Methods of screening for ligands of target molecules

• US 20030059811A1
• Filed: 06/13/2002
• Published: 03/27/2003
• Est. Priority Date: 06/14/2001
• Status: Abandoned Application

• Current Assignee: Anadys Pharmaceuticals Inc. (Roche Holding AG)
• Original Assignee: Anadys Pharmaceuticals Inc. (Roche Holding AG)
• Inventors: Djaballah, Hakim, Li, Bin, Coyle, Joseph, Patel, Rupal, Rongey, Scott, Wang, Mei Mei, Worland, Stephen
• Application Number:
  US10/170,758
• Publication Number:
  US 20030059811A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

The present invention provides methods of screening for ligands of target molecules. The methods of the present invention include assays in which a target molecule is subjected to denaturing conditions, and compounds are screened for the ability to alter the susceptibility of the target to unfolding. The methods of the present invention use fluorescence detection to determine that degree of unfolding of a target molecule. In some aspects of the present invention, fluorescence resonance energy transfer (FRET) is detected. In other aspects of the invention, fluorescence polarization (FP) is detected. In preferred embodiments, a target molecule such as a target protein is heated to a temperature, called T_ATLAS, at which at least a portion of the target molecule unfolds, in the presence of a test compound. In some embodiments of the present invention, the degree of unfolding of the target molecule is determined by binding of a specific binding member specific for the unfolded form of a target molecule that is coupled to a fluorophore that can participate in FRET. In some other embodiments of the present invention, the degree of unfolding of a target molecule is determined by FRET detection of aggregates of the target molecule. In yet other embodiments of the present invention, the degree of unfolding of a target molecule is determined by detection of fluorescence polarization of aggregates of the target molecule. The present invention provides sensitive, high throughput screens for identifying ligands of target molecules that are not dependent on the identity or function of the target.

Claims

1. A method of screening to identify one or more ligands that bind to a target molecule, comprising the steps:

(a) providing a target molecule in solution in one or more wells;

(b) adding to said one or more wells one or more test compounds to provide one or more test wells comprising a target molecule and one or more test compounds;

(c) adding to said one or more test wells a first specific binding member that specifically binds the unfolded form of said target molecule, wherein said first specific binding member comprises a FRET donor or a FRET acceptor or can directly or indirectly bind a FRET donor or a FRET acceptor;

(d) subjecting said one or more test wells to conditions at which at least a portion of said target molecule is denatured;

(e) adding to said one or more test wells a second specific binding member that can bind said target protein at a site distinct from the binding site of said first specific binding member, wherein;

when said first specific binding member comprises or can directly or indirectly bind a FRET donor fluorophore, said second specific binding member comprises or can directly or indirectly bind a FRET acceptor, and when said first specific binding member comprises or can directly or indirectly bind a FRET acceptor, said second specific binding member comprises or can directly or indirectly bind a FRET donor;

(f) measuring fluorescence emission at one or more wavelengths from said test wells;

(g) making a comparison of fluorescence emission at one or more wavelengths of said one or more test wells with one or more reference values;

(h) using said comparison in step (g) to determine the extent to which said target molecule occurs in the unfolded state, the folded state, or both in said one or more test wells; and

(i) using the determination in part (h) to determine whether said one or more test compounds binds said target molecule, thereby identifying one or more ligands of said target molecule.

2. The method of claim 1, wherein said subjecting said one or more test wells to conditions at which at least a portion of said target molecule is denatured comprises heating said one or more test wells to one or more predetermined temperatures at which at least a portion of said target molecule is denatured.

3. The method of claim 2, wherein said at least one predetermined temperature is one predetermined temperature.

4. The method of claim 1, wherein said target molecule is a target protein.

5. The method of claim 4, wherein said target protein comprises an attached tag that is recognized by said second specific binding member

6. The method of claim 5, wherein said attached tag is a chemical moiety.

7. The method of claim 6, wherein said chemical moiety is DNP or biotin.

8. The method of claim 5, wherein said attached tag is an engineered peptide tag.

9. The method of claim 8, wherein said engineered peptide tag is a 6xHis tag, a FLAG tag, a myc tag, or a hemaglutinin tag.

10. The method of claim 1, wherein said first specific binding member is an antibody that specifically binds the unfolded form of said target protein.

11. The method of claim 10, wherein said antibody can directly or indirectly bind a FRET donor or a FRET acceptor.

12. The method of claim 11, wherein said antibody can indirectly bind a FRET donor or a FRET acceptor.

13. The method of claim 12, wherein said antibody is bound to biotin, and said FRET donor or FRET acceptor is bound to streptavidin.

