Fluorescence polarization assay

• US 20030082665A1
• Filed: 03/28/2002
• Published: 05/01/2003
• Est. Priority Date: 04/09/2001
• Status: Active Grant

• Current Assignee: Boehringer Ingelheim Pharmaceuticals Incorporated (C.H. Boehringer Sohn AG & Co. KG)
• Original Assignee: Boehringer Ingelheim Pharmaceuticals Incorporated (C.H. Boehringer Sohn AG & Co. KG)
• Inventors: Cardozo, Mario G., Kroe, Rachel Rebecca, Grygon, Christine Anne, Proudfoot, John Robert, Ingraham, Richard Harold
• Granted Patent: US 6,794,158 B2
• Time in Patent Office: Days
• US Class Current: 435/18
• CPC Class Codes: G01N 2333/914 Hydrolases (3)
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive

Abstract

This invention is directed to a fluorescence polarization assay useful in the detection and evaluation of soluble epoxide hydrolase (sEH) inhibitors. This invention also relates to novel fluorescent probes used in the fluorescence polarization assay, and methods of manufacturing such fluorescent probes.

This fluorescent probe of the invention is a compound having the following formula (I):

X-spacer-R₁-Y  (I)

wherein X is the radical of compound that binds to the active site of soluble epoxide hydrolase, Y is a fluorescent label, and “spacer” and R₁ are as defined herein.

Claims

1. A fluorescent probe that binds to the active site of soluble epoxide hydrolase, said probe having the formula (I):

X-spacer-R₁-Y 

 

(I)

wherein X is the radical of compound that binds to the active site of soluble epoxide hydrolase, Y is a fluorescent label, “

spacer”

is a direct bond or is a C₁-C₁₆ alkylene group, a C₂-C₁₆ alkenylene group or a C₂-C₁₆ alkynylene group, wherein any of the available —

CH₂—

groups present in the C₁-C₁₆ alkylene group, C₂-C₁₆ alkenylene group or C₂-C₁₆ alkynylene group can optionally be replaced with O, S(O)_p wherein p is 0 to 2, or N(R₂), R₁ is selected from the group consisting of O, S, —

N(R₂)C(O)—

, —

C(O)N(R₂)—

, —

N(R₂)C(S)—

, —

C(S)N(R₂)—

, —

N(R₂)C(S)NH—

, —

NHC(S)N(R₂), —

N(R₂)C(O)NH—

, —

NHC(O)N(R₂), —

SO₂NR₂—

, —

NR₂SO₂—

, —

CH₂N(R₂)—

, —

N(R₂)CH₂—

, —

CH₂S—

, —

SCH₂—

, —

C(O)CH₂S—

, —

SC(O)CH₂—

,

, —

NHCH₂CH₂S—

, —

SCH₂CH₂NH—

, —

NC(O)O—

, —

ONC(O)—

, —

C(O)O—

, —

OC(O)—

, —

NH—

N═

C(R₂)—

, —

C(R₂)═

N—

NH—

, —

NHCH(R₂)—

, —

CH(R₂)NH—

, and R₂ is selected from H or C_1-3alkyl.

2. A fluorescent probe according to claim 1, wherein the probe binds to the active site of mammalian soluble epoxide hydrolase.

3. A fluorescent probe according to claim 2, wherein the probe binds to the active site of human soluble epoxide hydrolase or murine soluble epoxide hydrolase.

