Fluorogenic or fluorescent reporter molecules and their applications for whole-cell fluorescence screening assays for caspases and other enzymes and the use thereof

• US 7,270,801 B2
• Filed: 04/22/2004
• Issued: 09/18/2007
• Est. Priority Date: 10/10/1997
• Status: Expired due to Fees

• Current Assignee: Cytovia Incorporated (Immune Pharmaceuticals Inc.)
• Original Assignee: Cytovia Incorporated (Immune Pharmaceuticals Inc.)
• Inventors: Weber, Eckard, Cai, Sui Xiong, Keana, John F. W., Zhang, Han-Zhong, Drewe, John A.
• Primary Examiner(s): GITOMER, RALPH J
• Application Number:
  US10/829,381
• Publication Number:
  US 20040191844A1
• Time in Patent Office: 1,244 Days
• Field of Search: 424/9.2, 424/9.6
• US Class Current: 424/9.2
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: G01N 15/1456 without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1488 Methods for deciding
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

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, dipeptidyl peptidase IV, calpain, aminopeptidase, HIV protease, adenovirus protease, HSV-1 protease, HCMV protease and HCV protease.

Claims

1. 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 a reporter compound having the Formula II:

R₁-(AA)_n-Asp-y-Asp-(AA)_n-R₁ 

 

(II)

or the Formula V;

R₁-(AA)_n-Asp-y-R₆ 

 

(V)

or a biologically acceptable salt of said compound or a tautomer of said compound or a biologically acceptable salt of said tautomer, wherein;

R₁ is an N-terminal protecting group;

each AA independently is a residue of an α

-amino acid or β

-amino acid, or a methyl, acetoxymethyl or methoxy ester of a carboxyl-containing α

-amino acid or β

-amino acid;

each n independently is 0-5;

y is a fluorogenic or fluorescent moiety comprising rhodamine; and

R₆ is a blocking group which is not an amino acid or a derivative of an amino acid;

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 compound, 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.

2. The method of claim 1, wherein R₆ is a blocking group which is selected from the group consisting of C_1-12alkyloxycarbonyl, CH₃(OCH₂CH₂)_qOCO—

, CH₃(CH₂)_r(OCH₂CH₂)_sOCO—

, C_1-12(alkylthio)carbonyl, aryl(C_1-12)alkoxycarbonyl, H₂NCO—

, (CH₃)₂NCO—

, (CH₃CH₂)₂NCO—

, (CH₃(CH₂)_v)(CH₃)NCO—

, C_1-12alkylsulfonyl, C_1-12 haloalkylsulfonyl, aryl(C_1-12)alkylsulfonyl, Cl₃CCH₂OCO—

, unsubstituted benzoyl, benzylcarbonyl, phenylsulfonyl and tosyl;

wherein q is 2-4, r is 0-5, s is 1-4, and v is 1-9.

3. The method of claim 1, wherein (AA)_n-Asp is selected from the group consisting of HD, AD, TD, VD, LD, PD, ND, ED, YD, ID, EHD, VAD, ETD, EVD, HVD, ELD, GPD, EPD, GTD, LND, EED, SLD, VPD, EAD, SYD, LPD, EID, 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, LEVD SEQ ID NO;

9, 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 method of claim 1, wherein said compound is of formula III:

5. The method of claim 4, wherein said compound is selected from the group consisting of:

(Z-YVAD)₂-Rhodamine 110, SEQ ID NO;

2;

(Z-DEVD)₂-Rhodamine 110, SEQ ID NO;

5;

(Z-VAD)₂-Rhodamine 110;

(Z-YVAD(OAM))₂-Rhodamine 110, SEQ ID NO;

2;

(Z-LE(OAM)HD(OAM))₂-Rhodamine 110, SEQ ID NO;

3;

(Z-D(OAM)E(OAM)TD(OAM))₂-Rhodamine 110, SEQ ID NO;

4;

(Z-D(OAM)E(OAM)VD(OAM))₂-Rhodamine 110, SEQ ID NO;

5;

(Z-D(OMe)E(OMe)VD(OAM))₂-Rhodamine 110, SEQ ID NO;

5; and

(Z-D(OMe)E(OMe)VD)₂-Rhodamine 110 SEQ IS NO;

5;

wherein Z is benzyloxycarbonyl.

6. The method of claim 1, wherein said compound is of formula VII:

7. The method of claim 6, wherein R₁ is t-butyloxycarbonyl, acetyl, hexanoyl, octanoyl or benzyloxycarbonyl;

and R₂, R₃, R₄ and R₅ are hydrogen.

8. The method of claim 6, wherein said compound is selected from the group consisting of:

N-(Z-YVAD)-N′

-acetyl-Rhodamine 110, SEQ ID NO;

2;

N-(Z-DEVD)-N′

-acetyl-Rhodamine 110, SEQ ID NO;

5;

N-(Z-VD)-N′

-acetyl-Rhodamine 110;

N-(Z-AD)-N′

-acetyl-Rhodamine 110;

N-(Z-VAD)-N′

-acetyl-Rhodamine 110;

N-(Z-DEVD)-N′

-ethoxycarbonyl-Rhodamine 110, SEQ ID NO;

5;

N-(Ac-DEVD)-N′

-ethoxycarbonyl-Rhodamine 110, SEQ ID NO;

5;

N-(Ac-DEVD)-N′

-hexyloxycarbonyl-Rhodamine 110, SEQ ID NO;

5;

N-(Ac-DEVD)-N′

-octyloxycarbonyl-Rhodamine 110, SEQ ID NO;

5;

N-(Ac-DEVD)-N′

-decyloxycarbonyl-Rhodamine 110, SEQ ID NO;

5;

N-(Ac-DEVD)-N′

-dodecyloxycarbonyl-Rhodamine 110, SEQ ID NO;

5; and

N-(Ac-DEVD)-N′

-(ethylthio)carbonyl-Rhodamine 110, SEQ ID NO;

5;

wherein Z is benzyloxycarbonyl.