14. The method of claim 13, wherein step (e) further comprises adding said FRET donor or FRET acceptor bound to streptavidin.

15. The method of claim 12, wherein said second specific binding member is an antibody.

16. The method of claim 14, wherein said second specific binding member is an antibody that is directly bound to a FRET donor or FRET acceptor.

17. The method of claim 1, wherein said FRET donor is terbium, Alexa 488, Alexa 568, Alexa 594, Alexa 647, Cy3, BODIPY FL, fluorescein, IEDANS, EDANS, or Europium cryptate.

18. The method of claim 20, wherein said FRET donor is Europium cryptate.

19. The method of claim 1, wherein said FRET acceptor is fluorescein, GFP, TMR, Cy3, R phycoerythrin, Cy5, APC, Alexa 555, Alexa 647, Alexa 647, Alexa 594, Cy5, BODIPY FL, TMR, DABCYL, XL-665, or allophycocyanin.

20. The method of claim 19, wherein said FRET acceptor is XL-665.

21. The method of claim 1, wherein said one or more wavelengths is two wavelengths.

22. The method of claim 3, further comprising expressing said fluorescence emission as a ratio of fluorescence emission at two wavelengths.

23. The method of claim 1, wherein said reference value is one or more measurements or calculated values from one or more control wells.

24. The method of claim 1, wherein said reference value is one or more measurements or calculated values from one or more standard wells.

25. A method of screening to identify one or more ligands that bind to a target molecule, comprising the steps:

(a) providing a population of a target molecule, wherein at least a portion of said population is labeled with a first specific binding member, wherein said first specific binding member can bind a single attached tag of said target molecule and wherein said first specific binding member comprises or can directly or indirectly bind a FRET donor or a FRET acceptor;

(b) contacting an aliquot of said population of a target molecule with at least one test compound in one or more test wells;

(c) subjecting said one or more test wells to conditions at which at least a portion of said target protein is denatured;

(d) adding to said one or more test wells a second specific binding member that binds said single attached tag of said target molecule, wherein said second specific binding member comprises or can bind an acceptor or donor fluorophore, wherein;

when said first specific binding member comprises or can directly or indirectly bind a FRET donor, said second specific binding member comprises or can directly or indirectly bind a FRET acceptor, and when said first specific binding member comprises or can directly or indirectly bind a FRET acceptor, said second specific binding member comprises or can directly or indirectly bind a FRET donor;

(e) measuring fluorescence emission at one or more wavelengths from said one or more test wells;

(f) comparing fluorescence emission at said one or more wavelengths of said one or more test wells with one or more reference values;

(g) determining the extent to which said target molecule occurs in the unfolded state, the folded state, or both in said one or more test wells; and

(h) using the determination in part (g) to determine whether one or more test compounds binds said target molecule, thereby identifying one or more ligands of said target molecule.

26. The method of claim 25, wherein said at least a portion is approximately 50% of said population.

27. The method of claim 25, wherein said at least a portion is at least 80% of said population.

28. The method of claim 25, wherein said subjecting said one or more test wells to conditions at which at least a portion of said target molecule is denatured comprises heating said one or more test wells to one or more predetermined temperatures at which at least a portion of said target molecule is denatured.

29. The method of claim 28, wherein said at least one predetermined temperature is one predetermined temperature.

30. The method of claim 25, wherein said target molecule is a target protein that comprises a single attached tag.

31. The method of claim 30, wherein said single attached tag is a chemical moiety.

32. The method of claim 31, wherein said chemical moiety is DNP or biotin.

33. The method of claim 30, wherein said single attached tag is an engineered peptide tag.

34. The method of claim 33, wherein said engineered peptide tag is a 6xHis tag, a FLAG tag, a myc tag, or a hemaglutinin tag.

35. The method of claim 25, wherein said first specific binding member is an antibody.

36. The method of claim 35, wherein said antibody comprises or can directly or indirectly bind a donor fluorophore.

37. The method of claim 36, wherein said antibody comprises a donor fluorophore.

38. The method of claim 37, wherein said donor fluorophore is, terbium, Alexa 488 , Alexa 568, Alexa 594, Alexa 647, Cy3, BODIPY FL, fluorescein, IEDANS, EDANS, or Europium.

39. The method of claim 38, wherein said second specific binding member comprises or can directly or indirectly bind an acceptor fluorophore.

