Profiling of protease specificity using combinatorial fluorogenic substrate libraries

• US 6,680,178 B2
• Filed: 05/25/2001
• Issued: 01/20/2004
• Est. Priority Date: 06/02/2000
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Ellman, Jonathan A., Backes, Bradley J., Harris, Jennifer L., Craik, Charles S.
• Primary Examiner(s): Leary, Louise N.
• Application Number:
  US09/866,132
• Publication Number:
  US 20020022243A1
• Time in Patent Office: 970 Days
• Field of Search: 435/24, 435/23, 435/34, 435/4, 530/300, 530/324, 530/350, 530/334, 530/345, 525/50, 525/54.1, 549/288, 702/22, 702/30
• US Class Current: 435/23
• CPC Class Codes: C07K 1/047 Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C07K 5/101 the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
• CPC Class Codes: C07K 5/1024 with the first amino acid being heterocyclic
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase

Abstract

A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of enzymes, such as proteases. The substrates contain a fluorogenic-leaving group, such as 7-amino-4-carbamoylmethyl-coumarin (ACC). Substrates incorporating the ACC leaving group show comparable kinetic profiles as those with the traditionally used 7-amino-4-methyl-coumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries using solid-phase synthesis techniques. The approximately 3-fold increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137,180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed. Employing this screening method the substrate specificities of a diverse array of proteases were profiled, including the serine proteases thrombin, plasmin, factor Xa, uPA, tPA, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases allowing for the design of selective substrates and potent inhibitors.

Claims

1. A material having a fluorogenic moiety linked to a solid support, said material having the structure:

wherein;

R¹, R², R³, R⁴, R⁵ and R⁶ are members independently selected from the group consisting of H, halogen, —

NO₂, —

CN, —

C(O)_mR⁷, —

C(O)NR⁸R⁹, —

S(O)_tR¹⁰, —

SO₂NR¹¹R¹², —

OR¹³, substituted or unsubstituted alkyl, —

R¹⁴—

SS, and —

NHR¹⁵

with the proviso that at least one of R¹, R², R³, R⁴, R⁵ and R⁶ is —

R¹⁴—

SS and at least one of R¹, R², R³, R ⁴, R⁵ and R⁶ is —

NHR¹⁵,

wherein;

R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁸ and R¹⁹ are members independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted aryl;

R¹⁴ is a linking group adjoining said fluorogenic moiety and said solid support;

R¹⁵ is a member selected from the group consisting of amine protecting groups, —

C(O)-AA and —

C(O)—

P;

wherein;

P is a peptide sequence;

AA is an amino acid residue;

m is a member selected from the group consisting of the integers 1 and 2;

t is a member selected from the group consisting of the integers from 0 to 2; and

SS is a solid support.

2. The material according to claim 1, wherein said linking group is a member selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and substituted or unsubstituted aryl.

3. The material according to claim 1, wherein P is a peptide sequence comprising the structure:

4. The material according to claim 1, wherein R¹⁵ has the structure:

5. The material according to claim 1, having the structure:

6. The material according to claim 5, having the structure:

wherein, Z is a member selected from the group consisting of —

O—

, and —

—

NR¹⁶—

; and

c is a member selected from the integers from 0 to 6.

7. A material according to claim 6, having the structure:

8. A method of assaying for the presence of an enzymatically active protease in a sample, said method comprising:

(a) contacting said sample with a material according to claim 3 in such a manner whereby said fluorogenic moiety is released from said peptide sequence upon action of said protease, thereby producing a fluorescent moiety; and

(b) observing whether said sample undergoes a detectable change in fluorescence, said detectable change being an indication of the presence of said enzymatically active protease in said sample.

9. The method according to claim 8, wherein said protease is a member selected from the group consisting of aspartic protease, cysteine protease, metalloprotease and serine protease.

10. The method according to claim 8, wherein said protease is a protease of a microorganism.

11. The method according to claim 10, wherein said microorganism is a member selected from the group consisting of bacteria, fungi, yeast, viruses, and protozoa.

12. The method according to claim 8, wherein said sample is a clinical sample from a subject.

13. The method according to claim 8, further comprising (c) quantifying said fluorescent moiety, thereby quantifying said protease.

