Compositions for the detection of enzyme activity in biological samples and methods of use thereof

• US 7,312,302 B2
• Filed: 06/04/2001
• Issued: 12/25/2007
• Est. Priority Date: 02/20/1997
• Status: Expired due to Fees

• Current Assignee: OncoImmunin, Inc.
• Original Assignee: Oncolmmunin Incorporated
• Inventors: Packard, Beverly S., Komoriya, Akira
• Primary Examiner(s): KAM, CHIH MIN
• Application Number:
  US09/874,350
• Publication Number:
  US 20040096926A1
• Time in Patent Office: 2,395 Days
• Field of Search: 514/13, 514/2, 530/300, 530/324, 530/326, 435/23, 435/24, 435/7.72
• US Class Current: 530/300
• 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 2333/96425 from mammals
• CPC Class Codes: G01N 2333/96469 Interleukin 1-beta convertase-like enzymes
• CPC Class Codes: G01N 33/533 with fluorescent label
• 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-teminal amide group or free carboxylic acid group.

4. The composition of claim 1, comprising an amino acid sequence selected from the group consisting of KDPJGYVHDAPVGJPKGY, KDPJGYVHDAPVPKGY (SEQ ID NO:

171), and KDPYVHDAPVGJPKGY (SEQ ID NO;

172).

5. The composition of claim 4, wherein said composition has a terminal blocking group.

6. The composition of claim 4, wherein said composition has a terminal 9-fluoreneacetyl (Fa) group.

7. The composition of claim 4, wherein said composition has a terminal 9-fluorenylmethoxycarbonyl (Fmoc) group.

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

9. The composition of claim 8, wherein said F¹ and F² have an excitation wavelength between about 315 nm and about 800 nm.

10. The composition of claim 8, wherein said composition bears a hydrophobic group.

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-dicarboxyphenyl)-3,6-bis(dimethylamino)xanthyliumhalide or other anion, 9-(2,6-dicarboxyphenyl)-3,6-bis(dimethylamino)xanthyliumhalide or other anion, 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-dicarboxyphenyl)-3,6-bisamino-xanthylium halide or other anion (Rh110), 9-(2,6-dicarboxyphenyl)-3,6-bisamino-xanthylium halide or other anion (Rh110), 9-(2,5-dicarboxyphenyl)-3-amino-6-hydroxy-xanthylium halide or other anion (Blue Rh), 9-(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 15, wherein said hydrophobic group is selected from the group consisting of:

Fmoc, 9-fluoreneacetyl group (Fa), 1-fluorenecarboxylic group, 9-fluorenecarboxylic group, and 9-fluorenone-1-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mint), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), Tosyl (Tos), 4,4-dimethoxybenzhydryl (Mbh), 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-dimethyl-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).

17. The composition of claim 16, wherein said hydrophobic group is Fmoc.

18. The composition of claim 16, wherein said hydrophobic group is Fa.

19. The composition of claim 16, wherein said hydrophobic group is attached to the amino terminus of the molecule.

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

21. The composition of claim 20, wherein the carboxyl terminal amino acid in which the carboxylic acid group is replaced with an amide.

22. The composition of claim 20, wherein:

r is zero; and

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

23. The composition of claim 20, comprising an amino acid sequence selected from the group consisting of KDBYVHDAPVPKGY (SEQ ID NO:

218), KDBGYVHDAPVGPKGY (SEQ ID NO;

219), KDBJGYVHDAPVGJPKGY (SEQ ID NO;

220), and KDBJG(dY)VHDAPVGJPKGY (SEQ ID NO;

221).

24. The composition of claim 23, wherein said composition has a terminal blocking group.

25. The composition of claim 23, wherein said composition has a terminal Fa group.

26. The composition of claim 23, wherein said composition has a terminal Fmoc group.

27. The composition of claim 20, wherein F¹ and F² are the same fluorophore.

28. The composition of claim 27, wherein F¹ and F² have an excitation wavelength between about 315 nm and about 800 nm.

29. The composition of claim 20, 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.

30. The composition of claim 20, 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.

31. The composition of claim 20, wherein said fluorophore is selected from the group consisting of rhodamine X, 9-(2,5-dicarboxyphenyl)-3,6-bis(dimethylamino)xanthyliumhalide or other anion, 9-(2,6-dicarboxyphenyl)-3,6-bis(dimethylamino)xanthyliumhalide or other anion, 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-dicarboxyphenyl)-3,6-bisamino-xanthylium halide or other anion (Rh110), 9-(2,6-dicarboxyphenyl)-3,6-bisamino-xanthylium halide or other anion (Rh110), 9-(2,5-dicarboxyphenyl)-3-amino-6-hydroxy-xanthylium halide or other anion (Blue Rh), 9-(2,6-dicarboxyphenyl)-3-amino-6-hydroxy-xanthylium halide or other anion (Blue Rh), carboxytetramethyirhodamine, 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.

32. The composition of claim 20, wherein said fluorophore comprises a carbocyanine dye.

33. The composition of claim 20, wherein said composition bears a hydrophobic group.

34. The composition of claim 33, wherein said hydrophobic group is selected from the group consisting of:

Fmoc, 9-fluoreneacetyl group (Fa), 1-fluorenecarboxylic group, 9-fluorenecarboxylic group, and 9-fluorenone-1-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mint), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), Tosyl (Tos), 4,4-dimethoxybenzhydryl (Mbh), 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-dimethyl-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).

35. The composition of claim 34, wherein said hydrophobic group is Fmoc.

36. The composition of claim 34, wherein said hydrophobic group is Fa.

37. The composition of claim 34, wherein said hydrophobic group is attached to the amino terminus of the molecule.