Fluorogenic kinase assays and substrates

1. A substrate compound comprising two or more hydrophobic moieties capable of integrating the compound into a micelle, one or more non-proteinaceous fluorescent moiety(ies), and an enzyme recognition moiety, wherein one of said two or more hydrophobic moieties is located N-terminal to said enzyme recognition moiety and another of said two or more hydrophobic moieties is located C-terminal to said enzyme recognition moiety.

2. The substrate compound of claim 1 which has a net neutral charge in aqueous solution at a pH of about pH 7.6.

3. The substrate compound of claim 1 in which the enzyme recognition moiety comprises a protein kinase recognition sequence including at least one unphosphorylated residue capable of being phosphorylated by a protein kinase.

4. The substrate compound of claim 3 in which the at least one unphosphorylated residue is tyrosine, serine or threonine.

5. The substrate compound of claim 3 in which the protein kinase recognition sequence is recognized by a TK kinase, an AGC kinase, a CAMK kinase, a CMGC kinase, an STE kinase, a TKL kinase, a CKI kinase or a kinase belonging to the group “other.”

6. The substrate compound of claim 3 in which the protein kinase recognition sequence is recognized by a protein kinase A, a protein kinase C, a Src kinase, a Lyn kinase, a Fyn kinase, an Akt kinase, a MAP kinase, a MAPKAP2 kinase, cAMP dependent kinase, a PKB kinase, or a p38βII kinase.

7. The substrate compound of claim 3 in which the protein kinase recognition sequence comprises a peptide sequence selected from the group consisting of:

-R-R-X-S/T-Z-;

(SEQ ID NO:1)

-R-X-X-S/T-F-F-;

(SEQ ID NO:2)

-S/T-P-X-R/K-;

(SEQ ID NO:3)

-P-X-S/T-P-;

(SEQ ID NO:4)

-K-K-K-K-R-F-S-F-K-;

(SEQ ID NO:5)

-X-R-X-X-S-X-R-X-;

(SEQ ID NO:6)

-L-R-R-L-S-D-S-N-F-;

(SEQ ID NO:7)

-K-K-L-N-R-T-L-T-V-A-;

(SEQ ID NO:8)

-E-E-I-Y-E/G-X-F-;

(SEQ ID NO:9)

-E-I-Y-E-X-I/V-;

(SEQ ID NO:10)

-I-Y-M-F-F-F-;

(SEQ ID NO:11)

-Y-M-M-M-;

(SEQ ID NO:12)

-E-E-E-Y-F-;

(SEQ ID NO:13)

-L-R-R-A-S-L-G-;

(SEQ ID NO:14)

-R-Q-G-S-F-R-A-;

(SEQ ID NO:15)

-R-I-G-E-G-T-Y-G-V-V-R-R-;

(SEQ ID NO:16)

-R-P-R-T-S-S-F-;

(SEQ ID NO:17)

-P-R-T-P-G-G-R-;

(SEQ ID NO:18)

-R-L-N-R-T-L-S-V-;

(SEQ ID NO:19)

-R-R-I-P-L-S-P-;

(SEQ ID NO:20)

and

analogs and conservative mutants thereof, wherein X represents any residue and Z represents a hydrophobic residue.

8. The substrate compound of claim 3 which has a net neutral charge in aqueous solution at a pH of about pH 7.6.

9. The substrate compound of claim 3 which has the structure:

wherein:

each q is, independently of the other, 0 or 1;

each p is, independently of the other, an integer ranging from 1 to 6;

R 1 is a substituted or unsubstituted, saturated or unsaturated, linear or branched alkyl containing from 6 to 30 carbon atoms;

R 2 is a substituted or unsubstituted, saturated or unsaturated, linear or branched alkyl containing from 6 to 30 carbon atoms;

L 1 represents a linker;

X n comprises the protein kinase recognition sequence;

L 2 represents a linker; and

Dye represents a fluorescent dye.

10. The substrate compound of claim 9 in which both q's are 1 and L 1 and L 2 are each —[(CH 2 CH 2 O) n —CH 2 ] m —, where each n is, independently of the other, an integer ranging from 1 to 6 and each m is, independently of the other, an integer ranging from 1 to 6.

11. The substrate compound of claim 10 in which each n is 2 and each m is, independently of the other, selected from 2, 3 and 4.

12. The substrate compound of claim 9 in which Dye comprises a fluorescein or a rhodamine dye.

13. The substrate compound of claim 9 in which X n is a peptide selected from the group consisting of:

-R-R-X-S/T-Z-

(SEQ ID NO:1);

-R-X-X-S/T-F-F-

(SEQ ID NO:2);

-S/T-P-X-R/K-

(SEQ ID NO:3);

-P-X-S/T-P-

(SEQ ID NO:4);

-K-K-K-K-R-F-S-F-K-

(SEQ ID NO:5);

-X-R-X-X-S-X-R-X-

(SEQ ID NO:6);

-L-R-R-L-S-D-S-N-F-

(SEQ ID NO:7);

-K-K-L-N-R-T-L-T-V-A-

(SEQ ID NO:8);

-E-E-I-Y-E/G-X-F-

(SEQ ID NO:9);

-E-I-Y-E-X-I/V-

(SEQ ID NO:10);

-I-Y-M-F-F-F-

(SEQ ID NO:11);

-Y-M-M-M-

(SEQ ID NO:12);

-E-E-E-Y-F-

(SEQ ID NO:13);

-L-R-R-A-S-L-G-

(SEQ ID NO:14);

-R-Q-G-S-F-R-A-

(SEQ ID NO:15);

-R-I-G-E-G-T-Y-G-V-V-R-R-

(SEQ ID NO:16);

-R-P-R-T-S-S-F-

(SEQ ID NO:17);

-P-R-T-P-G-G-R-

(SEQ ID NO:18);

-R-L-N-R-T-L-S-V-

(SEQ ID NO:19); and

-R-R-I-P-L-S-P-

(SEQ ID NO:20).