4. A fluorescent probe according to claim 1, having the following formula (II):

5. A fluorescent probe according to claim 4, having the following formula (III):

6. A fluorescent probe according to claim 5, wherein:

F is selected from N(Rs) and O;

G is a direct bond or is C_1-3alkylene;

Z is O;

“

spacer”

is a C_1-6alkylene group;

R₁ is —

N(R₂)C(O)—

, —

C(O)N(R₂)—

, —

N(R₂)C(S)—

or —

C(S)N(R₂)—

, where R₂ is H or C_1-3alkyl;

R₃ is C_1-6alkyl, C_2-6 alkenyl, C_2-6 alkynyl, C_5-7 cycloalkyl, or phenyl, each being optionally substituted with one or more C_1-3alkyl, halogen, CN, C_1-3alkoxy or C_1-3alkylthio;

R₄ and R₅ are both hydrogen; and

R₆ is selected from C_1-6alkylene, C_2-6 alkenylene, C_2-6 alkynylene, C_5-7cycloalkylene, phenyl or a C_6-12 bridged carbocyclic ring system, optionally having one to three double bonds in the ring system, each being optionally substituted with one or more C_1-3alkyl, halogen, CN, C_1-3alkoxy or C_1-3alkylthio.

7. A fluorescent probe according to claim 6, said probe having the following formula:

8. A fluorescent probe according to claim 1, wherein Y is a fluorescent label that fluoresces at a wavelength above about 450 nm.

9. A fluorescent probe according to claim 8, wherein the fluorescent label is selected from the group consisting of rhodamine and rhodamine derivatives, fluorescein and fluorescein derivatives, 4,4-difluor-4-bora-3a,4a-diaza-s-indacene and its derivatives, and cyanine dyes.

10. A fluorescent probe according to claim 9, wherein the fluorescent label is selected from the group consisting of rhodamine, tetramethyl rhodamine, carboxytetramethylrhodamine, carboxy-X-rhodamine, fluorescein, fluorinated fluoresceins, fluoresceinamine, carboxyfluorescein, alpha-iodoacetamidofluorescein, 4′

-aminomethylfluorescein, 4′

-N-alkylaminomethylfluorescein, 5-aminomethylfluorescein, 6-aminomethylfluorescein, 2,4-dichloro-1,3,5-triazin-2-yl-aminofluorescein, 4-chloro-6-methoxy-1,3,5-triazln-2-yl-aminofluorescein, fluoresceinisothiocyanate, 4,4-difluor-4-bora-3a,4a-diaza-s-indacene and its derivatives and cyanine dyes.

11. A method for preparing the fluorescent probe of formula (II) according to claim 4 wherein R₁ is —

N(R₂)C(O)—

, D is —

N(R₄)—

, E is —

C(═

Z)— and

F is N(R₅), said method comprising steps (a) and (b1) or steps (a) and (b2);

(a) reacting a compound of formula (IV) with a compound of formula (V) under coupling conditions to produce a compound of formula (VI);

(b1) reacting a compound of formula VI produced in step (a) with an isocyanate R₃N═

C═

O or an isothiocyanate R₃N═

C═

S, in the presence of a base in an inert solvent, to produce a compound of formula Ia;

or

(b2) reacting a compound of formula (VI) produced in step (a) with a carbamoyl chloride R₃R₄N(C═

O)Cl where R₄ is C_1-6alkyl or a thiocarbamoyl chloride R₃R₄N(C═

S)Cl where R₄ is C_1-6alkyl, in the presence of a base in an inert solvent, to produce a compound of formula IIb where R₄ is C_1-6alkyl;

wherein, except where otherwise noted above, Y, G, spacer, Z, R₂, R₃, R₄, R₅ and R₆ are as defined in claim 4.

12. A fluorescence polarization assay for determining whether a compound inhibits soluble epoxide hydrolase, said assay comprising:

(a) determining the fluorescence polarization values of a fluorescent probe according to claim 1 and said fluorescent probe bound to soluble epoxide hydrolase to obtain a range of fluorescence polarization values and selecting a reference fluorescence polarization value falling within that range;

(b) mixing the fluorescent probe with soluble epoxide hydrolase in a buffered aqueous solution;

(c) mixing a test compound with the mixture obtained in step (b) and incubating the resulting mixture of fluorescent probe, soluble epoxide hydrolase and test compound;

(d) measuring the fluorescence polarization value of the incubated mixture obtained in step (c) to obtain a test fluorescence polarization value; and

(e) determining the difference between the test fluorescence polarization value and the reference fluorescence polarization value;

wherein the difference in fluorescence polarization values obtained in step (e) indicates whether the test compound inhibits soluble epoxide hydrolase.

