Fluorescence dyes and their applications for whole-cell fluorescence screening assays for caspases, peptidases, proteases and other enzymes and the use thereof

• US 6,984,718 B2
• Filed: 05/06/2002
• Issued: 01/10/2006
• Est. Priority Date: 07/21/1998
• Status: Expired due to Fees

• Current Assignee: Cytovia Incorporated (Immune Pharmaceuticals Inc.)
• Original Assignee: Cytovia Incorporated (Immune Pharmaceuticals Inc.)
• Inventors: Cai, Sui Xiong, Drewe, John A., Zhang, Han-Zhong, Yang, Wu
• Primary Examiner(s): Weber, Jon
• Assistant Examiner(s): KAM, CHIH MIN
• Application Number:
  US10/138,375
• Publication Number:
  US 20030208037A1
• Time in Patent Office: 1,345 Days
• Field of Search: 530/300, 530/402, 514/2, 436/546, 436/89, 435/18, 435/24, 435/7.4
• US Class Current: 530/300
• CPC Class Codes: C07D 311/82 Xanthenes
• CPC Class Codes: C07D 493/10 Spiro-condensed systems
• CPC Class Codes: C07K 5/06191 containing heteroatoms different from O, S, or N
• CPC Class Codes: C07K 5/0827 containing heteroatoms different from O, S, or N
• CPC Class Codes: C07K 5/1027 containing heteroatoms different from O, S, or N
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: Y10T 436/142222 Hetero-O [e.g., ascorbic acid, etc.]

Abstract

The present invention relates to novel fluorescent dyes, novel fluorogenic and fluorescent reporter molecules and new enzyme assay processes that can be used to detect the activity of caspases and other enzymes involved in apoptosis in whole cells, cell lines and tissue samples derived from any living organism or organ. The reporter molecules and assay processes can be used in drug screening procedures to identify compounds which act as inhibitors or inducers of the caspase cascade in whole cells or tissues. The reagents and assays described herein are also useful for determining the chemosensitivity of human cancer cells to treatment with chemotherapeutic drugs. The present invention also relates to novel fluorogenic and fluorescent reporter molecules and new enzyme assay processes that can be used to detect the activity of type 2 methionine aminopeptidase, HIV protease, adenovirus protease, HSV-1 protease, HCMV protease and HCV protease.

Claims

1. A compound having the following structural formula:

or a biologically acceptable salt or pro-reporter molecule thereof, wherein;

X is a peptide comprising aspartic acid and an N-terminal protecting group;

R₈ and R₉ are each independently selected from the group consisting of hydrogen, alkyl and aryl;

R₁₀ and R₁₁ are each independently selected from the group consisting of hydrogen and alkyl;

R₁-R₅ are each independently hydrogen, fluoro, chloro, bromo, iodo, haloalkyl, aryl, cycloalkyl, alkyl, alkenyl, alkynyl, aralkyl, aralkenyl, aryl alkynyl, hydroxyalkyl, nitro, amino, cyano, acylamino, acyl, hydroxy, acyloxy, alkoxy, alkylthio, and carboxy;

provided that at least one of R₁, R₃ and R₅ is selected from the group consisting of fluoro and chloro; and

provided that said N-terminal protecting group is not an acetyl group.

2. The compound of claim 1, wherein each of R¹-R⁵ are fluoro.

3. The compounds of claim 1, wherein each of R⁸-R¹¹ are hydrogen.

4. The compound of claim 1, wherein X is a substrate for a caspase enzyme.

5. The compound of claim 4, wherein said substrate is an N-blocked peptide selected from the group consisting of WEHD (SEQ ID NO:

1), YVAD (SEQ ID NO;

2), LEHD (SEQ ID NO;

3), DETD (SEQ ID NO;

4), DEVD (SEQ ID NO;

5), DEHD (SEQ ID NO;

6), VEHD (SEQ ID NO;

7), LETD (SEQ ID NO;

8), SHVD (SEQ ID NO;

10), DELD (SEQ ID NO;

11), DGPD (SEQ ID NO;

12), DEPD (SEQ ID NO;

13), DGTD (SEQ ID NO;

14), DLND (SEQ ID NO;

15), DEED (SEQ ID NO;

16), DSLD (SEQ ID NO;

17), DVPD (SEQ ID NO;

18), DEAD (SEQ ID NO;

19), DSYD (SEQ ID NO;

20), ELPD (SEQ ID NO;

21), VEID (SEQ ID NO;

26), IETD (SEQ ID NO;

24), IEPD SEQ ID NO;

23) and VEPD (SEQ ID NO;

27).

6. The compound of claim 1, wherein X is a substrate for granzyme B.

7. The compound of claim 1, wherein X is a N-blocked tetrapeptide selected from the group consisting of IEPD (SEQ ID NO:

23) and VEPD (SEQ ID NO;

27).

8. The compound of claim 1, wherein R₆ is a N-terminal protecting group selected from the consisting of t-butyloxycarbonyl, octanoyl, dodecanoyl, benzyloxycarbonyl and pentaflurobenzoyl.

9. A compound having the following structural formula:

or a biologically acceptable salt or pro-reporter molecule thereof, wherein

R₆ is a N-terminal protecting group;

each AA is independently an amino acid;

n is an integer from 0-5;

m is an interger from 0-3; and

R₁-R₅ are each independently hydrogen, fluoro, chloro, bromo, iodo, haloalkyl, aryl, cycloalkyl, alkyl, alkenyl, alkynyl, aralkyl, aralkenyl aryl alkynyl, hydroxyalkyl, nitro, amino, cyano, acylamino, acyl, hydroxy, acyloxy, alkoxy, alkylthio, and carboxy;

provided that at least one of R₁, R₃ and R₅ is selected from the group consisting of fluoro and chloro; and

provided that said N-terminal protecting group is not an acetyl group.