14. The substrate compound of claim 3 in which each hydrophobic moiety comprises a substituted or unsubstituted, saturated or unsaturated hydrocarbon containing from 6 to 30 carbon atoms.

15. The substrate compound of claim 14 in which the hydrocarbon is a linear, branched or cyclic, saturated or unsaturated alkyl.

16. The substrate compound of claim 3 in which the one or more non-proteinaceous fluorescent moiety(ies) comprises a dye selected from a xanthene dye, a rhodamine dye, a fluorescein dye, a cyanine dye, a phthalocyanine dye, a squaraine dye and a bodipy dye.

17. The substrate compound of claim 3 in which the one or more non-proteinaceous fluorescent moiety(ies) comprises a fluorescence donor moiety and a fluorescence acceptor moiety.

18. The substrate compound of claim 17 in which the fluorescence donor moiety comprises a fluorescein dye.

19. The substrate compound of claim 17 in which the fluorescence acceptor moiety comprises a fluorescein or a rhodamine dye.

20. The substrate compound of claim 19 in which the fluorescence donor moiety comprises a fluorescein dye.

21. The substrate compound of claim 3 in which the one or more non-proteinaceous fluorescent moiety(ies) comprises fewer than 150 atoms.

22. The substrate compound of claim 3 in which one hydrophobic moiety is linked to the enzyme recognition moiety through the one or more non-proteinaceous fluorescent moiety(ies), optionally via a linker, and another hydrophobic moiety is linked to the enzyme recognition moiety through one or more optional linkers.

23. The substrate compound of claim 3 in which two of the two or more hydrophobic moieties are linked to one another through the one or more non-proteinaceous fluorescent moiety(ies).

24. The substrate compound of claim 3 in which one of the two or more hydrophobic moieties, the one or more non-proteinaceous fluorescent moiety(ies) and the enzyme recognition moiety are linked to each other via a trivalent linker.

25. The substrate compound of claim 3 in which one of the two or more hydrophobic moieties is linked to the one or more non-proteinaceous fluorescent moiety(ies) by a linker that does not include a part of the enzyme recognition moiety.

26. The substrate compound of claim 3 in which at least one of the two or more hydrophobic moieties is linked to the one or more non-proteinaceous fluorescent moiety(ies) by a linker that includes at least a part of the enzyme recognition moiety.

27. The substrate compound of claim 1 in which the enzyme recognition moiety comprises a phosphatase recognition sequence including at least one phosphorylated residue capable of being dephosphorylated by a phosphatase.

28. The substrate compound of claim 27 which has a net neutral charge in aqueous solution at a pH of about pH 7.6.

29. A method of detecting phosphorylation activity of one or more protein kinases in a sample, comprising the steps of:

contacting the sample with a composition comprising a protein kinase substrate which comprises (1) a protein kinase recognition sequence according to claim 3 containing at least one unphosphorylated residue capable of being phosphorylated by a protein kinase, (2) two or more hydrophobic moieties capable of integrating the substrate into a micelle, and (3) one or more non-proteinaceous fluorescent moiety(ies), under conditions effective to allow phosphorylation of said residue when the protein kinase is present in the sample, thereby increasing a fluorescence signal produced by the one or more non-proteinaceous fluorescent moiety(ies); and

detecting a fluorescence signal, where an increase in the fluorescence signal indicates the presence and/or quantity of protein kinase phosphorylation activity in the sample.

30. A method of identifying a compound that modulates phosphorylation activity of a protein kinase, comprising the steps of:

contacting the protein kinase with a composition comprising a protein kinase substrate which comprises (1) a protein kinase recognition sequence according to claim 3 containing at least one unphosphorylated residue capable of being phosphorylated by a protein kinase, (2) two or more hydrophobic moieties capable of integrating the substrate into a micelle, and (3) one or more non-proteinaceous fluorescent moiety(ies), in the presence of a candidate compound and under conditions effective to allow phosphorylation of said residue by the protein kinase, thereby increasing a fluorescence signal produced by the one or more non-proteinaceous fluorescent moiety(ies); and

detecting a fluorescence signal, where a change in the fluorescence signal as compared to a control reaction or a standard curve indicates that the candidate compound modulates the activity of the protein kinase.

31. A substrate compound comprising two or more hydrophobic moieties capable of integrating the compound into a micelle, one or more fluorescent moiety(ies), and an enzyme recognition moiety, wherein one of said two or more hydrophobic moieties is located N-terminal to said enzyme recognition moiety and another of said two or more hydrophobic moieties is located C-terminal to said enzyme recognition moiety, and wherein said substrate compound has a molecular weight of less than 5000 daltons.