13. An assay according to claim 12, wherein the range of fluorescence polarization values in step (a) is obtained by periodically adding increasing amounts of soluble epoxide hydrolase to an amount of fluorescent probe in a buffered aqueous solution and periodically measuring the fluorescence polarization value of this mixture.

14. An assay according to claim 12, wherein the reference fluorescence polarization value is selected such that the difference between the reference fluorescence polarization value and the polarization value of free fluorescent probe is equal to about 50% to 100% of the difference between the polarization value of fluorescent probe bound to soluble epoxide hydrolase and the polarization value of free fluorescent probe.

15. The assay according to claim 12, wherein the difference in fluorescence polarization values obtained in step (e) is a positive value.

16. The assay according to claim 12, wherein the difference in fluorescence polarization values obtained in step (e) is a negative value.

17. The assay according to claim 12, wherein the soluble epoxide hydrolase is mammalian soluble epoxide hydrolase.

18. The assay according to claim 17, wherein the soluble epoxide hydrolase is human or murine soluble epoxide hydrolase.

19. The assay according to claim 12, wherein the fluorescent probe has the following formula (II):

20. The assay according to claim 19, wherein the fluorescent probe has the following formula (III):

21. The assay according to claim 20, wherein the fluorescent probe has the following formula:

22. An assay for screening a library of test compounds to determine whether the compounds inhibit soluble epoxide hydrolase, said assay comprising:

(a) determining the fluorescence polarization values of a fluorescent probe according to claim 1 and said fluorescent probe bound to soluble epoxide hydrolase to obtain a range of fluorescence polarization values and selecting a reference fluorescence polarization value falling within that range;

(b) mixing the fluorescent probe with soluble epoxide hydrolase in a buffered aqueous solution;

(c) adding test compounds to a plurality of containers;

(d) adding the mixture obtained in step (b) to said plurality of containers, and incubating the resulting mixtures of fluorescent probe, soluble epoxide hydrolase and test compounds;

(e) measuring the fluorescence polarization values of the incubated mixtures obtained in step (d) to obtain test fluorescence polarization values; and

(f) determining the differences between the test fluorescence polarization values and the reference fluorescence polarization value;

wherein the differences in fluorescence polarization values obtained in step (f) indicate whether the test compounds inhibit soluble epoxide hydrolase.

23. An assay according to claim 22, wherein the range of fluorescence polarization values in step (a) is obtained by periodically adding increasing amounts of soluble epoxide hydrolase to an amount of fluorescent probe in buffered aqueous solution and periodically measuring the fluorescence polarization value of this mixture.

24. An assay according to claim 22, wherein the reference fluorescence polarization value is selected such that the difference between the reference fluorescence polarization value and the polarization value of free fluorescent probe is equal to about 50% to 100% of the difference between the polarization value of fluorescent probe bound to soluble epoxide hydrolase and the polarization value of free fluorescent probe.

25. The assay according to claim 22, wherein at least some of the differences in fluorescence polarization values obtained in step (f) are positive values.

26. The assay according to claim 22, wherein at least some of the differences in fluorescence polarization values obtained in step (f) are negative values.

27. The assay according to claim 22, wherein the soluble epoxide hydrolase is mammalian soluble epoxide hydrolase.

28. The assay according to claim 27, wherein the soluble epoxide hydrolase is human or murine soluble epoxide hydrolase.

29. The assay according to claim 22, wherein the fluorescent probe has the following formula (II):

30. The assay according to claim 29, wherein the fluorescent probe has the following formula (III):

31. The assay according to claim 30, wherein the fluorescent probe has the following formula: