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

• US 20030208037A1
• Filed: 05/06/2002
• Published: 11/06/2003
• Est. Priority Date: 07/21/1998
• Status: Active Grant

• Current Assignee: Cytovia Incorporated (Immune Pharmaceuticals Inc.)
• Original Assignee: Cytovia Incorporated (Immune Pharmaceuticals Inc.)
• Inventors: Yang, Wu, Cai, Sui Xiong, Drewe, John A., Zhang, Han-Zhong
• Granted Patent: US 6,984,718 B2
• Time in Patent Office: Days
• US Class Current: 530/330
• 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:

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

3. The compound of claim 2, wherein said substrate 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).

4. The compound of claim 1, wherein each of R₁-R₅ are fluoro, or four of the R₁-R₅ are fluoro.

5. The compound of claim 1, wherein Y is Rhodamine 110.

41. A method for detecting an enzyme involved in the apoptosis cascade in one or more cells, comprising:

(a) contacting said one or more cells with the reporter compound of claim 1 under conditions whereby said reporter compound is taken into said one or more cells, and

(b) recording the fluorescence of said one or more cells,

wherein a change in fluorescence within said one or more cells compared to control cells which have not been so contacted is an indication of the presence of the enzyme.

42. The method of claim 41, wherein said enzyme is an intracellular caspase.

43. A method for measuring the activity of an enzyme involved in the apoptosis cascade in one or more cells, comprising:

(a) contacting said one or more cells with the reporter compound of claim 1 under conditions whereby said reporter compound is taken into said one or more cells, and

(b) recording the fluorescence of said one or more cells,

wherein the relative change in fluorescence within said one or more cells compared to control cells which have not be so contacted is a measure of the activity of the enzyme.

44. The method of claim 43, wherein said enzyme is an intracellular caspase.

45. A method for determining whether a test substance has an effect on an enzyme involved in the apoptosis cascade in one or more test cells, comprising:

(a) contacting said one or more test cells with said test substance and the reporter compound of claim 1 under conditions whereby said reporter compound is taken into said one or more cells, and

(b) recording the fluorescence of said one or more test cells compared to control cells which have only been contacted with said reporter compound,

wherein a change in fluorescence within said one or more test cells compared to said control cells is an indication that said test substance has an effect on said enzyme.

46. The method of claim 45, wherein said enzyme is an intracellular caspase.

47. The method according to claim 45, wherein said one or more test cells is contacted with said test substance prior to contacting said test cells with said reporter compound.

48. The method according to claim 45, wherein said one or more test cells is contacted with said test substance after contacting with said reporter compound.

49. The method according to claim 45, wherein said one or more test cells is contacted substantially simultaneously with said test substance and said reporter compound.

50. The method according to claim 45, wherein said contacting step further includes contacting said one or more test cells with at least one second test substance in the presence of said first test substance.

51. The method according to claim 45, wherein said one or more test cells are derived from a single-cell organism.

52. The method accoding to claim 45, wherein said one or more test cells is derived from a multi-cellular organism.

53. The method according to claim 52, wherein said multi-cellular organism is selected from the group consists of a mammal, a primate, an invertebrate animal, an insect and a plant.

54. The method according to claim 45, wherein the one or more test and control cells are derived from the group consisting of the hair, brain, peripheral nervous system, eye, ear, nose, mouth, tonsils, teeth, esophagus, lung, breast, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver, stomach, intestinal tract, pancreas, endocrine glands and tissues, kidney, bladder, reproductive organs or glands, joint, bone and skin of said multicellular organism.

55. The method according to claim 45, wherein the one or more test cells are cancerous.

56. The method according to claim 55, wherein said one or more cancerous test cells are derived from the group consisting of the brain, peripheral nervous system, eye, ear, nose, mouth, tonsils, teeth, esophagus, lung, breast, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver, stomach, intestinal tract, pancreas, endocrine glands or tissues, kidney, bladder, reproductive organs or glands, joints, bones and skin of said multi-cellular organism.

57. The method according to claim 55, wherein said one or more cancerous test cells are derived from a human in need of treatment with a chemotherapeutic drug and said test substance is a chemotherapeutic agent.

58. The method according to claim 45, wherein the test substance is a chemotherapeutic agent.

59. The method according to claim 45, wherein the test substance is a mixture of chemotherapeutic agents.

60. A method to determine the sensitivity of an animal with cancer to treatment with one or more chemotherapeutic agents, comprising:

(a) contacting cancer cells taken from said animal with said one or more chemotherapeutic agents and the reporter compound of claim 1 under conditions whereby said one or more agents, either interacts with an external receptor or is taken into said cancer cells, and

(b) recording the fluorescence of said cancer cells compared to control cells which have only been contacted with said reporter,

wherein a change in fluorescence of said cancer cells compared to said control cells is an indication that said cancer cells are chemosensitive to said one or more chemotherapeutic agents and that said animal is sensitive to said treatment.

61. The method of claim 60, wherein said animal is a human.

62. A method for monitoring the treatment of an animal with one or more chemotherapeutic agents, comprising:

(a) administering said one or more chemotherapeutic agents to said animal,

(b) contacting cells taken from said animal after said administering with the reporter compound of claim 1 under conditions whereby said reporter compound is taken into said cells, and

(c) recording the fluorescence of said cells contacted with said reporter compound compared to contol cells which have been taken from said animal before said administering,

wherein a change in fluorescence of said cells taken from said animal compared to said control cells is an indication that said animal is sensitive to said chemotherapeutic agents.

63. The method according to claim 62, wherein said animal suffers from a malady related to apoptotic cell death.

64. A method for determining whether a test substance inhibits or prevents cell death in one or more test cells, comprising:

(a) contacting said one or more test cells with said test substance and the reporter compound of claim 1 under conditions whereby said reporter compound is taken into said one or more cells, and

(b) recording the fluorescence of said one or more test cells compared to control cells which have only been contacted with said reporter compound,

wherein a change in fluorescence within said one or more test cells compared to said control cells is an indication that said test substance influences cell death.

65. The method of claim 64, wherein said one or more test cells are nerve cells.

66. The method of claim 64, wherein said one or more test cells are selected from the group consisting of myocardial cells, immune cells, cells of an organ to be transplanted, spermatozoa, egg, cell lines which produce a recombinant protein, hair cells, skin cells and nerve cells.

67. A method for determining whether a test substance causes or enhances cell death in one or more test cells, comprising:

(a) contacting said one or more test cells with said test substance and the reporter compound of claim 1 under conditions whereby said reporter compound is taken into said one or more cells, and

(b) recording the fluorescence of said one or more test cells compared to control cells which have only been contacted with said reporter compound,

wherein an increase of fluorescence within said one or more test cells compared to said control cells is an indication that said test substance causes or enhances cell death.

68. The method of claim 67, wherein said one or more test cells are selected from the group consisting of cancer cells, yeast, fungi and bacteria.

6. A compound having the following structural formula:

7. The compound of claim 6, wherein each of R₁-R₅ are fluoro.

8. The compounds of claim 6, wherein each of R₈-R₁₁ are hydrogen.

9. The compound of claim 6, wherein X is a substrate for a caspase enzyme.

10. The compound of claim 9, wherein said substrate is an N-blocked peptide consisting of C-terminal Asp and at least one amino acids 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).

11. The compound of claim 9, wherein X is a substrate for granzyme B.

12. The compound of claim 6, wherein X is a N-blocked tetrapeptide selected from the group consisting of IEPD (SEQ ID NO:

23) and VEPD (SEQ ID NO;

     27).

13. A compound having the following structural formula:

14. The compound of claim 13, wherein R₆ is a N-terminal protecting group selected from the group consisting of t-butyloxycarbonyl, acetyl, octanoyl, dodecanoyl, benzyloxycarbonyl and pentafluorobenzoyl.

15. The compound of claim 13, 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).

16. The compound of claim 13, 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).

17. The compound of claim 13, 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).

18. The compound of claim 13, wherein m=2 and AA is selected from the group consisting of G and A.

19. The compound of claim 13, wherein at least one of R₁-R₅ is fluoro.

20. The compound of claim 13, 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-(Ac-LETD)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     8),

N-(Ac-DEVD)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     5),

N-(Ac-IETD)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     24),

N-(Ac-LQTD)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     72),

N-(Ac-EETD)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     73),

N-(Ac-LEVD)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     9),

N-(Ac-AEHD)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     74),

N-(Ac-WEHD)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     71),

N-(Ac-YVAD)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     2),

N-(Ac-LEHD)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     3),

N-(Ac-D(OEt)E(OEt)VD(OEt))-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-(Ac-DEVD)-N′

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

     5),

N-(Ac-IETD)-N′

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

     24),

N-(Ac-LQTD)-N′

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

     72),

N-(Ac-EETD)-N′

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

     73),

N-(Ac-LEVD)-N′

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

     9),

N-(Ac-AEHD)-N′

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

     74),

N-(Ac-WEHD)-N′

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

     1),

N-(Ac-YVAD)-N′

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

     2),

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.

21. A method for the preparation of the compound of claim 13, comprising:

(a) reacting Rhodamine with a substituted benzoyl chloride to give N-substituted benzoyl-Rhodamine;

(b) condensing the N-substituted benzoyl-Rhodamine together with N-Z-Gly to give N-Z-Gly-N′

-substituted benzoyl-Rhodamine;

(c) removing the Z group to give N-Gly-N′

-substituted benzoyl-Rhodamine;

(d) condensing N-Gly-N′

-substituted benzoyl-Rhodamine with Z-(AA)_n-Asp(OBu-t) to give N-(Z-(AA)_n-Asp(OBu-t)-Gly)-N′

-substituted benzoyl-Rhodamine; and

(e) removing the OBu-t protecting group to give N-(Z-(AA)_n-Asp-Gly)-N′

-substituted benzoyl-Rhodamine.

22. A method for the preparation of the compound of claim 13, comprising:

(a) reacting Rhodamine with a substituted benzoyl chloride to give N-substituted benzoyl-Rhodamine;

(b) condensing N-substituted benzoyl-Rhodamine with Z-(AA)_n-Asp(OBu-t) to give N-(Z-(AA).-Asp(OBu-t))-N′

-substituted benzoyl-Rhodamine; and

(c) removing the OBu-t protecting group to give N-(Z-(AA)_n-Asp)-N′

-substituted benzoyl-Rhodamine.

23. The method of claim 21 or 22, wherein n=3 and -(AA)_n is selected from the group consisting of WEH, YVA, LEH, DET, DEV, DEH, VEH, LET, SHV, DEL, DGP, DEP, DGT, DLN, DEE, DSL, DVP, DEA, DSY, ELP, VED, IEP and IET.

24. A compound having the following structural formula:

25. The compound of claim 24, wherein at least one of R₁-R₅ are fluoro.

26. The compound of claim 24, wherein R₇ is H;

m=l;

(AA)_m is M;

n is an integer from 1-2; and

(AA)_n is selected from the group consisting of GG, GA, AG, G, and A.

27. The compound of claim 24, wherein the compound is selected from the group consisting of:

N-(MG)-N′

-pentafluorobenzoyl-Rhodamine 110,

N-(MA)-N′

-pentafluorobenzoyl-Rhodamine 110,

N-M-N′

-pentafluorobenzoyl-Rhodamine 110,

N-(MGG)-N′

-pentafluorobenzoyl-Rhodamine 110,

N-(MAG)-N′

-pentafluorobenzoyl-Rhodamine 110,

N-(MGA)-N′

-pentafluorobenzoyl-Rhodamine 110,

N-(MG)-N′

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

N-(MA)-N′

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

N-(MG)-N′

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

N-(MG)-N′

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

N-(MA)-N′

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

N-M-N′

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

N-(MG)-N′

-(2,4,6-trifluorobenzoyl)-Rhodamine 110, and

N-M-N′

-(2,4,6-trifluorobenzoyl)-Rhodamine 110,

28. The compound of claim 24, wherein R₇ is a N-terminal protecting group selected from the group consisting of t-butyloxycarbonyl, acetyl, octanoyl, dodecanoyl, benzyloxycarbonyl and pentafluorobenzoyl.