9. The method of claim 1, wherein said animal is a human.

10. A method for monitoring the treatment of an animal to treatment 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 a reporter compound having the Formula II:

R₁-(AA)_n-Asp-y-Asp-(AA)_n-R₁ 

 

(II)

or the Formula V;

R₁-(AA)_n-Asp-y-R₆ 

 

(V)

or a biologically acceptable salt of said compound or a tautomer of said compound or a biologically acceptable salt of said tautomer, wherein;

R₁ is an N-terminal protecting group;

each AA independently is a residue of an α

-amino acid or β

-amino acid, or a methyl, acetoxymethyl or methoxy ester of a carboxyl-containing α

-amino acid or β

-amino acid;

each n independently is 0-5;

y is a fluorogenic or fluorescent moiety comprising rhodamine; and

R₆ is a blocking group which is not an amino acid or a derivative of an amino acid;

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 control 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.

11. The method of claim 10, wherein R₆ is a blocking group which is selected from the group consisting of C_1-12alkyloxycarbonyl, CH₃(OCH₂CH₂)_qOCO—

, CH₃(CH₂)_r(OCH₂CH₂)_sOCO—

, C_1-12(alkylthio)carbonyl, aryl(C_1-12)alkoxycarbonyl, H₂NCO—

, (CH₃)₂NCO—

, (CH₃CH₂)₂NCO—

, (CH₃(CH₂)_v)(CH₃)NCO—

, C_1-12alkylsulfonyl, C_1-12 haloalkylsulfonyl, aryl(C_1-12)alkylsulfonyl, Cl₃CCH₂OCO—

, unsubstituted benzoyl, benzylcarbonyl, phenylsulfonyl and tosyl;

wherein q is 2-4, r is 0-5, s is 1-4, and v is 1-9.

12. The method of claim 10, wherein (AA)_n-Asp is selected from the group consisting of HD, AD, TD, VD, LD, PD, ND, ED, YD, ID, EHD, VAD, ETD, EVD, HVD, ELD, GPD, EPD, GTD, LND, EED, SLD, VPD, EAD, SYD, LPD, EID, 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, LEVD SEQ ID NO;

9, 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 method of claim 10, wherein said compound is of formula III:

14. The method of claim 13, wherein said compound is selected from the group consisting of:

(Z-YVAD)₂-Rhodamine 110, SEQ ID NO;

2;

(Z-DEVD)₂-Rhodamine 110, SEQ ID NO;

5;

(Z-VAD)₂-Rhodamine 110;

(Z-YVAD(OAM))₂-Rhodamine 110, SEQ ID NO;

2;

(Z-LE(OAM)HD(OAM))₂-Rhodamine 110, SEQ ID NO;

3;

(Z-D(OAM)E(OAM)TD(OAM))₂-Rhodamine 110, SEQ ID NO;

4;

(Z-D(OAM)E(OAM)VD(OAM))₂-Rhodamine 110, SEQ ID NO;

5;

(Z-D(OMe)E(OMe)VD(OAM))₂-Rhodamine 110, SEQ ID NO;

5; and

(Z-D(OMe)E(OMe)VD)₂-Rhodamine 110 SEQ IS NO;

5;

wherein Z is benzyloxycarbonyl.

15. The method of claim 10, wherein said compound is of formula VII:

16. The compound of claim 15, wherein R₁ is t-butyloxycarbonyl, acetyl, hexanoyl, octanoyl or benzyloxycarbonyl;

and R₂, R₃, R₄ and R₅ are hydrogen.

17. The method of claim 15, wherein said compound is selected from the group consisting of:

N-(Z-YVAD)-N′

-acetyl-Rhodamine 110, SEQ ID NO;

2;

N-(Z-DEVD)-N′

-acetyl-Rhodamine 110, SEQ ID NO;

5;

N-(Z-VD)-N′

-acetyl-Rhodamine 110;

N-(Z-AD)-N′

-acetyl-Rhodamine 110;

N-(Z-VAD)-N′

-acetyl-Rhodamine 110;

N-(Z-DEVD)-N′

-ethoxycarbonyl-Rhodamine 110, SEQ ID NO;

5;

N-(Ac-DEVD)-N′

-ethoxycarbonyl-Rhodamine 110, SEQ ID NO;

5;

N-(Ac-DEVD)-N′

-hexyloxycarbonyl-Rhodamine 110, SEQ ID NO;

5;

N-(Ac-DEVD)-N′

-octyloxycarbonyl-Rhodamine 110, SEQ ID NO;

5;

N-(Ac-DEVD)-N′

-decyloxycarbonyl-Rhodamine 110, SEQ ID NO;

5;

N-(Ac-DEVD)-N′

-dodecyloxycarbonyl-Rhodamine 110, SEQ ID NO;

5; and

N-(Ac-DEVD)-N′

-(ethylthio)carbonyl-Rhodamine 110, SEQ ID NO;

5;

wherein Z is benzyloxycarbonyl.

18. The method according to claim 10, wherein said animal suffers from a malady related to apoptotic cell death.