Method for Determining the Activity of a Protease in a Sample
First Claim
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1. A method for determining the activity of a protease in a sample comprising the steps of:
- (i) admixing said sample with a substrate, wherein the substrate has the formula (1a)
wherein;
R1 is a hydrocarbyl groupR2 is a first peptide moietyR3 is a second peptide moiety andX is selected from the group consisting of O, S and NH;
Y1 is a suitable substituent;
Y2 is a suitable substituent;
and(ii) determining the activity of said protease by detecting the presence of a reporter having the formula H—
X—
R1, wherein;
X is selected from the group consisting of O, S and NH; and
R1 is a hydrocarbyl group.
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Abstract
There is provided a method for determining the activity of a protease in a sample. The method comprises (i) admixing said sample with a substrate, wherein the substrate has the formula (1a) wherein: R1 is a hydrocarbyl group; R2 is a first peptide moiety; R3 is a second peptide moiety and X is selected from the group consisting of O, S and NH; Y1 is a suitable substituent; Y2 is a suitable substituent; and (ii) determining the activity of said protease by detecting the presence of a reporter having the formula H—X—R1, wherein: X is selected from the group consisting of O, S and NH; R1 is a hydrocarbyl group. The substrate and reporter are useful for determining the efficacy of protease-modulators and candidate protease-modulators and in the diagnosis of a disease or disorder in a subject.
11 Citations
36 Claims
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1. A method for determining the activity of a protease in a sample comprising the steps of:
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(i) admixing said sample with a substrate, wherein the substrate has the formula (1a) wherein; R1 is a hydrocarbyl group R2 is a first peptide moiety R3 is a second peptide moiety and X is selected from the group consisting of O, S and NH; Y1 is a suitable substituent; Y2 is a suitable substituent; and (ii) determining the activity of said protease by detecting the presence of a reporter having the formula H—
X—
R1, wherein;X is selected from the group consisting of O, S and NH; and R1 is a hydrocarbyl group. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 21, 22, 23, 24, 25, 28, 31, 32, 33, 34, 35)
wherein X denotes the remainder of the substrate of formula (1a) and/or the remainder of the reporter having the formula H—
X—
R1.
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17. The method according to claim 1 wherein said substrate is methyl 1-acetyl-L-prolyl-L-leucylglycyl-α
- -R-(4-nitrophenylamino)-glycyl-L-leucyl-β
-alaninate.
- -R-(4-nitrophenylamino)-glycyl-L-leucyl-β
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18. The method according to claim 17 wherein said reporter is 4-nitroaniline.
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21. A reporter having the formula H—
- X—
R1,wherein; R1 is a hydrocarbyl group and X is selected from the group consisting of O, S and NH; and wherein said reporter is derived from a substrate of the formula (1a) as defined in claim 1; but wherein said reporter is not 2-(4-Isobutyl-phenyl)-propionic acid.
- X—
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22. The reporter according to claim 21 wherein the hydrocarbyl group R1 is selected from the group consisting of an aryl, heteroaryl, aryloxyaryl, biaryl, alkyl, cycloalkyl, heterocycloalkyl group and derivatives thereof.
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23. The reporter according to claim 22 wherein R1 is selected from the group consisting of an aryl, heteroaryl, aryloxyaryl, biaryl and derivatives thereof.
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24. The reporter according to claim 23 wherein R1 is selected from the group consisting of:
wherein X denotes the remainder of the reporter.
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25. A kit for determining the activity of a protease in a sample comprising:
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(i) a substrate as defined in claim 1; and (ii) means for detecting a reporter as defined in claim 1 in a sample.
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28. A method for diagnosing a disease or disorder in a subject comprising the steps of obtaining a sample from said subject and determining the activity of a protease in said sample by the method according to claim 1.
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31. The method according to claim 28, wherein said disease is chronic obstructive pulmonary disease (COPD).
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32. A method for determining the efficacy of a protease-modulator;
- wherein said method comprises the steps of;
(i) admixing said protease-modulator with a protease and a substrate having the formula (1a) as defined in claim 1; (ii) determining the activity of said protease by detecting the presence of a reporter having the formula H—
X—
R1, as defined in claim 1.
- wherein said method comprises the steps of;
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33. A method for determining the efficacy of a candidate protease-modulator;
- wherein said method comprises the steps of;
(i) admixing said candidate protease-modulator with a protease and a substrate having the formula (1a) as defined in claim 1; and (ii) determining the activity of said protease by detecting the presence of a reporter having the formula H—
X—
R1, as defined in claim 1.
- wherein said method comprises the steps of;
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34. A method for identifying a protease-modulator;
- wherein said method comprises the steps of;
(i) admixing a candidate protease-modulator with a protease and a substrate having the formula (1a) as defined in claim 1; and (ii) determining the activity of said protease by detecting the presence of a reporter having the formula H—
X—
R1, as defined in claim 1.
- wherein said method comprises the steps of;
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35. A process comprising the steps of identifying a protease-modulator, preparing more of an identified protease-modulator and/or then formulating more of the identified protease-modulator;
- wherein said identification part comprises the steps of;
(i) admixing a candidate protease-modulator with a protease and a substrate having the formula (1a) as defined in claim 1; and (ii) determining the activity of said protease by detecting the presence of a reporter having the formula H—
X—
R1, as defined in claim 1.
- wherein said identification part comprises the steps of;
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19. A substrate having the formula (1a)
wherein: -
R1 is a hydrocarbyl group R2 is a first peptide moiety R3 is a second peptide moiety and X is selected from the group consisting of O, S and NH; Y1 is a suitable substituent; Y2 is a suitable substituent.
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20. A substrate capable of being cleaved at a peptide bond between a carbonyl group and a —
- NH—
CH(X—
R1)-group by a protease;wherein; R1 is a hydrocarbyl group and X is selected from the group consisting of O, S and NH.
- NH—
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26-27. -27. (canceled)
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29-30. -30. (canceled)
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36. (canceled)
Specification
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Current AssigneeAstrazeneca AB (Astrazeneca PLC)
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Original AssigneeAstrazeneca AB (Astrazeneca PLC)
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InventorsJolley, Keith, Blomgren, Anders, Hansson, Thomas, Eriksson, Anders, Lepisto, Matti, Von Wachenfeldt, Karin
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Application NumberUS12/296,568Publication NumberTime in Patent OfficeDaysField of SearchUS Class Current435/7.800CPC Class CodesC12Q 1/37 involving peptidase or prot...G01N 2333/96486 Metalloendopeptidases (3.4.24)G01N 2500/00 Screening for compounds of ...G01N 2800/122 Chronic or obstructive airw...
