Profiling of protease specificity using combinatorial fluorogenic substrate libraries
First Claim
1. A material having a fluorogenic moiety linked to a solid support, said material having the structure:
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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 Assignments
0 Petitions
Accused Products
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.
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Citations
112 Claims
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1. A material having a fluorogenic moiety linked to a solid support, said material having the structure:
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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 —
NHR15with 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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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4. The material according to claim 1, wherein R15 has the structure:
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5. The material according to claim 1, having the structure:
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6. The material according to claim 5, having the structure:
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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.
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7. A material according to claim 6, having the structure:
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8. A method of assaying for the presence of an enzymatically active protease in a sample, said method comprising:
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(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.
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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.
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10. The method according to claim 8, wherein said protease is a protease of a microorganism.
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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.
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12. The method according to claim 8, wherein said sample is a clinical sample from a subject.
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13. The method according to claim 8, further comprising (c) quantifying said fluorescent moiety, thereby quantifying said protease.
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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:
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(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.
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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:
- 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:
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73. A method of preparing a fluorogenic peptide, said method comprising:
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(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. - View Dependent Claims (74, 75, 76, 77, 78, 79, 80, 81, 82, 83)
(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.
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75. The method according to claim 73, further comprising between steps (b) and (c) capping aniline amine groups that have not reacted with pAA1.
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76. The method according to claim 75, wherein said capping utilizes a mixture comprising an active ester of a carboxylic acid.
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77. The method according to claim 76, wherein said active ester is the nitrotriazole ester of acetic acid.
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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.
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79. The method according to claim 78, wherein said mixture comprises at least 2 unique amino acids.
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80. The method according to claim 79, wherein said mixture comprises at least 6 unique amino acids.
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81. The method according to claim 80, wherein said mixture comprises at least 12 unique amino acids.
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82. The method according to claim 81, wherein said mixture comprises at least 20 unique amino acids.
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83. The method according to claim 78, wherein said mixture is an isokinetic mixture.
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84. A fluorogenic peptide comprising a fluorogenic moiety covalently bound to a peptide sequence, said peptide having the structure:
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85. A fluorogenic peptide having the structure:
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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 —
NR17R21wherein, R17 and R21 are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted aryl.
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- 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:
Specification