Kinase and phosphatase assays

• US 7,582,461 B2
• Filed: 07/29/2004
• Issued: 09/01/2009
• Est. Priority Date: 07/29/2003
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Beebe, Jane A., Werner, Elizabeth A., Somberg, Richard, Kleman-Leyer, Karen M., Lasky, David A., Klink, Tony A.
• Primary Examiner(s): Tsang; Cecilia
• Assistant Examiner(s): BRADLEY, CHRISTINA
• Application Number:
  US10/903,529
• Publication Number:
  US 20050054573A1
• Time in Patent Office: 1,860 Days
• Field of Search: None
• US Class Current: 435/194
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C07K 7/08 having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: G01N 2333/81 Protease inhibitors
• CPC Class Codes: G01N 2333/9121 with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases

Abstract

Compositions, methods, and kits for detecting and monitoring kinase or phosphatase activity are described. The compositions typically include a peptide, a detectable moiety, and a protease cleavage site. Modification of a peptide by a kinase or phosphatase alters the proteolytic sensitivity of the peptide, resulting in a change of a detectable property of the composition. Panel assays for determining substrates or modulators of kinase or phosphatase activity are also described.

Claims

1. A composition of matter comprising:

a) a peptide having a length from five to fifty amino acids and comprising a recognition motif for a serine/threonine kinase selected from the group consisting of PRAFSVG (SEQ ID NO;

     56) and RRQFSLR (SEQ ID NO;

     57); and

b) a first detectable moiety,

wherein the first detectable moiety is attached to the peptide.

3. The composition of claim 1, wherein the first detectable moiety is covalently attached to the peptide.

4. The composition of claim 1, wherein the peptide comprises a protease cleavage site.

5. The composition of claim 4, wherein the protease cleavage site is selected from the group consisting of a chymotrypsin protease cleavage site, a caspase 3 protease cleavage site, a cathepsin G protease cleavage site, a trypsin protease cleavage site, an elastase protease cleavage site, an endoproteinase Asp-N protease cleavage site, and an endoproteinase Glu-N protease cleavage site.

6. The composition of claim 1, wherein the composition exhibits an optical property, a magnetic property, or a radioactive property.

7. The composition of claim 6, wherein the composition exhibits a fluorescence excitation maximum in the range from 250 to 750 nm and a fluorescence emission maximum in the range from 450 to 800 nm.

8. The composition of claim 7, wherein the first detectable moiety is selected from the group consisting of 5-carboxyfluorescein, 6-carboxyfluorescein, 7-hydroxycoumarin-3-carboxamide, 6-chloro-7-hydroxycoumarin-3-carboxamide-, fluorescein-5-isothiocyanate, dichlorotriazinylaminofluorescein, tetramethylrhodamine-5-isothiocyanate, tetramethylrhodamine-6-isothiocyanate, succinimidyl ester of 5-carboxyfluorescein, succinimidyl ester of 6-carboxyfluorescein, 5-carboxytetramethylrhodamine, 6-carboxymethylrhodamine, and 7-amino-4-methylcoumarin-3-acetic acid.

9. An article of manufacture comprising:

a) packaging material;

b) a composition of matter of claim 1 associated with the packaging material; and

c) a protein kinase or protein phosphatase.

20. The composition of claim 1, wherein the peptide has a length from 8 to 50 amino acids.

21. The composition of claim 1, wherein the peptide has a length from 8 to 25 amino acids.

22. The composition of claim 1, wherein the peptide has a length from 8 to 15 amino acids.

23. The composition of claim 3, wherein the first detectable moiety is attached to the peptide via a linker.

24. The composition of claim 23, wherein the linker is selected from the group consisting of GABA, an amino acid, a small peptide, diaminopentanyl, and aminohexanoyl.

25. The composition of claim 1, wherein the recognition motif for a serine/threonine kinase is PRAFSVG (SEQ ID NO:

56), and wherein the serine/threonine kinase is selected from the group consisting of Akt1, Akt 2, Akt3, CaMKII, CDK7/CycH, IKKβ

, MAPKAP-K1α

/RSK1, MAPKAP-K1β

/RSK2, MAPKAP-K1γ

/RSK3, MSK1, PAK2, PIM1, PKA, PKG, REDK, and SGK1.

26. The composition of claim 1, wherein the recognition motif for a serine/threonine kinase is RRQFSLR (SEQ ID NO:

57), and wherein the serine/threonine kinase is selected from the group consisting of Akt1, Akt2, Akt3, CaMKII, CHK1, CHK2, MAPKAP-K1α

, MAPKAP-K1β

, MAPKAP-K1γ

, MSK1, p70 S6 Kinase, PAK2, PIM1, PKA, PKC α

, PKC β

I, PKC β

I, PKC γ

, PKCδ

, PKCε

, PKCζ

, PKCη

, PKCθ

, PKCι

, PKQ ROCK, ROCK2, and SGK1.

27. The composition of claim 1, wherein the peptide is selected from the group consisting of ARPRAFSVGK (SEQ ID NO:

59) and RRRQFSLRRKAK (SEQ ID NO;

     60).

2. A composition of matter comprising:

a) a peptide having a length from five to fifty amino acids and comprising a recognition motif for a serine/threonine kinase selected from the group consisting of PRAFSVG (SEQ ID NO;

     56) and RRQFSLR (SEQ ID NO;

     57); and

b) a first detectable moiety; and

c) a second detectable moiety;

wherein the first detectable moiety is attached to the peptide.