10. The compound of claim 9, wherein R₆ is a N-terminal protecting group selected from the group consisting of t-butyloxycarbonyl, acetyl, octanoyl, dodecanoyl, benzyloxycarbonyl and pentafluorobenzoyl.

11. The compound of claim 9, wherein n=3 and said (AA)n-Asp is a member selected from the group consisting of WEHD (SEQ ID NO:

1), YVAD (SEQ ID NO;

2), LEHD (SEQ ID NO;

3), DETD (SEQ ID NO;

4), DEVD (SEQ ID NO;

5), DEHD (SEQ ID NO;

6), VEHD (SEQ ID NO;

7), LETD (SEQ ID NO;

8), SHVD (SEQ ID NO;

10), DELD (SEQ ID NO;

11), DGPD (SEQ ID NO;

12), DEPD (SEQ ID NO;

13), DGTD (SEQ ID NO;

14), DLND (SEQ ID NO;

15), DEED (SEQ ID NO;

16), DSLD (SEQ ID NO;

17), DVPD (SEQ ID NO;

18), DEAD (SEQ ID NO;

19), DSYD (SEQ ID NO;

20), ELPD (SEQ ID NO;

21), VEID (SEQ ID NO;

26) and IETD (SEQ ID NO;

24).

12. The compound of claim 9, wherein R₆-(AA)n-Asp is an N-blocked peptide consisting of C-terminal Asp and at least one amino acid of a peptide chain selected from the group consisting of WEHD (SEQ ID NO:

1), YVAD (SEQ ID NO;

2), LEHD (SEQ ID NO;

3), DETD (SEQ ID NO;

4), DEVD (SEQ ID NO;

5), DEHD (SEQ ID NO;

6), VEHD (SEQ ID NO;

7), LETD (SEQ ID NO;

8), SHVD (SEQ ID NO;

10), DELD (SEQ ID NO;

11), DGPD (SEQ ID NO;

12), DEPD (SEQ ID NO;

13), DGTD (SEQ ID NO;

14), DLND (SEQ ID NO;

15), DEED (SEQ ID NO;

16), DSLD (SEQ ID NO;

17), DVPD (SEQ ID NO;

18), DEAD (SEQ ID NO;

19), DSYD (SEQ ID NO;

20), ELPD (SEQ ID NO;

21), VEID (SEQ ID NO;

26), IETD (SEQ ID NO;

24), IEPD (SEQ ID NO;

23) and VEPD (SEQ ID NO;

27).

13. The compound of claim 9, wherein n=3 and said (AA)_n-Asp is selected from the group consisting of IEPD (SEQ ID NO:

23) and VEPD (SEQ ID NO;

27).

14. The compound of claim 9, wherein (AA)_m is glycine.

15. The compound of claim 9, wherein at least one of R₁-R₅ is fluoro.

16. The compound of claim 9, wherein the compound is selected from the group consisting of:

N-(Z-YVAD)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

2),

N-(Z-DEVD)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

5),

N-(Z-VAD)-N′

-pentafluorobenzoyl-Rhodamine 110,

N-(Z-VD)-N′

-pentafluorobenzoyl-Rhodamine 110.

N-(PFB-DEVD)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

5),

N-(Octanoyl-DEVD)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

5),

N-(Z -DEVDG)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

76),

N-(FB-DEVDG)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

76),

N-(Z-YVAD)-N′

-(2,3,4,5-tetrafluorobenzoyl)-Rhodamine 110 (SEQ ID NO;

2),

N-(Z-DEVD)-N′

-(2,3,4,5-tetrafluorobenzoyl)-Rhodamine 110 (SEQ ID NO;

5),

N-(Z-DEVDG)-N′

-(2,3,4,5-tetrafluorobenzoyl)-Rhodamine 110 (SEQ ID NO;

76),

N-(Z-YVAD)-N′

-(3,4,5-trifluorobenzoyl)-Rhodamine 110 (SEQ ID NO;

2),

N-(Z-DEVD)-N′

-(3,4,5-trifluorobenzoyl)-Rhodamine 110 (SEQ ID NO;

5),

N-(Z-VAD)-N′

-(3,4,5-trifluorobenzoyl)-Rhodamine 110,

N-(Z-DEVD)-N′

-(3,4,5-trifluorobenzoyl)-Rhodamine 110 (SEQ ID NO;

76),

N-(Z-DEVD)-N′

-(2,4,6-trifluorobenzoyl)-Rhodamine 110 (SEQ ID NO;

5),

N-(Z-DEVDG)-N′

-(2,4,6-trifluorobenzoyl)-Rhodamine 110 (SEQ ID NO;

76),

N-(Z-YVAD(OAM))-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

2),

N-(Z-D(OAM)E(OAM)VD(OAM))-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

5),

N-(Z-E(OAM)VD(OAM))-N′

-pentafluorobenzoyl-Rhodamine 110,

N-(Z-D(OMe)E(OMe)VD(OAM))-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

5),

N-(Z-VD(OAM))-N′

-pentafluorobenzoyl-Rhodamine 110,

N-(Z-D(OAM)E(OAM)VD(OAM))-N′

-(2,3,4,5-tetrafluorobenzoyl)-Rhodamine 110 (SEQ ID NO;

5),

N-(Z-VD(OAM))-N′

-(2,3,4,5-tetrafluorobenzoyl)-Rhodamine 110,

N-(Z-D(OAM)E(OAM)VD(OAM))-N′

-(3,4,5-trifluorobenzoyl)-Rhodamine 110 (SEQ ID NO;

5), and

N-(Z-VD(OAM))-N′

-(3,4,5-trifluorobenzoyl)-Rhodamine 110.