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

• US 6,936,687 B1
• Filed: 09/10/1999
• Issued: 08/30/2005
• Est. Priority Date: 02/20/1997
• Status: Expired due to Fees

• Current Assignee: OncoImmunin, Inc.
• Original Assignee: ONCO Immunity As
• Inventors: Packard, Beverly S., Komoriya, Akira
• Primary Examiner(s): Weber, Jon
• Assistant Examiner(s): KAM, CHIH MIN
• Application Number:
  US09/394,019
• Time in Patent Office: 2,181 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 2333/96469 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 each end of which is conjugated to a fluorophore. 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 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:

wherein, P has the sequence Leu-Glu-His-Asp-Gly-Ile-Asn (SEQ ID NO;

212);

F¹ and F² are fluorophores and F¹ is attached to the amino terminal amino acid and F² is attached to the carboxyl terminal amino acid;

S¹ and S², when present, are peptide spacers ranging in length from 1 to about 50 amino acids and S¹, when present, is attached to the amino terminal amino acid and S², when present, is attached to the carboxyl terminal amino acid;

i, j, k, l, m, n, o, p, q, and r are independently 0 or 1;

aa¹ and aa¹⁰ are independently selected from the group consisting of lysine, ornithine and cysteine;

aa² and aa³ are independently selected from the group consisting of Asp, Glu, Lys, Ornithine, Arg, Citulline, homocitrulline, Ser, Pro, homoserine, Aib, Thr, and Tyr;

aa⁸ and aa⁹ are independently selected from the group consisting of Asp, Glu, Lys, Ornithine, Arg, Citulline, homocitrulline, Ser, homoserine, Thr, and Tyr;

aa⁵, aa⁴, aa⁶, and aa⁷ are independently selected from the group consisting of proline, 3,4-dehydroproline, hydroxyproline, alpha aminoisobutyric acid and N-methyl alanine;

X is selected from the group consisting of Gly, β

Ala, γ

Abu, Gly-Gly, Ahx, 8-aminocaprylic acid, β

Ala-Gly, β

Ala-β

Ala, γ

Abu-Gly, β

Ala-γ

Abu, Gly-Gly-Gly, γ

Abu-γ

Abu, Ahx-Gly, β

Ala-Gly-Gly, Ahx-β

Ala, β

Ala-β

Ala-Gly, Gly-Gly-Gly-Gly (SEQ ID NO;

223), Ahx-γ

Abu, β

Ala-β

Ala-β

Ala, γ

Abu-β

Ala-Gly, γ

Abu-γ

Abu-Gly, Ahx-Ahx, γ

Abu-γ

Abu-β

Ala, and Ahx-Ahx-Gly,

Y is selected from the group consisting of Gly, β

Ala, γ

Abu, Gly-Gly, Ahx, 8-aminocaprylic acid, Gly-β

Ala, β

Ala-β

Ala, Gly-γ

Abu, γ

Abu-β

Ala, Gly-Gly-Gly, γ

Abu-γ

Abu, Gly-Ahx, Gly-Gly-β

Ala, β

Ala-Ahx, Gly-β

Ala-β

Ala, Gly-Gly-Gly-Gly (SEQ ID NO;

223), γ

Abu-Ahx, β

Ala-β

Ala-β

Ala, Gly-β

Ala-γ

Abu, Gly-γ

Abu-γ

Abu, Ahx-Ahx, β

Ala-γ

Abu-γ

Abu, and Gly-Ahx-Ahx;

when i is 1, S¹ is joined to aa¹ by a peptide bond through a terminal alpha amino group of aa¹; and

when r is 1, S² is joined to aa¹⁰ by a peptide bond through a terminal alpha carboxyl group.

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.

4. The composition of claim 1, having the amino acid sequence Lys-Asp-Pro-Ahx-Gly-Leu-Glu-His-Asp-Gly-Ile-Asn-Gly-Ahx-Pro-Lys-Gly-Tyr (SEQ ID NO:

248).

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

6. The composition of claim 5, wherein said F¹ and F² have an excitation wavelength in the range of 315 nm to 750 nm.

7. 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 aa amino acid.

8. 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.

9. 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)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 (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, 9-(2-carboxyphenyl)-xanthylium, and a carbocyanine dye.

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

11. The composition of claim 4, wherein said composition bears a hydrophobic group.

12. The composition of claim 11, wherein said hydrophobic group is selected from the group consisting of:

Fmoc, 9-fluoreneacetyl group (Fa), 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-dimethyl-2,6-dioxocyclohexylidene)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).

13. The composition of claim 12, wherein said hydrophobic group is Fmoc.

14. The composition of claim 12, wherein said hydrophobic group is Fa.

15. The composition of claim 12, wherein said hydrophobic group is attached to the amino terminus of the molecule.