the known target receptor.

50. A method for identifying an unknown target receptor to which a known molecule is capable of binding in a bacterial cell, comprising:

(a) providing a dimeric molecule having a first ligand which has a specificity for the unknown target receptor covalently bonded to a second ligand capable of selectively binding to a receptor;

(b) introducing the dimeric molecule into a bacterial cell which expresses

a first fusion protein which comprises the unknown target receptor domain,

a second fusion protein which comprises the receptor domain to which the second ligand selectively binds, and

a reporter gene wherein expression of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein;

(c) permitting the dimeric molecule to bind to the first fusion protein and to the second fusion protein so as to activate the expression of the reporter gene;

(d) selecting which bacterial cell expresses the unknown target receptor; and

(e) identifying the unknown target receptor.

51. The method of claim 50, wherein the unknown target receptor is encoded by a DNA from the group consisting of genomicDNA, cDNA and syntheticDNA.

52. The method of claim 50, wherein the steps (b)-(e) of the method are iteratively repeated so as to identify the unkown target receptor.

53. The method of claim 50, wherein the dimeric molecule comprises a ligand capable of selectively binding to a receptor with a IC₅₀ of less than 100 μ

M.

54. The method of claim 50, wherein the dimeric molecule comprises a ligand capable of selectively binding to a receptor with a IC₅₀ of less than 100 nM.

55. The method of claim 50, wherein the dimeric molecule comprises a ligand capable of selectively binding to a receptor with a IC₅₀ of less than 1 nM.

56. The method of claim 50, wherein the dimeric molecule comprises a methotrexate moiety, FK506 moiety, an FK506 analog, a teracycline moiety, or a cephem moiety.

57. The method of claim 50, wherein the dimeric molecule comprises a methotrexate moiety.

58. The method of claim 50, wherein the dimeric molecule comprises an FK506 analog.

59. The method of claim 58, wherein the FK506 analog has the structure:

60. The method of claim 50, wherein the first fusion protein further comprises a DNA binding domain, and the second fusion protein further comprises a transcription activation domain.

61. The method of claim 50, wherein the first fusion protein further comprises a transcription activation domain, and the second fusion protein further comprises a DNA binding domain.

62. The method of claim 60 or 61, wherein the transcription activation domain is α

NTD.

63. The method of claim 60 or 61, wherein the DNA-binding domain is λ

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

64. The method of claim 50, wherein the first or the second fusion protein comprises a receptor domain of dihydrofolate reductase (“

DHFR”

), glucocorticoid receptor, FKBP12, FKBP mutants, tetracycline repressor, or a penicillin binding protein.

65. The method of claim 64, wherein the DHFR is the E. coli DHFR (“

eDHFR”

).

66. The method of claim 50, wherein the first fusion protein is DHFR-λ

cI or FKBP12-λ

cI.

67. The method of claim 50, wherein the second fusion protein is DHFR-α

NTD or FKBP12-α

NTD.

68. The method of claim 50, wherein the reporter gene is Lac Z, araBAD, aadA, his3, β

-lactamase, GFP, luciferase, TetR, KanR, Cm.

69. The method of claim 68, wherein the reporter gene is Lac Z.

74. A process for screening a cDNA library for a nucleic acid that encodes a receptor to which a known molecule binds, comprising providing a cDNA library, providing a dimeric molecule having two ligands, of which one ligand is the known molecule, and identifying the nucleic acid that encodes the receptor to which the known molecule binds by the method of claim 50.

75. An isolated nucleic acid identified by the process of claim 74, wherein the isolated nucleic acid was not previously known to encode a receptor to which the known molecule binds.

76. The isolated nucleic acid of claim 75, wherein the isolated nucleic acid encodes an enzyme or a portion thereof.

70. A kit for identifying a molecule that binds to a known target in a bacterial cell from a pool of candidate molecules, comprising

(a) a host bacterial cell containing a reporter gene that is expressed only when bound to a DNA-binding domain and when in the proximity of a transcription activation domain;

(b) a first vector containing a promoter that functions in the host bacterial cell and a DNA encoding a DNA-binding domain;

(c) a second vector containing a promoter that functions in the host bacterial cell and a DNA encoding a transcription activation domain;

(d) a dimeric small molecule which comprises a moiety that binds to a known receptor domain covalently linked to a candidate molecule;

(e) a means for inserting into the first vector or the second vector a DNA encoding the known receptor domain in such a manner that the known receptor domain and an expression product of the first or second vector are expressed as a fusion protein;

(f) a means for inserting into the first vector or the second vector a DNA encoding the known target receptor in such a manner that the known target receptor and an expression product of the first or second vector are expressed as a fusion protein; and

(g) a means for transfecting the host cell with the first vector, and the second vector,

wherein binding of the dimeric small molecule to the known target receptor results in a measurably greater expression of the reporter gene then in the absence of such binding.