29. The compound of claim 28, wherein (AA)_n-(AA)_m is selected from the group consisting of SQNY-PIV (SEQ ID NO:

28), ARVL-AEA (SEQ ID NO;

     29), ATIM-MQR (SEQ ID NO;

     30), RQAN-FLG (SEQ ID NO;

     31), PGNF-LQS (SEQ ID NO;

     32), SFSF-PQI (SEQ ID NO;

     33), TLNF-PIS (SEQ ID NO;

     34), AETF-YVD (SEQ ID NO;

     35) or RKVL-FLD (SEQ ID NO;

     36);

SQNY-PI (SEQ ID NO;

     117), ARVL-AE (SEQ ID NO;

     118), ATIM-MQ (SEQ ID NO;

     119), RQAN-FL (SEQ ID NO;

     120), PGNF-LQ (SEQ ID NO;

     121), SFSF-PQ (SEQ ID NO;

     122), TLNF-PI (SEQ ID NO;

     123), AETF-YV (SEQ ID NO;

     124) or RKVL-FL (SEQ ID NO;

     125);

SQNY-P (SEQ ID NO;

     126), ARVL-A (SEQ ID NO;

     127), ATIM-M (SEQ ID NO;

     128), RQAN-F (SEQ ID NO;

     129), PGNF-L (SEQ ID NO;

     130), SFSF-P (SEQ ID NO;

     131), TLNF-P (SEQ ID NO;

     132), AETF-Y (SEQ ID NO;

     133) and RKVL-F (SEQ ID NO;

     134).

30. The compound of claim 24, wherein the compound is selected from the group consisting of:

N-(Ac-SLNFPIV)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     80),

N-(Ac-SLNFPI)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     81),

N-(Ac-SLNFP)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     82),

N-(Ac-LNFPIV)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     83),

N-(Ac-LNFPI)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     84),

N-(Ac-LNFP)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     85),

N-(Ac-RGFP)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     37),

N-(Z-LNFPIV)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     83),

N-(Z-LNFPI)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     84),

N-(Z-LNFP)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     85),

N-(Z-RGFP)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     37),

N-(Z-RQANFPLG)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     31),

N-(Z-RQANFL)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     86),

N-(Z-RQANF)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     87),

N-(Z-RKVLFLD)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     36),

N-(Z-RKVLFL)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     88),

N-(Z-RKVLF)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     89),

N-(Z-ARVLFLG)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     90),

N-(Z-ARVLFL)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     91),

N-(Z-ARVLF)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     92),

N-(Z-SQNYFLG)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     93),

N-(Z-SQNYFL)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     94), and

N-(Z-SQNYF)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     95).

31. The compound of claim 28, wherein (AA)n-(AA)m is selected from the group consisting of LRGGG (SEQ ID NO:

101), LRGGA (SEQ ID NO;

     102), MRGGG (SEQ ID NO;

     96), MRGGA (SEQ ID NO;

     135), IRGGG (SEQ ID NO;

     97), IRGGA (SEQ ID NO;

     136), LVGGG (SEQ ID NO;

     98), LVGGA (SEQ ID NO;

     137), MVGGG (SEQ ID NO;

     99), MVGGA (SEQ ID NO;

     138), IVGGG (SEQ ID NO;

     100), IVGGA (SEQ ID NO;

     139), LRGG (SEQ ID NO;

     55), MRGG (SEQ ID NO;

     56), IRGG (SEQ ID NO;

     57), LVGG (SEQ ID NO;

     58), MVGG (SEQ ID NO;

     59) and IVGG (SEQ ID NO;

     60).

32. The compound of claim 24, wherein the compound is selected from the group consisting of:

N-(Ac-LRGGG)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     101),

N-(Ac-MRGGG)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     96),

N-(Ac-IRGGG)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     97),

N-(Ac-LVGGG)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     98),

N-(Ac-MVGGG)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     99),

N-(Ac-IVGGG)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     100),

N-(Ac-LRGGA)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     102),

N-(Ac-LRGG)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     35),

N-(Z-LRGGG)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     101),

N-(Z-LRGGA)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     102), and

N-(Z-LRGG)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     55).

33. The compound of claim 24, wherein the compound is selected from the group consisting of:

N-(Ac-LVLASSS)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     103),

N-(Ac-LVLASS)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     104),

N-(Ac-LVLAS)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     105),

N-(Ac-LVLA)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     62),

N-(Z-LVLASSS)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     103),

N-(Z-LVLASS)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     104),

N-(Z-LVLAS)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     105), and

N-(Z-LVLA)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     62).

34. The compound of claim 24, wherein the compound is selected from the group consisting of:

N-(AZ-VVNASS)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

106 ),

N-(Ac-VVNAS)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     107),

N-(Ac-VVNA)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     64),

N-(Ac-Tbg-Tbg-NASS)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     108),

N-(Ac-Tbg-Tbg-NAS)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     109),

N-(Ac-Tbg-Tbg-NA)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     110),

N-(Z-Tbg-Tbg-NASS)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     108),

N-(Z-Tbg-Tbg-NAS)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     109), and

N-(Z-Tbg-Tg-NA)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     110).

35. The compound of claim 24, wherein the compound is selected from the group consisting of:

N-(Ac-DDIVPCSMST)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     111),

N-(Ac-DIVPCSMST)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     112),

N-(Ac-IVPCSMST)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     113),

N-(Ac-IVPCSMS)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     114),

N-(Ac-IVPCSM)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     115),

N-(Ac-IVPCS)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     116),

N-(Ac-IVPC)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     69),

N-(Z-IVPCSM)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     113),

N-(Z-IVPCSMS)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     114),

N-(Z-IVPCS)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     115), and

N-(Z-IVPCS)-N′

-pentafluorobenzoyl-Rhodamine 110 (SEQ ID NO;

     116).

69. A method for detecting HIV protease in one or more cells, comprising:

(a) contacting said cells with the reporter compound of claim 24 or 30 under conditions whereby the reporter compound is taken into the cells, and

(b) recording the fluorescence of said cells,

wherein a change in fluorescence within the cells compared to control cells which have not been so contacted is an indication of the presence of the HIV protease.

70. A method for measuring the activity of HIV protease in one or more HIV infected cells, comprising:

(a) contacting said one or more HIV infected cells with the reporter compound of claim 24 under conditions whereby the reporter compound is taken into said one or more HIV infected cells, and

(b) recording the fluorescence of said one or more cells,

wherein a change in fluorescence within said one or more HIV infected cells compared to control cells which have not been so contacted is a measure of the activity of the HIV protease.

71. A method for determining whether a test substance has an effect on the activity of HIV protease in one or more HIV infected cells, comprising:

(a) contacting said infected test cells with said test substance and the reporter compound of claim 24 under conditions whereby said reporter compound is taken into said infected test cells, and

(b) recording the fluorescence of said infected test cells compared to infected control cells which have only been contacted with said reporter compound,

wherein a change in fluorescence within said infected test cells compared to said infected control cells is an indication that said test substance has an effect on the HIV protease.

72. A method for measuring the activity of type 2 methionine aminopeptidase in endothelial cells, comprising:

(a) contacting endothelial test cells with the reporter compound of claim 24 under conditions whereby the reporter compound is taken into said endothelial test cells, and

(b) recording the fluorescence of said cells,

wherein a change in fluorescence within said endothelial test cell compared to endothelial control cells which have not been so contacted is a measure of the activity of the type 2 methionine aminopeptidase.

73. A method for determining whether a test substance has an effect on the activity of type 2 methionine aminopeptidase in endothelial test cells, comprising:

(a) contacting said test cells with said test substance and the reporter compound of claim 24 under conditions whereby said reporter compound is taken into said test cells, and

(b) recording the fluorescence of said test cells compared to control cells which have only been contacted with said reporter,

wherein a change in fluorescence within the test cells compared to said control cells is an indication that said test substance has an effect on the type 2 methionine aminopeptidase.

36. A compound having the following structural formula:

37. The compound of claim 36, wherein at least three of R₁-R₅ is fluoro.

38. The compound of claim 36, wherein R₁-R₅ are all fluoro.

39. The compound of claim 36, wherein R₈-R₁₁ are hydrogen.

40. The compound of claim 36, wherein the compound is selected from the group consisting of:

N-pentafluorobenzoyl-Rhodamine 110,

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

N-(2,4,6-trifluorobenzoyl)-Rhodamine 110,

N-(4-fluoro-3-trifluoromethylbenzoyl)-Rhodamine 110,

N-(2,3,5,6-tetrafluorobenzoyl)-Rhodamine 110,

N-(2,3,4-trifluorobenzoyl)-Rhodamine 110,

N-(2,4,5-trifluorobenzoyl)-Rhodamine 110,

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

N-pentafluorobenzoyl-Rhodamine 116,

N-pentafluorobenzoyl-Rhodamine 19,

N-(2,3,4,5-tetrafluorobenzoyl)-Rhodamine 116,

N-(2,3,4,5-tetrafluorobenzoyl)-Rhodamine 19,

N-(2,4,6-trifluorobenzoyl)-Rhodamine 116, and

N-(2,4,6-trifluorobenzoyl)-Rhodamine 19.