Fluorogenic peptide substrate arrays for highly multiplexed, real-time monitoring of kinase activities

• US 20100248975A1
• Filed: 12/29/2006
• Published: 09/30/2010
• Est. Priority Date: 12/29/2006
• Status: Abandoned Application

• Current Assignee: Intel Corporation
• Original Assignee: Intel Corporation
• Inventors: Sundberg, Steven, Yu, Jiangtao, Tiwari, Gunjan
• Application Number:
  US11/647,579
• Publication Number:
  US 20100248975A1
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C07K 17/14 Peptides being immobilised on, or in, an inorganic carrier
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: G01N 2500/04 Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Abstract

The embodiments of the invention relate to a biomolecule microarray comprising a plurality of different polypeptides, where the polypeptides contain a kinase recognition sequence connected via a linker sequence to a metal binding amino acid that undergoes chelation enhanced fluorescence upon binding to a divalent cation. The embodiments further relate to kits comprising the microarray, and methods of using the microarray to screen for kinase activity, and to screen for inhibitors of kinase activity.

Claims

1. A positionally addressable array comprising 10 or more different polypeptides immobilized on a substrate, wherein the polypeptides comprise a kinase recognition sequence joined by a linker sequence to a metal binding amino acid comprising a compound having the formula (I)

2. The array of claim 1, wherein the metal binding amino acid comprises a compound having the formula (II)

3. The array of claim 1, wherein the metal binding amino acid comprises a compound of the formula (III)

4. The array of claim 1, wherein the linker sequence comprises a beta-turn sequence.

5. The array of claim 4, wherein the beta-turn sequence comprises the sequence Xaa-Pro or Pro-Xaa, where Xaa is any amino acid.

6. The array of claim 5, wherein Xaa is Gly.

7. The array of claim 1, wherein the polypeptide comprises the sequence (Xaa)_y-(Xaa)_p-(Xaa)_z-Xaa-Pro-Sox (SEQ ID NO:

1) or Sox-Pro-Xaa-(Xaa)_z-(Xaa)_p-(Xaa)_y (SEQ ID NO;

2),

wherein y=4-20 amino acids,

z=0-4 amino acids, and

Xaa is any amino acid except that (Xaa)_p is selected from the group consisting of serine, threonine and tyrosine.

8. The array of claim 7, wherein the polypeptide comprises the sequence (Xaa)_y-Tyr-(Xaa)_z-Xaa-Pro-Sox (SEQ ID NO:

3).

9. The array of claim 7, wherein the polypeptide comprises the sequence Sox-Pro-Xaa-(Xaa)_z-Ser-(Xaa)_y (SEQ ID NO:

4) or Sox-Pro-Xaa-(Xaa)_z-Thr-(Xaa)_y (SEQ ID NO;

5).

10. The array of claim 1, wherein the array comprises 100 or more different polypeptides.

11. The array of claim 1, wherein the polypeptides are present on the array at a density of between 5000 polypeptides/cm² and 50,000 polypeptides/cm².

12. The array of claim 1, further comprising a buffer capable of permitting the activity of a kinase, and comprising a divalent cation and a high energy donor source.

13. The array of claim 12, wherein the high energy donor source is ATP.

14. A method of using the array of claim 1, comprising contacting the array with a kinase capable of phosphorylating a kinase recognition sequence, wherein the phosphorylated polypeptide has increased affinity for a divalent cation than the unphosphorylated peptide, and wherein the divalent cation interacts with and increases the fluorescence of the metal binding amino acid.

15. A kit comprising the positionally addressable array of claim 1, and instructions means for its use.

16. A kit comprising the positionally addressable array of claim 1, and a kinase.

17. A kit comprising the positionally addressable array of claim 1, and a kinase inhibitor.

18. A method for identifying a target of a kinase, comprising

contacting the kinase with a positionally addressable array comprising at least 10 different polypeptides immobilized on a substrate, wherein the polypeptide comprises a kinase recognition site joined by a beta-turn sequence to a metal binding amino acid, and

identifying a polypeptide on the positionally addressable array that shows greater than an approximately 2 fold increase in fluorescence after being contacted with the kinase, wherein the increase in fluorescence indicates that the polypeptide is a target for the kinase.

19. The method of claim 18, wherein the beta-turn sequence comprises the sequence Xaa-Pro or Pro-Xaa, where Xaa is any amino acid.

20. The method of claim 18, wherein the polypeptide comprises the sequence (Xaa)_y-(Xaa)_p-(Xaa)_z-Xaa-Pro-Sox (SEQ ID NO:

1) or Sox-Pro-Xaa-(Xaa)_z-(Xaa)_p-(Xaa)_y (SEQ ID NO;

2),

wherein y=4-20 amino acids,

z=0-4 amino acids, and

Xaa is any amino acid except that (Xaa)_p is selected from the group consisting of serine, threonine and tyrosine.

21. The method of claim 18, wherein the metal binding amino acid comprises a compound of the formula (III)

22. The method of claim 18, wherein the array comprises 100 or more different polypeptides.

23. The method of claim 18, wherein the polypeptides are present on the array at a density of between 5000 proteins/cm² and 50,000 proteins/cm².

24. The method of claim 18, where the array is contacted with the kinase in the presence of a buffer capable of permitting the activity of a kinase, comprising a divalent cation and ATP.

25. A method for identifying a compound that modulates kinase activity, comprising

contacting a kinase with a positionally addressable array comprising at least 10 different polypeptides immobilized on a substrate, wherein the polypeptide comprises a kinase recognition site joined by a linker polypeptide to a metal binding amino acid, wherein said contact is in the presence of a test compound, a divalent cation, and ATP, and

determining whether fluorescence of the polypeptide is modulated in the presence of the test compound, wherein a change in fluorescence is an indication that the test compound modulates kinase activity.

26. The method of claim 25, wherein the beta-turn sequence comprises the sequence Xaa-Pro or Pro-Xaa, where Xaa is any amino acid.

27. The method of claim 25, wherein the polypeptide comprises the sequence (Xaa)_y-(Xaa)_p-(Xaa)_z-Xaa-Pro-Sox (SEQ ID NO:

1) or Sox-Pro-Xaa-(Xaa)_z-(Xaa)_p-(Xaa)_y (SEQ ID NO;

2),

wherein y=4-20 amino acids,

z=0-4 amino acids, and

Xaa is any amino acid except that (Xaa)_p is selected from the group consisting of serine, threonine and tyrosine.

28. The method of claim 25, wherein the metal binding amino acid comprises a compound of the formula (III)

29. The method of claim 25, wherein the array comprises 100 or more different polypeptides.

30. The method of claim 25, wherein the polypeptides are present on the array at a density of between 5000 proteins/cm² and 50,000 proteins/cm².

31. The method of claim 25, wherein the test compound inhibits kinase activity.