Fluorescent phospholipase assays and compositions

• US 20050026235A1
• Filed: 06/25/2004
• Published: 02/03/2005
• Est. Priority Date: 06/30/2003
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Graham, Ronald J.
• Application Number:
  US10/877,421
• Publication Number:
  US 20050026235A1
• Time in Patent Office: Days
• US Class Current: 435/21
• CPC Class Codes: C12Q 1/44 involving esterase
• CPC Class Codes: G01N 33/92 involving lipids, e.g. cholesterol , lipoproteins, or their receptors steroid hormones G01N33/743

Abstract

The present disclosure provides methods, compositions, and kits for carrying out phospholipase assays. In some aspects, the methods comprise the use of a lipid complex comprising a fluorescently-labeled phospholipase substrate and detecting an increase in fluorescence due to phospholipase-mediated cleavage of the substrate.

Claims

1. A method of analyzing a sample for the presence of a phospholipase, the method comprising:

contacting a sample suspected of containing a phospholipase with a lipid complex comprising a fluorescently-labeled phospholipase substrate under conditions effective to permit the phospholipase to cleave the fluorescently-labeled phospholipase substrate, wherein the fluorescently-labeled phospholipase substrate comprises a hydrophobic moiety, a phosphate moiety and a fluorescent moiety covalently linked to the phosphate moiety, either directly or via an optional linker; and

detecting the fluorescence of the fluorescent moiety as a function of time, wherein an increase in fluorescence as a function of time correlates with the presence of the phospholipase in the sample.

2. The method of claim 1 in which the fluorescently-labeled phospholipase substrate is present in the lipid complex in an amount effective to quench the fluorescence signal of the fluorescent moiety.

3. The method of claim 1 in which the fluorescently-labeled phospholipase substrate is cleavable by a phospholipase selected from a phospholipase A₁, a phospholipase A₂, a phospholipase B, a phospholipase C and a phospholipase D.

4. The method of claim 1 in which the fluorescent moiety is selected from a xanthene fluorophore, a rhodamine fluorophore, a fluorescein fluorophore, a cyanine fluorophore and a bodipy fluorophore.

5. The method of claim 1 in which the fluorescently-labeled phospholipase substrate has the formula:

6. The method of claim 5 in which L comprises a linker having the formula —

S¹—

y—

S²—

, wherein S¹ and S² are spacing moieties and can be the same length or different lengths, and wherein Y comprises a linkage moiety.

7. The method of claim 5 in which one of R¹ or R² is an alkenyl and the other is an alkanyl.

8. The method of claim 5 in which L is derived from ethanolamine or serine.

9. The method of claim 5 in which D is a fluorescein or a rhodamine fluorophore.

10. The method of claim 5 in which the fluorescently-labeled phospholipase substrate is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Oregon Green 488, 5-isomer) or 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(Oregon Green 500, 5-isomer).

11. The method of claim 1 in which the lipid complex is a liposome.

12. The method of claim 1 in which the lipid complex is a micelle.

13. The method of claim 11 in which the liposome further comprises cholesterol.

14. The method of claim 11 in which the liposome encapsulates a fluorescent dye.

15. The method of claim 14 in which the fluorescent dye is capable of quenching the fluorescence of the fluorescent moiety.

16. The method of claim 1 in which the fluorescence signal is detected at one or more discrete time points.

17. The method of claim 1 in which the fluorescence signal is detected substantially continuously.

83. The kit of claim 76 further including instructions for carrying out the method of claim 1.

84. The method of any of claims 1, 18 or 35 in which the fluorescently-labeled substrate is capable of fluorescing in aqueous medium.

18. A method of detecting a cleavage activity of a phospholipase, the method comprising:

contacting a phospholipase with a lipid complex comprising a fluorescently-labeled phospholipase substrate under conditions effective to permit the phospholipase to cleave the phospholipase substrate, wherein the phospholipase substrate comprises a hydrophobic moiety, a phosphate moiety and a fluorescent moiety covalently linked to the phosphate moiety either directly or via an optional linker; and

detecting the fluorescence of the fluorescent moiety as a function of time, wherein an increase in fluorescence correlates with the presence of a cleavage activity of the phospholipase.

19. The method of claim 18 in which the fluorescently-labeled phospholipase substrate is present in the lipid complex in an amount effective to quench the fluorescence signal of the fluorescent moiety.

20. The method of claim 18 in which the fluorescently-labeled phospholipase substrate is cleavable by a phospholipase selected from a phospholipase A₁, a phospholipase A₂, a phospholipase C and a phospholipase D.

21. The method of claim 18 in which the fluorescent moiety is selected from a xanthene fluorophore, a rhodamine fluorophore, a fluorescein fluorophore, a cyanine fluorophore and a bodipy fluorophore.

22. The method of claim 18 in which the fluorescently-labeled phospholipase substrate has the formula:

23. The method of claim 22 in which L comprises a linker having the formula —

S¹—

Y—

S²—

, wherein S¹ and S² are spacing moieties and can be the same length or different lengths, and wherein Y comprises a linkage moiety.

24. The method of claim 22 in which one of R¹ or R² is an alkenyl and the other is an alkanyl.

25. The method of claim 22 in which L is derived from ethanolamine or serine.

26. The method of claim 22 in which D is a fluorescein or a rhodamine fluorophore.

27. The method of claim 22 in which the fluorescently-labeled phospholipase substrate is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Oregon Green 488, 5-isomer) or 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(Oregon Green 500, 5-isomer).

28. The method of claim 18 in which the lipid complex is a liposome.

29. The method of claim 18 in which the lipid complex is a micelle.

30. The method of claim 28 in which the liposome further comprises cholesterol.

31. The method of claim 28 in which the liposome encapsulates a fluorescent dye.

32. The method of claim 31 in which the fluorescent dye is capable of quenching the fluorescence of the fluorescent moiety.

33. The method of claim 18 in which the fluorescence signal is detected at discrete time points.

34. The method of claim 18 in which the fluorescence signal is detected substantially continuously.

35. A method of identifying a modulator of a phospholipase, comprising the steps of:

contacting a phospholipase with a lipid complex comprising a fluorescently-labeled phospholipase substrate in the presence of a candidate compound under conditions effective to permit the phospholipase to cleave the phospholipase substrate, wherein the phospholipase substrate comprises a hydrophobic moiety, a phosphate moiety and a fluorescent moiety covalently linked to the phosphate moiety either directly or via an optional linker; and

detecting the fluorescence of the fluorescent moiety as a function of time, wherein an increase or decrease in the quantity or rate of accumulation of fluorescence as compared to a control identifies the compound as a modulator of the phospholipase

36. The method of claim 35 in which the fluorescently-labeled phospholipase substrate is present in the lipid complex in an amount effective to quench the fluorescence signal of the fluorescent moiety.

37. The method of claim 35 in which the fluorescently-labeled phospholipase substrate is cleavable by a phospholipase selected from a phospholipase A₁, a phospholipase A₂, a phospholipase C and a phospholipase D.

38. The method of claim 35 in which the fluorescent moiety is selected from a xanthene fluorophore, a rhodamine fluorophore, a fluorescein fluorophore, a cyanine fluorophore and a bodipy fluorophore.

39. The method of claim 35 in which the fluorescently-labeled phospholipase substrate has the formula:

40. The method of claim 39 in which L comprises a linker having the formula —

S¹—

Y—

S²—

, wherein S¹ and S² are spacing moieties and can be the same length or different lengths, and wherein Y comprises a linkage moiety.

41. The method of claim 39 in which one of R¹ or R² is an alkenyl and the other is an alkanyl.

42. The method of claim 39 in which L is derived from ethanolamine or serine.

43. The method of claim 39 in which D is a fluorescein or a rhodamine fluorophore.

44. The method of claim 39 in which the fluorescently-labeled phospholipase substrate is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Oregon Green 488, 5-isomer) or 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(Oregon Green 500, 5-isomer).

45. The method of claim 35 in which the lipid complex is a liposome.

46. The method of claim 35 in which the lipid complex is a micelle.

47. The method of claim 45 in which the liposome further comprises cholesterol.

48. The method of claim 45 in which the liposome encapsulates a fluorescent dye.

49. The method of claim 48 in which the fluorescent dye is capable of quenching the fluorescence of the fluorescent moiety.

