COMPOSITIONS FOR THE DETECTION OF ENZYME ACTIVITY IN BIOLOGICAL SAMPLES AND METHODS OF USE THEREOF

• US 20080199898A1
• Filed: 11/16/2007
• Published: 08/21/2008
• Est. Priority Date: 02/20/1997
• Status: Active Grant

• Current Assignee: OncoImmunin, Inc.
• Original Assignee: OncoImmunin, Inc.
• Inventors: Packard, Beverly S., Komoriya, Akira
• Granted Patent: US 7,879,574 B2
• Time in Patent Office: Days
• US Class Current: 435/24
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C07K 7/08 having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 2333/96469 Interleukin 1-beta convertase-like enzymes
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

The present invention provides for novel reagents whose fluorescence increases in the presence of particular proteases. The reagents comprise a characteristically folded peptide backbone conjugated to two fluorophores such that the fluorophores are located opposite sides of a cleavage site. When the folded peptide is cleaved, as by digestion with a protease, the fluorophores provide a high intensity fluorescent signal at a visible wavelength. Because of their high specificity and their high fluorescence signal in the visible wavelengths, these protease indicators are particularly well suited for detection of protease activity in biological samples, in particular in frozen tissue sections. Thus this invention also provides for methods of detecting protease activity in situ in frozen sections.

Claims

1. A fluorogenic composition for the detection of the activity of a protease, said composition having the formula:

2. The composition of claim 1, wherein the carboxyl terminal amino acid in which the carboxylic acid group is replaced with an amide.

3. The composition of claim 1, wherein:

r is zero; and

aa¹⁰ has a C-terminal amide group or free carboxylic acid group.

5. The composition of claim 1, wherein said composition has a terminal blocking group.

6. The composition of claim 1, wherein said composition has a terminal Fa group.

7. The composition of claim 1, wherein said composition has a terminal Fmoc group.

9. The composition of claim 1, wherein F¹ and F² are the same fluorophore.

10. The composition of claim 9, wherein said F¹ and F² have an excitation wavelength between about 315 nm and about 800 nm.

11. The composition of claim 1, wherein the F¹ molecule is attached through either an α

-amino group of the aa¹ amino acid or through a side chain amino group of the aa¹ amino acid, or through a sulfhydryl group of a side chain of the aa¹ amino acid.

12. The composition of claim 1, wherein the F² molecule is attached either through a side chain amino group of the aa¹⁰ amino acid, through a carboxyl group of the aa¹⁰ amino acid, or through a sulfhydryl group of a side chain of the aa¹⁰ amino acid.

13. The composition of claim 1, wherein said fluorophore is selected from the group consisting of rhodamine X, 9-(2,5 (or 2,6)-dicarboxyphenyl)-3,6-bis(dimethylamino)xanthyliumhalide or other anion (TMR), 9-(2,5)-dicarboxyphenyl)-2,7-dimethyl-3,6-bis(ethylamino)xanthylium halide or other anion (Rh6G), 9-(2,6)-dicarboxyphenyl)-2,7-dimethyl-3,6-bis(ethylamino)xanthylium halide or other anion, 9-(2,5 (or 2,6)-dicarboxyphenyl)-3,6-bisamino-xanthylium halide or other anion (Rh110), 9-(2,5 (or 2,6)-dicarboxyphenyl)-3-amino-6-hydroxy-xanthylium halide or other anion (Blue Rh), carboxytetramethylrhodamine, carboxyrhodamine-X, diethylaminocoumarin, 9-(2,5-dicarboxyphenyl)-3,6-bis-(dimethylamino)xanthylium chloride (5-TMR), 9-(2,6-dicarboxyphenyl)-3,6-bis-(dimethylamino)xanthylium chloride (6-TMR), 9-(2-carboxyphenyl)-2,7-dimethyl-3,6-bis(ethylamino)xanthylium, 9-(2-carboxyphenyl)-3,6-bis(dimethylamino)xanthylium, and 9-(2-carboxyphenyl)-xanthylium.

