US 6,680,178 B2 · Profiling of protease specificity using combinatorial fluorogenic substrate libraries

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.

Claims112

Dependent claims

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.

11 more elements

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

R—P
wherein:
P is a peptide sequence having the structure:
—C(O)-AA1-AA2-(AAi)J-2
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
R is a fluorogenic moiety 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, 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, R4, 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;
R20 is either present or absent and is a member selected from the group consisting of substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
Y is an organic functional group;
m is a member selected from the group consisting of the integers 1 and 2;
and
t is a member selected from the group consisting of the integers from 0 to 2.

17 more elements

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:

R—P
wherein:
for each of said first peptide and said second peptide, P is independently selected from peptide sequences having the structure:
—C(O)-AA1-AA2-(AAi)J-2
wherein:
each of AA1 through AAi is an amino acid residue which is a member independently selected from the group consisting of natural amino acid residues, unnatural amino acid residues and modified amino acid residues;
each J is independently selected and denotes the number of amino acid residues forming said first peptide sequence and said second peptide sequence and is a member selected from the group consisting of the numbers from 2 to 10;
each i is independently selected and denotes the position of said amino acid residue 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
for each of said first peptide and said second peptide R is independently selected from fluorogenic moieties 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, 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, R4, 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 the solid support;
R20 is either present or absent and is a member selected from the group consisting of substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
Y is a member selected from the group consisting of organic functional groups and methyl;
m is a member selected from the group consisting of the integers from 1 to 2;
t is a member selected from the group consisting of the integers from 0 to 2;
R17 is a member selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted aryl;
and
SS is a solid support.

20 more elements

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.

11 more elements

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

R—P
wherein:
P is a peptide sequence having the structure:
—C(O)-AA1-AA2-(AAi)J-2
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
R is a fluorogenic moiety 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)mR6, —C(O)NR7R8, —S(O)tR9, —SO2NR10R11, —OR12, —NR18R19, substituted or unsubstituted alkyl, —NHC(O)—P, and —R20—Y, with the proviso that at least one of R1, R2, R3, R4, R5 and R6 is —R20—Y and at least one of R1, R2, R3, R4, R5 and R6 is —NHC(O)—P,
wherein:
R6, R7, R8, R9, R10, R11, R12, R18 and R19 are members independently selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted aryl;
R20 is either present or absent and is a member selected from the group consisting of substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
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;
m is a member selected from the group consisting of the integers 1 and 2;
and
t is a member selected from the group consisting of the integers from 0 to 2.

17 more elements

R—P
wherein:
for each of said first peptide and said second peptide, P is independently selected from peptide sequences having the structure:
—C(O)-AA1-AA2-(AAi)J-2
wherein:
each of AA1 through AAi is an amino acid residue which is a member independently selected from the group consisting of natural amino acid residues, unnatural amino acid residues and modified amino acid residues;
each J is independently selected and denotes the number of amino acid residues forming said first peptide sequence and said second peptide sequence and is a member selected from the group consisting of the numbers from 2 to 10;
each i is independently selected and denotes the position of said amino acid residue relative to AA1 and when Jis greater than 2, i is a member selected from the group consisting of the numbers from 3 to 10;
and
for each of said first peptide and said second peptide R is independently selected from fluorogenic moieties having the structure:
wherein:
R1, R2, R3, and R4 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, substituted or unsubstituted alkyl, —R14—SS, and —NHR15;
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, R4, 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 the solid support wherein said linking group is a member selected from the group consisting of substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
R20 is either present or absent and is a member selected from the group consisting of substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
Y is a member selected from the group consisting of organic functional groups and methyl;
m is a member selected from the group consisting of the integers from 1 to 2;
t is a member selected from the group consisting of the integers from 0 to 2;
R17 is a member selected from the group consisting of H, substituted or unsubstituted alkyl and substituted or unsubstituted aryl;
and
SS is a solid support.

21 more elements

Dependencies are inferred from the claim text.

Profiling of protease specificity using combinatorial fluorogenic substrate libraries

Life of this patent

Profiling of protease specificity using combinatorial fluorogenic substrate libraries

Life of this patent

Abstract

Claims112