10. The composition of claim 2, wherein the first detectable moiety is covalently attached to the peptide.

11. The composition of claim 2, wherein the peptide comprises a protease cleavage site.

12. The composition of claim 11, wherein the protease cleavage site is selected from the group consisting of a chymotrypsin protease cleavage site, a caspase 3 protease cleavage site, a cathepsin G protease cleavage site, a trypsin protease cleavage site, an elastase protease cleavage site, an endoproteinase Asp-N protease cleavage site, and an endoproteinase Glu-N protease cleavage site.

13. The composition of claim 2, wherein the composition exhibits an optical property, a magnetic property, or a radioactive property.

14. The composition of claim 13, wherein the composition exhibits a fluorescence excitation maximum in the range from 250 to 750 nm and a fluorescence emission maximum in the range from 450 to 800 nm.

15. The composition of claim 14, wherein the first detectable moiety is selected from the group consisting of 5-carboxyfluorescein, 6-carboxyfluorescein, 7-hydroxycoumarin-3-carboxamide, 6-chloro-7-hydroxycoumarin-3-carboxamide-, fluorescein-5-isothiocyanate, dichlorotriazinylaminofluorescein, tetramethylrhodamine-5-isothiocyanate, tetramethylrhodamine-6-isothiocyanate, succinimidyl ester of 5-carboxyfluorescein, succinimidyl ester of 6-carboxyfluorescein, 5-carboxytetramethylrhodamine, 6-carboxymethylrhodamine, and 7-amino-4-methylcoumarin-3-acetic acid.

16. The composition of claim 2, wherein the first detectable moiety and the second detectable moiety form a dark quenching resonance energy transfer pair.

17. The composition of claim 2, wherein the first detectable moiety and the second detectable moiety form a fluorescence resonance energy transfer pair.

18. The composition of claim 17, wherein the first detectable moiety is 7-hydroxycoumarin-3-carboxamide and the second detectable moiety is 5-carboxyfluorescein.

19. An article of manufacture comprising:

a) packaging material;

b) a composition of matter of claim 2 associated with the packaging material; and

c) a protein kinase or protein phosphatase.

32. The composition of claim 2, wherein the peptide has a length from 8 to 50 amino acids.

33. The composition of claim 2, wherein the peptide has a length from 8 to 25 amino acids.

34. The composition of claim 2, wherein the peptide has a length from 8 to 15 amino acids.

35. The composition of claim 10, wherein the first detectable moiety is attached to the peptide via a linker.

36. The composition of claim 35, wherein the linker is selected from the group consisting of GABA, an amino acid, a small peptide, diaminopentanyl, and aminohexanoyl.

37. The composition of claim 2, wherein the recognition motif for a serine/threonine kinase is PRAFSVG (SEQ ID NO:

56), and wherein the serine/threonine kinase is selected from the group consisting of Akt1, Akt2, Akt3, CaMKII, CDK7/CycH, IKKβ

, MAPKAP-K1α

/RSK1, MAPKAP-K1β

/RSK2, MAPKAP-K1γ

/RSK3, MSK1, PAK2, PIM1, PKA, PKG, REDK, and SGK1.

38. The composition of claim 2 wherein the recognition motif for a serine/threonine kinase is RRQFSLR (SEQ ID NO:

57), and wherein the serine/threonine kinase is selected from the group consisting of Akt1, Akt2, Akt3, CaMKII, CHK 1, CHK 2, MAPKAP-K1α

, MAPKAP-K1β

, MAPKAP-K1γ

, MSK1, p70 S6 Kinase, PAK2, PIM1, PKA, PKC α

, PKC β

I, PKC β

I, PKC γ

, PKCδ

, PKCε

, PKCζ

, PKCη

, PKCθ

, PKCι

, PKQ ROCK, ROCK 2, and SGK 1.

39. The composition of claim 2, wherein the peptide is selected from the group consisting of ARPRAFSVGK (SEQ ID NO:

59) and RRRQFSLRRKAK (SEQ ID NO;

     60).

28. A composition of matter comprising:

a) a peptide having a length from five to fifty amino acids and comprising a protein phosphatase recognition motif selected from the group consisting of PRAFS*VG (SEQ ID NO;

     106) and RRQFS*LR (SEQ ID NO;

     107), wherein S* represents a phosphorylated serine; and

b) a first detectable moiety,

wherein the first detectable moiety is attached to the peptide.

29. The composition of claim 28, wherein the protein phosphatase recognition motif is PRAFS*VG (SEQ ID NO:

106), and wherein the protein phosphatase is selected from the group consisting of PP1α

, PP2A, and PP2B.

30. The composition of claim 28, wherein the protein phosphatase recognition motif is RRQFS*LR (SEQ ID NO:

107), and wherein the protein phosphatase is selected from the group consisting of PP1α

, PP2A, and PP2B.

31. The composition of claim 28, wherein the peptide has a sequence selected from the group consisting of ARPRAFS*VGK (SEQ ID NO:

109) and RRRQFS*LRRKAK (SEQ ID NO;

     110), wherein S* represents a phosphorylated serine.

40. A composition of matter comprising:

a) a peptide having a length from five to fifty amino acids and comprising a protein phosphatase recognition motif selected from the group consisting of PRAFS*VG (SEQ ID NO;

     106) and RRQFS*LR

(SEQ ID NO;

     107), wherein S* represents a phosphorylated serine;

b) a first detectable moiety,

wherein the first detectable moiety is attached to the peptide; and

c) a second detectable moiety.

41. The composition of claim 40, wherein the protein phosphatase recognition motif is PRAFS*VG (SEQ ID NO:

106), and wherein the protein phosphatase is selected from the group consisting of PP1α

, PP2A, and PP2B.

42. The composition of claim 40, wherein the protein phosphatase recognition motif is RRQFS*LR (SEQ ID NO:

107), and wherein the protein phosphatase is

selected from the group consisting of PP1α

, PP2A, and PP2B.