14. The composition of claim 1, wherein said fluorophore comprises a carbocyanine dye.

15. The composition of claim 1, wherein said composition bears a hydrophobic group.

16. The composition of claim 1, wherein said composition bears a hydrophobic group.

17. The composition of claim 16, wherein said hydrophobic group is selected from the group consisting of:

Fmoc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylic group, and 9-fluorenone-1-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4=-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys), 1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom), t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), and Trifluoroacetyl (TFA).

18. The composition of claim 17, wherein said hydrophobic group is Fmoc.

19. The composition of claim 17, wherein said hydrophobic group is Fa.

20. The composition of claim 17, wherein said hydrophobic group is attached to the amino terminus of the molecule.

39. A method of detecting the activity of a protease, said method comprising contacting said protease with a composition of claim 1 or claim 21.

40. The method of claim 39, wherein said contacting is in a histological section.

41. The method of claim 39, wherein said contacting is in a cell culture.

42. The method of claim 39, wherein said contacting is in a tissue section.

43. The method of claim 39, wherein said contacting is contacting a seeded or cultured adherent cell.

44. The method of claim 39, wherein said contacting is in a cell suspension derived from a biological sample selected from the group consisting of a tissue, blood, urine, saliva, lymph, biopsy.

45. The method of claim 39, wherein said detecting is by a method selected from the group consisting of fluorescence microscopy, fluorescence microplate reader, absorption microplate reader, flow cytometry, fluorometry, absorption spectroscopy, and confocal fluorescent microplate reader.

46. A method of delivering a molecule into a cell, said method comprising:

providing a molecule according to claim 1 attached to a hydrophobic group or to at least one fused ring structure; and

contacting said cell with said molecule whereby said molecule enters said cell.

47. The method of claim 46, wherein said hydrophobic group is selected from the group consisting of:

selected from the group consisting of;

Fmoc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylic group, and 9-fluorenone-1-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys), 1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom), t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), and Trifluoroacetyl (TFA).

48. The method of claim 46, wherein, said fluorophores are selected from the group consisting of rhodamine X, 9-(2,5 (or 2,6)-dicarboxyphenyl)-3,6-bis(dimethylamino)xanthyliumhalide or other anion (TMR), 9-(2,5)-dicarboxyphenyl)-2,7-dimethyl-3,6-bis(ethylamino)xanthylium halide or other anion (Rh6G), 9-(2,6)-dicarboxyphenyl)-2,7-dimethyl-3,6-bis(ethylamino)xanthylium halide or other anion, 9-(2,5 (or 2,6)-dicarboxyphenyl)-3,6-bisamino-xanthylium halide or other anion (Rh110), 9-(2,5 (or 2,6)-dicarboxyphenyl)-3-amino-6-hydroxy-xanthylium halide or other anion (Blue Rh), carboxytetramethylrhodamine, carboxyrhodamine-X, diethylaminocoumarin, 9-(2,5-dicarboxyphenyl)-3,6-bis-(dimethylamino)xanthylium chloride (5-TMR), 9-(2,6-dicarboxyphenyl)-3,6-bis-(dimethylamino)xanthylium chloride (6-TMR), 9-(2-carboxyphenyl)-2,7-dimethyl-3,6-bis(ethylamino)xanthylium, 9-(2-carboxyphenyl)-3,6-bis(dimethylamino)xanthylium, and 9-(2-carboxyphenyl)-xanthylium.

49. The method of claim 46, wherein, said fluorophores are selected from the group consisting of:

of carboxytetramethylrhodamine, carboxyrhodamine-X, diethylaminocoumarin, rhodamine 110, and a carbocyanine dye.

50. The method of claim 46, wherein, said cell is a mammalian cell.

51. A method of screening a test agent for the ability to modulate the activity of a protease, said method comprising:

contacting a protease or a cell comprising a protease with said test agent;

contacting said protease with a fluorogenic composition of claims 1 or 21; and

detecting a signal or lack of signal produced by said fluorogenic composition where a difference in the signal produced by the protease or cell contacted with said test agent compared to a control in which the protease or cell is contacted by said test agent at a lower concentration indicates that said test agent modulates activity of said protease.