40. The method of claim 39, wherein said second specific binding member comprises an acceptor fluorophore.

41. The method of claim 40, wherein said acceptor fluorophore is fluorescein, GFP, TMR, Cy3, R phycoerythrin, Cy5, APC, Alexa 555, Alexa 647, Alexa 647, Alexa 594, Cy5, BODIPY FL, TMR, DABCYL, or XL665.

42. The method of claim 25, wherein in steps (e) and (f), said one or more wavelengths is two wavelengths.

43. The method of claim 42, further comprising, after step (e), calculating a ratio between fluorescence emission at said two wavelengths from said one or more test wells, and wherein in step (f), comparing fluorescence emission at two wavelengths comprises comparing a ratio of fluorescence emission at two wavelengths.

44. The method of claim 43, wherein said one or more reference values is one or more ratios between fluorescence emission at said two wavelengths from one or more control wells.

45. The method of claim 43, wherein said one or more reference values is one or more ratios between fluorescence emission at said two wavelengths from one or more standard wells.

46. A method of screening to identify one or more ligands that bind to a target molecule, comprising the steps:

(a) providing a first population of a target molecule that comprises or can bind a FRET donor or a FRET acceptor;

(b) adding to said first population of said target molecule a second population of said target molecule that comprises or can bind a FRET donor or a FRET acceptor, to generate a mixed donor/acceptor population of said target molecule, wherein;

when said first specific binding member comprises or can directly or indirectly bind a FRET donor, said second specific binding member comprises or can directly or indirectly bind a FRET acceptor, and when said first specific binding member comprises or can directly or indirectly bind a FRET acceptor, said second specific binding member comprises or can directly or indirectly bind a FRET donor;

(c) contacting an aliquot of said mixed donor/acceptor population of said target molecule with at least one test compound in one or more test wells;

(d) subjecting said one or more test wells to conditions at which at least a portion of said target protein is denatured;

(e) measuring fluorescence emission at one or more wavelengths from said one or more test wells;

(f) comparing fluorescence emission at one or more wavelengths of said one or more test wells with one or more reference values;

(g) determining the extent to which the target molecule occurs in the unfolded state, the folded state, or both, in said one or more test wells; and

(h) using the determination in part (g) to determine whether one or more test compounds binds said target molecule, thereby identifying one or more ligands of said target molecule.

47. The method of claim 46, wherein said subjecting said one or more test wells to conditions at which at least a portion of said target molecule is denatured comprises heating said one or more test wells to one or more predetermined temperatures.

48. The method of claim 47, wherein said at least one predetermined temperature is one predetermined temperature.

49. The method of claim 46, wherein said target molecule is a target protein.

50. The method of claim 49, wherein said target protein comprises an attached tag.

51. The method of claim 50, wherein said attached tag is a chemical moiety.

52. The method of claim 51, wherein said chemical moiety is DNP or biotin.

53. The method of claim 50, wherein said attached tag is an engineered peptide tag.

54. The method of claim 53, wherein said engineered peptide tag is a 6xHis tag, a FLAG tag, a myc tag, or a hemaglutinin tag.

55. The method of claim 46, wherein said FRET donor or said FRET acceptor is directly bound to said first population of said target protein.

56. The method of claim 53, wherein said FRET donor or said FRET acceptor is indirectly bound to said first population of said target protein.

57. The method of claim 55, wherein said FRET donor or said FRET acceptor is bound to said first population of said target protein via a specific binding member that recognizes said engineered peptide tag of said target protein.

58. The method of claim 53, wherein said FRET donor or FRET acceptor is indirectly bound to said second population of said target protein.

59. The method of claim 58, wherein said FRET donor or FRET acceptor is bound to said second population of said target protein via a specific binding member that recognizes said engineered peptide tag of said target protein.

60. The method of claim 46, wherein in steps (e) and (f), said one or more wavelengths is two wavelengths.

61. The method of claim 46, further comprising, after step (e), calculating a ratio between fluorescence emission at said two wavelengths from said one or more test wells, and wherein in step (f), comparing fluorescence emission at two wavelengths comprises comparing a ratio of fluorescence emission at two wavelengths.

62. The method of claim 61, wherein said reference value comprises at least one ratio between fluorescence emission at said two wavelengths.

63. The method of claim 62, wherein said one or more reference values is one or more ratios between fluorescence emission at said two wavelengths from one or more control wells.

64. The method of claim 62, wherein said one or more reference values is one or more ratios between fluorescence emission at said two wavelengths from one or more standard wells.

