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

• US 7,879,574 B2
• Filed: 11/16/2007
• Issued: 02/01/2011
• Est. Priority Date: 02/20/1997
• Status: Expired due to Fees

• Current Assignee: OncoImmunin, Inc.
• Original Assignee: OncoImmunin, Inc.
• Inventors: Packard, Beverly S., Komoriya, Akira
• Primary Examiner(s): KAM, CHIH MIN
• Application Number:
  US11/941,766
• Publication Number:
  US 20080199898A1
• Time in Patent Office: 1,173 Days
• Field of Search: 514/13, 514/2, 530/300, 530/324, 530/326, 435/23, 435/24, 435/7.72
• US Class Current: 435/7.72
• 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 method of delivering a peptide into a cell, said method comprising:

providing a peptide attached to a pair of fluorophores of the same species that form an H dimer; and

contacting said cell with said peptide whereby said peptide enters said cell.

2. The method of claim 1, wherein said peptide further comprise a hydrophobic group, and wherein said hydrophobic group is selected from the group consisting of:

9-fluorenylmethoxycarbonyl (Fmoc), 9-fluoreneacetyl group (Fa), 1-fluorenecarboxylic group, 9-florenecarboxylic group,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).

3. The method of claim 1, wherein said fluorophores are selected from the group consisting of rhodamine X, 9-(2,5-dicarboxyphenyl)-3,6-bis(dimethylamino)xanthylium halide or other anion, 9-(2,6-dicarboxyphenyl)-3,6-bis(dimethylamino)xanthylium halide 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, 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, 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.

4. The method of claim 1, wherein, said fluorophores are selected from the group consisting of:

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

5. The method of claim 1, wherein, said cell is a mammalian cell.

6. The method of claim 1, wherein said peptide has the formula:

7. The method of claim 6, wherein P is selected from the group consisting of IETDSGV (SEQ ID NO:

208), SEVNLDAEF (SEQ ID NO;

209), and YVHDAPV (SEQ ID NO;

210).

8. The method of claim 6, wherein the carboxyl terminal amino acid in which the carboxylic acid group is replaced with an amide.

9. The method of claim 6, wherein:

r is zero; and

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

10. The method of claim 6, wherein F¹ and F² are the same fluorophore.

11. The method of claim 10, wherein F¹ and F² have an excitation wavelength between about 315 nm and about 800 nm.

12. The method of claim 6, wherein the F¹ 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.

13. The method of claim 6, wherein the F² 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.

14. The method of claim 6, wherein said fluorophores are selected from the group consisting of rhodamine X, 9-(2,5-dicarboxyphenyl)-3,6-bis(dimethylamino)xanthylium halide or other anion, 9-(2,6-dicarboxyphenyl)-3,6-bis (dimethylamino)xanthylium halide or other anion, 9-(2,5dicarboxyphenyl)-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, 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, 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.

15. The method of claim 6, wherein said fluorophore is a carbocyanine dye.

16. The method of claim 6, wherein said peptide further bears a hydrophobic group.

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

18. The method of claim 16, wherein said hydrophobic group is 9-fluoreneacetyl group (Fa).

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

20. The method of claim 1, wherein said fluorophores are selected from the group consisting of carboxytetramethylrhodamine, carboxyrhodamine-X, carboxyrhodamine 110, diethylaminocoumarin, and carbocyanine dyes.