58. The method of claim 51, wherein said detecting comprises detecting an intracellular signal.

59. The method of claim 51, wherein said detecting comprises microscopy.

60. The method of claim 51, wherein said detecting comprises flow cytometry.

61. The method of claim 51, wherein said detecting comprises high-throughput screening of whole cells.

4. (canceled)

8. (canceled)

21. A fluorogenic composition for the detection of the activity of a protease, said composition having the formula:

22. The composition of claim 21, wherein the carboxyl terminal amino acid in which the carboxylic acid group is replaced with an amide.

23. The composition of claim 21, wherein:

r is zero; and

aa¹⁰ has a C-terminal amide group or free carboxylic acid group.

25. The composition of claim 21, wherein said composition has a terminal blocking group.

26. The composition of claim 21, wherein said composition has a terminal Fa group.

27. The composition of claim 21, wherein said composition has a terminal Fmoc group.

28. The composition of claim 21, wherein F¹ and F² are the same fluorophore.

29. The composition of claim 28, wherein F¹ and F² have an excitation wavelength between about 315 nm and about 800 nm.

30. The composition of claim 21, wherein the F¹ molecule is attached through either an α

-amino group of the aa¹ amino acid or through a side chain amino group of the aa¹ amino acid, or through a sulfhydryl group of a side chain of the aa¹ amino acid.

31. The composition of claim 21, wherein the F² molecule is attached either through a side chain amino group of the aa¹⁰ amino acid, through a carboxyl group of the aa¹⁰ amino acid, or through a sulfhydryl group of a side chain of the aa¹⁰ amino acid.

32. The composition of claim 21, wherein said fluorophore is selected from the group consisting of rhodamine X, 9-(2,5 (or 2,6)-dicarboxyphenyl)-3,6-bis(dimethylamino)xanthyliumhalide or other anion (TMR), 9-(2,5)-dicarboxyphenyl)-2,7-dimethyl-3,6-bis(ethylamino)xanthylium halide or other anion (Rh6G), 9-(2,6)-dicarboxyphenyl)-2,7-dimethyl-3,6-bis(ethylamino)xanthylium halide or other anion, 9-(2,5 (or 2,6)-dicarboxyphenyl)-3,6-bisamino-xanthylium halide or other anion (Rh110), 9-(2,5 (or 2,6)-dicarboxyphenyl)-3-amino-6-hydroxy-xanthylium halide or other anion (Blue Rh), carboxytetramethylrhodamine, carboxyrhodamine-X, diethylaminocoumarin, 9-(2,5-dicarboxyphenyl)-3,6-bis-(dimethylamino)xanthylium chloride (5-TMR), 9-(2,6-dicarboxyphenyl)-3,6-bis-(dimethylamino)xanthylium chloride (6-TMR), 9-(2-carboxyphenyl)-2,7-dimethyl-3,6-bis(ethylamino)xanthylium, 9-(2-carboxyphenyl)-3,6-bis(dimethylamino)xanthylium, and 9-(2-carboxyphenyl)-xanthylium.

33. The composition of claim 21, wherein said fluorophore comprises a carbocyanine dye.

34. The composition of claim 21, wherein said composition bears a hydrophobic group.

35. The composition of claim 34, wherein said hydrophobic group is selected from the group consisting of:

Fmoc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylic group, and 9-fluorenone-1-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4=-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys), 1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom), t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), and Trifluoroacetyl (TFA).

36. The composition of claim 21, wherein said hydrophobic group is Fmoc.

37. The composition of claim 21, wherein said hydrophobic group is Fa.

38. The composition of claim 21, wherein said hydrophobic group is attached to the amino terminus of the molecule.

24. (canceled)

52-57. -57. (canceled)