14. A method of assaying for the presence of a selected microorganism in a sample by probing the sequence specificity of peptide cleavage by a protease of said microorganism, said method comprising:

(a) contacting a sample suspected of containing said selected microorganism with a material according to claim 3, wherein said peptide comprises a sequence that is selectively cleaved by said protease of said selected microorganism, thereby releasing the fluorogenic moiety from the peptide sequence;

(b) detecting the cleavage by detecting fluorescence arising from a fluorescent moiety produced by cleavage of said fluorogenic moiety from said peptide sequence, thereby confirming said presence of said selected microorganism in said sample.

15. The method according to claim 14, further comprising (c) quantifying said fluorescence, thereby quantifying said protease of said microorganism.

16. A fluorogenic peptide comprising a fluorogenic moiety covalently bound to a peptide sequence, said peptide having the structure:

17. The fluorogenic peptide according to claim 16, wherein said organic functional group is a member selected from the group consisting of —

COOR¹⁷, CONR¹⁷R²¹, —

C(O)R¹⁷R²¹, —

OR¹⁷, —

SR¹⁷, —

C(O)SR¹⁷ and —

NR¹⁷R²¹

wherein, R¹⁷ and R²¹ are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted aryl.

18. The fluorogenic peptide according to claim 16, having the structure:

19. The fluorogenic peptide according to claim 18, having the structure:

wherein;

c is a member selected from the group consisting of the integers from 0 to 6.

20. The fluorogenic peptide according to claim 19, having the structure:

21. The fluorogenic peptide according to claim 16, wherein said peptide sequence comprises a peptide bond that is cleaved by a protease releasing said fluorogenic moiety from said peptide sequence, thereby producing a fluorescent moiety and a peptide moiety.

22. The fluorogenic peptide according to claim 21, wherein said peptide bond is formed between a carboxyl of the carboxy-terminus amino acid residue and an amine group of said fluorogenic moiety.

23. A method of assaying for the presence of an enzymatically active protease in a sample, said method comprising:

(a) contacting a sample suspected of containing said protease with a peptide according to claim 16 in such a manner whereby said fluorogenic moiety is released from said peptide sequence upon action of said protease, thereby producing a fluorescent moiety; and

(b) observing whether said sample undergoes a detectable change in fluorescence, said detectable change being an indication of the presence of said enzymatically active protease in said sample.

24. The method according to claim 23, wherein said protease is a member selected from the group consisting of aspartic protease, cysteine protease, metalloprotease and serine protease.

25. The method according to claim 23, wherein said protease is a protease of a microorganism.

26. The method according to claim 25, wherein said microorganism is a member selected from the group consisting of bacteria, fungi, yeast, viruses, and protozoa.

27. The method according to claim 23, wherein said sample is a clinical sample from a subject.

28. The method according to claim 27, wherein said subject is a human.

29. The method according to claim 23, further comprising (c) quantifying said fluorescent moiety, thereby quantifying said protease.

30. A method of assaying for the presence of a selected microorganism in a sample by probing the sequence specificity of peptide cleavage by a protease of said microorganism, said method comprising:

(a) contacting a sample suspected of containing said selected microorganism with a material according to claim 16, wherein said peptide comprises a sequence that is selectively cleaved by a protease of a selected microorganism, thereby releasing said fluorogenic moiety from said peptide sequence;

(b) detecting said cleavage by detecting fluorescence arising from a fluorescent moiety produced by cleavage of said fluorogenic moiety from said peptide sequence, thereby confirming said presence of said selected microorganism in said sample.

31. The method according to claim 30, further comprising (c) quantifying said fluorescence, thereby quantifying said protease of said microorganism.

32. A library of fluorogenic peptides comprising at least a first peptide having a first peptide sequence covalently attached to a first fluorogenic moiety and a second peptide having a second peptide sequence covalently attached to a second fluorogenic moiety, said first peptide and said second peptide having the structure:

33. The library according to claim 32, wherein said linking group is a member selected from the group consisting of substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl.

34. The library according to claim 32, wherein said organic functional group is a member selected from the group consisting of —

COOR¹⁷, CONR¹⁷R²¹, —

C(O)R¹⁷R²¹, —

OR¹⁷, —

SR¹⁷, —

C(O)SR¹⁷, and —

NR¹⁷R²¹

wherein, R¹⁷ and R²¹ are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted aryl.