71. A kit for identifying an unknown target receptor to which a molecule is capable of binding in a bacterial cell, comprising

(a) a host bacterial cell containing a reporter gene that is expressed only when bound to a DNA-binding domain and when in the proximity of a transcription activation domain;

(b) a first vector containing a promoter that functions in the host bacterial cell and a DNA encoding a DNA-binding domain;

(c) a second vector containing a promoter that functions in the host bacterial cell and a DNA encoding a transcription activation domain;

(d) a dimeric small molecule which comprises a moiety that binds a known receptor domain covalently linked to a moiety against which the unknown target is to be screened for binding;

(e) a means for inserting into the first vector or the second vector a DNA encoding the known receptor domain in such a manner that the known receptor domain and an expression product of the first or second vector are expressed as a fusion protein;

(f) a means for inserting into the first vector or the second vector a DNA encoding the unknown target receptor in such a manner that the unknown target receptor and an expression product of the first or second vector are expressed as a fusion protein; and

(g) a means for transfecting the host cell with the first vector, and the second vector,

wherein binding of the dimeric small molecule to the unknown target receptor results in a measurably greater expression of the reporter gene then in the absence of such binding.

77. A transgenic bacterial cell comprising

(a) a dimeric small molecule which comprises a methotrexate moiety covalently linked to a moiety capable of binding a receptor domain;

(b) nucleotide sequences which upon transcription encode

i) a first fusion protein comprising a DHFR domain and a first fragment of an enzyme, and

ii) a second fusion protein comprising the receptor domain and a second fragment of the enzyme,

wherein activity of the enzyme is conditioned on the proximity of the first fragment of the enzyme to the second fragment of the enzyme.

78. The bacterial cell of claim 77, wherein the moiety capable of binding the receptor domain is capable of binding with a IC₅₀ of less than 100 μ

M.

79. The bacterial cell of claim 77, wherein the moiety capable of binding the receptor domain is capable of binding with a IC₅₀ of less than 100 nM.

80. The bacterial cell of claim 77, wherein the moiety capable of binding the receptor domain is capable of binding with a IC₅₀ of less than 1 nM.

81. A method for identifying a molecule that binds a known target receptor in a bacterial cell from a pool of candidate molecules, comprising:

(a) forming a dimeric molecule by covalently bonding each molecule in the pool of candidate molecules to a methotrexate moiety;

(b) introducing the dimeric molecule into a bacterial cell culture comprising bacterial cells that express

a first fusion protein which comprises the known target receptor domain against which the candidate molecule is screened, and a first fragment of an enzyme, and

a second fusion protein which comprises a DHFR receptor domain and a second fragment of the enzyme;

(c) permitting the dimeric molecule to bind to the first fusion protein and to the second fusion protein, bringing the first fragment and the second fragment of the enzyme in to proximity so as to reconstitute the activity of the enzyme;

(d) selecting the bacterial cell that exhibits the activity of the enzyme; and

(e) identifying the small molecule that binds the known target receptor.

82. A method for identifying an unknown target receptor to which a known molecule is capable of binding in a bacterial cell, comprising:

(a) providing a dimeric molecule having a first ligand which has a specificity for the unknown target receptor covalently bonded to a methotrexate moiety;

(b) introducing the dimeric molecule into a bacterial cell which expresses

a first fusion protein which comprises the unknown target receptor domain, and a first fragment of an enzyme, and

a second fusion protein which comprises a DHFR receptor domain and a second fragment of the enzyme;

(c) permitting the dimeric molecule to bind to the first fusion protein and to the second fusion protein, bringing the first fragment and the second fragment of the enzyme in to proximity so as to reconstitute the activity of the enzyme;

(d) selecting the bacterial cell that exhibits the activity of the enzyme; and

(e) identifying the unknown target receptor.