65. A method of screening to identify one or more ligands that binds to a target molecule, comprising the steps:

(a) labeling at least a portion of a population of a target molecule with at least one fluorophore;

(b) dispensing aliquots of said population of said target molecule in one or more test wells;

(c) adding to said one or more test wells one or more test compounds;

(d) subjecting said one or more test wells to conditions at which at least a portion of said target protein is denatured;

(e) measuring fluorescence polarization from said one or more test wells;

(f) comparing said fluourescence polarization measurements from said one or more test wells with a reference value;

(g) determining the extent to which the target molecule occurs in the unfolded state, the unfolded state, or both in the plurality of test wells and in said one or more control wells or control values; and

(h) using the determination in part (g) to determine whether one or more test compounds binds said target molecule, thereby identifying one or more ligands of said target molecule.

66. The method of claim 65, wherein said subjecting said one or more test wells to conditions at which at least a portion of said target molecule is denatured comprises heating said one or more test wells to one or more predetermined temperatures.

67. The method of claim 66, wherein said at least one predetermined temperature is one predetermined temperature.

68. The method of claim 65, wherein said target molecule is a target protein.

69. The method of claim 68, wherein said target protein is indirectly bound to a fluorophore.

70. The method of claim 68, wherein said target protein is directly bound to a fluorophore.

71. The method of claim 65, wherein said reference value is an average of fluorescence polarization measurements from two or more control wells.

72. The method of claim 65, wherein said reference value is an average of fluorescence polarization measurements from two or more standard wells.

74. The method of claim 70, wherein said subjecting said one or more test wells to conditions at which at least a portion of said target molecule is denatured comprises heating said one or more test wells to one or more predetermined temperatures.

75. The method of claim 71, wherein said at least one predetermined temperature is one predetermined temperature.

76. The method of claim 70, wherein said target molecule is a target protein.

77. The method of claim 70, wherein said specific binding member is indirectly bound to a fluorophore.

78. The method of claim 70, wherein said specific binding member is directly bound to a fluorophore.

79. The method of claim 78, wherein said specific binding member is coupled to a bead or particle.

73. A method of screening to identify one or more ligands that bind to a target molecule comprising the steps:

(a) providing a target molecule in solution in one or more test wells;

(b) adding to said one or more test wells one or more test compounds;

(c) adding to said one or more test wells at least one specific binding member that specifically binds the unfolded form of said target molecule, wherein said at least one first specific binding member comprises a fluorophore or can directly or indirectly bind a fluorophore;

(d) subjecting said one or more test wells conditions at which at least a portion of said target molecule is denatured;

(e) measuring fluorescence polarization from said one or more test wells;

(f) comparing said fluourescence polarization from said one or more test wells with a reference value;

(g) determining the extent to which said target molecule occurs in the unfolded state, the folded state, or both; and

(h) using the determination in part (g) to determine whether one or more test compounds binds said target molecule, thereby identifying one or more ligands of said target molecule.

80. A method of screening to identify one or more ligands that binds to a target molecule, comprising the steps:

(a) labeling at least a portion of a population of a target molecule with at least one fluorophore;

(b) dispensing aliquots of said population of said target molecule in one or more test wells;

(c) adding to said one or more test wells one or more test compounds;

(d) adding to said one or more test wells at least one specific binding member that specifically binds the unfolded form of said target molecule;

(e) subjecting said one or more test to conditions at which at least a portion of said target protein is denatured;

(f) measuring fluorescence polarization from said one or more test wells;

(g) comparing said fluourescence polarization from said one or more test wells with a reference value;

(h) determining the extent to which the target molecule occurs in the unfolded state, the folded state, or both in said one or more test; and

(i) using the determination in part (h) to determine whether one or more test compounds binds said target molecule, thereby identifying one or more ligands of said target molecule.

81. The method of claim 80, wherein said subjecting said one or more test wells to conditions at which at least a portion of said target molecule is denatured comprises heating said one or more test wells to one or more predetermined temperatures.

82. The method of claim 81, wherein said at least one predetermined temperature is one predetermined temperature.

83. The method of claim 80, wherein said target molecule is a target protein.

84. The method of claim 80, wherein said target molecule is indirectly bound to a fluorophore.

85. The method of claim 80, wherein said target molecule is directly bound to a fluorophore.

86. The method of claim 80, wherein said at least one specific binding member is coupled to a bead or particle.

87. The method of claim 80, wherein at least one specific binding member is at least two specific binding members.

88. The method of claim 87, wherein said at least two specific binding members comprises at least one primary antibody and at least one secondary antibody.