35. The library of fluorogenic peptides according to claim 32, wherein R—

P has the structure;

36. The library of fluorogenic peptides according to claim 35, wherein R—

P has the structure;

wherein,

c is a member selected from the group consisting of the numbers from 0 to 6.

37. The library of fluorogenic peptides according to claim 36, wherein R—

P has the structure;

38. The library according to claim 32, wherein said fluorogenic moiety of said first peptide and said fluorogenic moiety of said second peptide are different fluorogenic moieties.

39. The library according to claim 32, wherein said first peptide sequence and said second peptide sequence are identical.

40. The library according to claim 32, wherein said first peptide sequence and said second peptide sequence are different.

41. The library according to claim 40, wherein an amino acid residue selected from the group consisting of AA¹, AA², AAⁱ and combinations thereof of said first peptide is a different amino acid residue than an amino acid residue at a corresponding position relative to AA¹ of said second peptide.

42. The library according to claim 32, wherein AA¹ of said first peptide sequence and AA¹ of said second peptide sequence are identical amino acid residues.

43. The library according to claim 32, wherein AA¹ of said first peptide sequence and AA¹ of said second peptide sequence are different amino acid residues.

44. The library according to claim 32, wherein AA² of said first peptide sequence and AA² of said second peptide sequence are identical amino acid residues.

45. The library according to claim 32, wherein AA² of said first peptide sequence and AA² of said second peptide sequence are different amino acid residues.

46. The library according to claim 32, wherein AAⁱ of said first peptide sequence and AAⁱ of said second peptide sequence are identical amino acid residues.

47. The library according to claim 32, wherein AAⁱ of said first peptide sequence and AAⁱ of said second peptide sequence are different amino acid residues.

48. The library according to claim 42, comprising at least six peptides having different peptide sequences, wherein AA¹ is a different amino acid residue in each of said different peptide sequences.

49. The library according to claim 48, comprising at least twelve peptides having different peptide sequences wherein AA¹ is a different amino acid residue in each of said different peptide sequences.

50. The library according to claim 49, comprising at least twenty peptides having different peptide sequences wherein AA¹ is a different amino acid residue in each of said different peptide sequences.

51. The library according to claim 32, wherein AA¹ is a member selected from the group consisting of Lys, Arg, Leu and combinations thereof.

52. The library according to claim 32, wherein J is a member selected from the numbers from 4 to 8.

53. The library of peptides according to claim 32, wherein at least one of said first peptide and said second peptide is cleavable by a protease into a fluorescent moiety and the peptide sequence.

54. The library according to claim 32, comprising at least 10 peptides, wherein each of the peptide sequences is a different peptide sequence.

55. The library according to claim 54, comprising at least 100 peptides, wherein each of the peptide sequences is a different peptide sequence.

56. The library according to claim 55, comprising at least 1,000 peptides, wherein each of the peptide sequences is a different peptide sequence.

57. The library according to claim 56, comprising at least 10,000 peptides, wherein each of the peptide sequences is a different peptide sequence.

58. The library according to claim 57, comprising at least 100,000 peptides, wherein each of the peptide sequences is a different peptide sequence.

59. The library according to claim 58 comprising at least 1,000,000 peptides, wherein each of the peptide sequences is a different peptide sequence.

60. The library according to claim 32, wherein said first peptide is located at a first region of a substrate and said second peptide is located at a second region of a substrate.

61. A method of determining a peptide sequence specificity profile of an enzymatically active protease, said method comprising:

(a) contacting said protease with a library of peptides according to claim 32 in such a manner whereby the fluorogenic moiety is released from the peptide sequence, thereby forming a fluorescent moiety;

(b) detecting said fluorescent moiety;

(c) determining the sequence of said peptide sequence, thereby determining said peptide sequence specificity profile of said protease.

62. The method according to claim 61, further comprising (d) quantifying said fluorescent moiety, thereby quantifying said protease.

63. A database comprising at least one set of peptide sequence specificity data for a protease determined using a library according to claim 32.

64. The database according to claim 63, wherein said database is an electronic database.

65. The database according to claim 64, wherein said database is distributed on a wide area network.

66. A database comprising at least one set of peptide sequence specificity data for a protease determined using a method according to claim 61.

67. The database according to claim 63, wherein said database is an electronic database.

68. The database according to claim 64, wherein said database is distributed on a wide area network.

69. The method according to claim 61, wherein said protease is a member selected from the group consisting of aspartic protease, cysteine protease, and serine protease.

70. The method according to claim 61, wherein said protease is a protease of a microorganism.

71. The method according to claim 70, wherein said microorganism is a member selected from the group consisting of bacteria, fungi, yeast, viruses, and protozoa.

72. The method according to claim 61, further comprising (d) quantifying the fluorescent moiety, thereby quantifying said protease.

73. A method of preparing a fluorogenic peptide, said method comprising:

(a) providing a first conjugate comprising a fluorogenic moiety covalently bonded to a solid support, said conjugate having the structure;

wherein,

R¹, R², R³, R⁴, R⁵ and R⁶ are members independently selected from the group consisting of H, halogen, —

NO₂, —

CN, —

C(O)_mR⁷, —

C(O)NR⁸R⁹, —

S(O)_tR¹⁰, —

SO₂NR¹¹R¹², —

OR¹³, —

NR¹⁸R¹⁹, and substituted or unsubstituted alkyl, with the proviso that at least one of R¹, R², R³, R⁴, R⁵ and R⁶ is —

NH₂;

R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁸ and R¹⁹ are members independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted aryl;

m is a member selected from the group consisting of the numbers from 1 to 2;

t is a member selected from the group consisting of the numbers from 0 to 2;

R⁵ and R⁶ are members independently selected from the group consisting of H and —

R¹⁴—

C(O)NH—

SS, wherein at least one of R⁵ and R⁶ is —

R¹⁴—

C(O)NH—

SS;

R¹⁴ is a member selected from the group consisting of substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;

SS is a solid support;

(b) contacting said first conjugate with a first protected amino acid moiety (pAA¹) and an activating agent, thereby forming a peptide bond between a carboxyl group of pAA¹ and the aniline nitrogen of said first conjugate;

(c) deprotecting said pAA¹, thereby forming a second conjugate having a reactive AA¹ amine moiety;

(d) contacting said second conjugate with a second protected amino acid (pAA²) and an activating agent, thereby forming a peptide bond between a carboxyl group of pAA2 and said reactive AA¹ amine moiety; and

(e) deprotecting said pAA², thereby forming a third conjugate having a reactive AA² amine moiety.

74. The method according to claim 73, further comprising:

(f) contacting said third conjugate with a third protected amino acid (pAA³) and an activating agent, thereby forming a peptide bond between a carboxyl group of pAA³ and said reactive AA² amine moiety; and

(e) deprotecting said pAA³, thereby forming a fourth conjugate having a reactive AA³ amine moiety.

75. The method according to claim 73, further comprising between steps (b) and (c) capping aniline amine groups that have not reacted with pAA¹.

76. The method according to claim 75, wherein said capping utilizes a mixture comprising an active ester of a carboxylic acid.

77. The method according to claim 76, wherein said active ester is the nitrotriazole ester of acetic acid.

78. The method according to claim 74, wherein a member selected from the group consisting of pAA¹, pAA², pAA³ and combinations thereof comprises a mixture of protected amino acids differing in the identity of the amino acid portion of the protected amino acids.

79. The method according to claim 78, wherein said mixture comprises at least 2 unique amino acids.

80. The method according to claim 79, wherein said mixture comprises at least 6 unique amino acids.

81. The method according to claim 80, wherein said mixture comprises at least 12 unique amino acids.

82. The method according to claim 81, wherein said mixture comprises at least 20 unique amino acids.

83. The method according to claim 78, wherein said mixture is an isokinetic mixture.

84. A fluorogenic peptide comprising a fluorogenic moiety covalently bound to a peptide sequence, said peptide having the structure:

85. A fluorogenic peptide having the structure:

wherein;

each of AA¹ through AAⁱ is an amino acid residue which is a member independently selected from the group of natural amino acid residues, unnatural amino acid residues and modified amino acid residues;

J denotes the number of amino acid residues forming said peptide sequence and is a member selected from the group consisting of the numbers from 2 to 10, such that J-2 is the number of amino acid residues in the peptide sequence exclusive of AA¹-AA²;

i denotes the position of said amino acid residue in sequence relative to AA¹ and when J is greater than 2, i is a member selected from the group consisting of the numbers from 3 to 10; and

Y is a member selected from the group consisting of —

COOR¹⁷, CONR¹⁷R²¹, —

C(O)R¹⁷R²¹, —

OR¹⁷, —

SR¹⁷, —

C(O)SR¹⁷ and —

NR¹⁷R²¹

wherein, R¹⁷ and R²¹ are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted aryl.