50. The method of claim 35 in which the fluorescence signal is detected at discrete time points.

51. The method of claim 35 in which the fluorescence signal is detected substantially continuously.

52. The method of claim 35 which is carried out to identify an activator of the phospholipase.

53. The method of claim 35 which is carried out to identify an antagonist of the phospholipase.

54. A method of identifying a modulator of a phospholipase, comprising the steps of:

contacting a phospholipase, a lipid complex comprising a fluorescently-labeled phospholipase substrate and a candidate compound under conditions effective to permit the phospholipase to cleave the phospholipase substrate, wherein the phospholipase substrate comprises a hydrophobic moiety, a phosphate moiety and a fluorescent moiety covalently linked to the phosphate moiety either directly or via an optional linker; and

detecting the fluorescence of the fluorescent moiety as a function of time, wherein an increase or decrease in the quantity or rate of accumulation of fluorescence as compared to a control reaction identifies the compound as a modulator of the phospholipase.

55. A composition comprising a phospholipase inhibitor and a lipid complex, said lipid complex comprising a phospholipid and a fluorescently-labeled phospholipase substrate, wherein the phospholipase substrate comprises a hydrophobic moiety, a phosphate moiety and a fluorescent moiety covalently attached to the phosphate moiety either directly or by way of an optional linker, and is included in the lipid complex in an amount effective to quench the fluorescence signal of the fluorescent moiety.

56. The lipid complex of claim 55 in which the fluorescent moiety is selected from a xanthene fluorophore, a fluorescein fluorophore, a rhodamine fluorophore and a cyanine fluorophore.

57. The lipid complex of claim 55 in which the fluorescent moiety is an energy transfer fluorophore.

58. The lipid complex of claim 55 which is in the form of a liposome.

59. The lipid complex of claim 58 which further includes cholesterol.

60. The lipid complex of claim 59 in which the cholesterol is in the range of 5 mol % to 80 mol %.

61. The lipid complex of claim 59 in which the amount of the cholesterol ranges from 10 to 50 mol %.

62. The lipid complex of claim 55 which encapsulates a fluorescent dye capable of quenching the fluorescent signal of the fluorescent moiety.

63. The lipid complex of claim 55 in which the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, cardiolipin, and mixtures thereof.

64. The lipid complex of claim 55 in which the amount of the phospholipase substrate ranges from 1 to 100 mol %.

65. The lipid complex of claim 55 comprising cholesterol and dioleoyl-phosphtidylcholine.

66. The lipid complex of claim 55 in which the phospholipid substrate comprises the formula:

67. The complex of claim 66 in which L comprises a linker having the formula —

S¹—

Y—

S²—

, wherein S¹ and S² are spacing moieties and can be the same length or different lengths, and wherein Y comprises a linkage moiety.

68. The lipid complex of claim 66 in which D is a fluorescein or a rhodamine fluorophore.

69. The lipid complex of claim 66 in which one of R¹ or R² is a saturated alkyl and the other is an unsaturated alkyl.

70. The lipid complex of claim 66 in which one of R¹ or R² is an alkanyl and the other is an alkenyl.

71. The lipid complex of claim 66 in which the fluorescently-labeled phospholipase substrate is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Oregon Green 488, 5-isomer) or 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(Oregon Green 500, 5-isomer).

85. The composition of claim 55 in which the fluorescently-labeled substrate is capable of fluorescing in aqueous medium.

72. A kit for use in a phospholipase reaction, said kit comprising:

at least one fluorescently labeled phospholipase cleavage substrate for detecting a target phospholipase and a buffer for preparing a reaction mixture that facilitates the phospholipase reaction.

73. The kit of claim 72 further including a phospholipase.

74. The kit of claim 73 wherein the phospholipase substrate is included in a lipid complex.

75. The kit of claim 74 wherein the lipid complex includes a fluorescent dye.

76. The kit of claim 75 wherein the lipid complex includes a fluorescence quenching compound.

77. The kit of claim 72 further including a modulator of a phospholipase activity.

78. The kit of claim 77 wherein said modulator is a phospholipase antagonist.

79. The kit of claim 78 wherein said antagonist is tricyclodecan-9-yl-xanthogenate or MJ33.

80. The kit of claim 77 wherein said modulator is a phospholipase activator.

81. The kit of claim 76 wherein said phospholipase is an enzyme standard.

82. The kit of claim 76 wherein said phospholipase is selected from the group consisting of a phospholipase A₁, a phospholipase A₂, a phospholipase C, a phospholipase D, and mixtures thereof.

86. The kit of claim 72 in which the fluorescently-labeled substrate is capable of fluorescing in aqueous medium.