86. A library of fluorogenic peptides comprising at least a first peptide having a first peptide sequence covalently attached to a first fluorogenic moiety and a second peptide having a second peptide sequence covalently attached to a second fluorogenic moiety, said first peptide and said second peptide having the structure:

87. The library according to claim 86, wherein said organic functional group is a member selected from the group consisting of —

COOR¹⁷, CONR¹⁷R²¹, —

C(O)R¹⁷R²¹, —

OR¹⁷, —

SR¹⁷, —

C(O)SR¹⁷, and —

NR¹⁷R²¹

wherein, R¹⁷ and R²¹ are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted aryl.

88. The library of fluorogenic peptides according to claim 86, wherein R—

P has the structure;

89. The library of fluorogenic peptides according to claim 88, wherein R—

P has the structure;

wherein,

c is a member selected from the group consisting of the numbers from 0 to 6.

90. The library of fluorogenic peptides according to claim 89, wherein R—

P has the structure;

91. The library according to claim 86, wherein said fluorogenic moiety of said first peptide and said fluorogenic moiety of said second peptide are different fluorogenic moieties.

92. The library according to claim 86, wherein said first peptide sequence and said second peptide sequence are identical.

93. The library according to claim 86, wherein said first peptide sequence and said second peptide sequence are different.

94. The library according to claim 93, wherein an amino acid residue selected from the group consisting of AA¹, AA², AAⁱ and combinations thereof of said first peptide is a different amino acid residue than an amino acid residue at a corresponding position relative to AA¹ of said second peptide.

95. The library according to claim 86, wherein AA¹ of said first peptide sequence and AA¹ of said second peptide sequence are identical amino acid residues.

96. The library according to claim 86, wherein AA¹ of said first peptide sequence and AA¹ of said second peptide sequence are different amino acid residues.

97. The library according to claim 86, wherein AA² of said first peptide sequence and AA² of said second peptide sequence are identical amino acid residues.

98. The library according to claim 86, wherein AA² of said first peptide sequence and AA² of said second peptide sequence are different amino acid residues.

99. The library according to claim 86, wherein AAⁱ of said first peptide sequence and AAⁱ of said second peptide sequence are identical amino acid residues.

100. The library according to claim 86, wherein AAⁱ of said first peptide sequence and AAⁱ of said second peptide sequence are different amino acid residues.

101. The library according to claim 99, comprising at least six peptides having different peptide sequences, wherein AA¹ is a different amino acid residue in each of said different peptide sequences.

102. The library according to claim 101, comprising at least twelve peptides having different peptide sequences wherein AA¹ is a difterent amino acid residue in each of said different peptide sequences.

103. The library according to claim 102, comprising at least twenty peptides having different peptide sequences wherein AA¹ is a different amino acid residue in each of said different peptide sequences.

104. The library according to claim 86, wherein AA¹ is a member selected from the group consisting of Lys, Arg, Leu and combinations thereof.

105. The library according to claim 86, wherein J is a member selected from the numbers from 4 to 8.

106. The library of peptides according to claim 86, wherein at least one of said first peptide and said second peptide is cleavable by a protease into a fluorescent moiety and the peptide sequence.

107. The library according to claim 86, comprising at least 10 peptides, wherein each of the peptide sequences is a different peptide sequence.

108. The library according to claim 107, comprising at least 100 peptides, wherein each of the peptide sequences is a different peptide sequence.

109. The library according to claim 108, comprising at least 1,000 peptides, wherein each of the peptide sequences is a different peptide sequence.

110. The library according to claim 109, comprising at least 10,000 peptides, wherein each of the peptide sequences is a different peptide sequence.

111. The library according to claim 110, comprising at least 100,000 peptides, wherein each of the peptide sequences is a different peptide sequence.

112. The library according to claim 111 comprising at least 1,000,000 peptides, wherein each of the peptide sequences is a different peptide sequence.