METHOD FOR DETECTION AND QUANTIFICATION OF PLK1 EXPRESSION AND ACTIVITY

• US 20110076693A1
• Filed: 05/15/2009
• Published: 03/31/2011
• Est. Priority Date: 05/16/2008
• Status: Abandoned Application

• Current Assignee: United States Department Of Health And Human Services
• Original Assignee: The United States of America As Represented By The Secretary of Agriculture
• Inventors: Lee, Kyung S., Park, Jung Eun
• Application Number:
  US12/992,887
• Publication Number:
  US 20110076693A1
• Time in Patent Office: Days
• US Class Current: 435/7.4
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 17/00 Carrier-bound or immobilised peptides carrier-bound or immobilised enzymes C12N11/00; Preparation thereof
• CPC Class Codes: C12N 9/1229 Phosphotransferases with a phosphate group as acceptor (2.7.4)
• 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)
• CPC Class Codes: G01N 2800/56 Staging of a disease; Further complications associated with the disease
• CPC Class Codes: G01N 33/57484 involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites

Abstract

Isolated peptide substrates of Plk1 and nucleic acids encoding these peptides are disclosed. The peptides include two to ten repeats of the amino acid sequence set forth as X₁X₂AX₃X₄X₅PLHSTX₆X₇X₈X₉X₁₀X₁₁X₁₂ (SEQ ID NO: 1), in which within each repeat X₁, X₂, X₆, X₇, X₈, X₉, X₁₀, X₁₁, and X₁₂ are each independently any amino acid or no amino acid, and X₃ and X₄ are each independently any amino acid. Methods of using these peptides to detect Plk1 activity in a sample are also disclosed. In some examples, the method includes contacting a sample with a disclosed peptide substrate of Plk1 in the presence of adenosine triphosphate, or an analog thereof, for a period of time sufficient for Plk1 to phosphorylate the PBIPtide. The presence and/or amount of the phosphorylated and/or the unphosphorylated peptide is detected, thereby detecting and/or quantitating Plk1 kinase activity in the sample.

Claims

1. An isolated peptide comprising two to ten consecutive repeats of the amino acid sequence set forth as X₁X₂AX₃X₄X₅PLHSTX₆X₇X₈X₉X₁₀X₁₁X₁₂ (SEQ ID NO:

1), in which within each repeat X₁ is independently any amino acid or no amino acid, X₂ is independently any amino acid or no amino acid, X₃ is independently any amino acid, X₄ is independently any amino acid, X₅ is independently any amino acid, X₆ is independently any amino acid, X₇ is independently any amino acid, X₈ is independently any amino acid or no amino acid, X₉ is independently any amino acid or no amino acid, X₁₀ is independently any amino acid or no amino acid, X₁₁ is independently any amino acid or no amino acid, and X₁₂ is independently any amino acid or no amino acid, and wherein the two to ten consecutive repeats of SEQ ID NO;

1 are joined a peptide linker between two and ten amino acids in length, wherein the peptide linker separates consecutive repeats of SEQ ID NO;

1.

2. The isolated peptide of claim 1, wherein X₁ is independently any amino acid or no amino acid, X₂ is independently any amino acid or no amino acid, X₃ is phenylalanine, X₄ is aspartic acid, X₅ is proline, X₆ is independently any amino acid, X₇ is independently any amino acid, X₈ is independently any amino acid or no amino acid, X₉ is independently any amino acid or no amino acid, X₁₀ is independently any amino acid or no amino acid, X₁₁ is independently any amino acid or no amino acid, and X₁₂ is independently any amino acid or no amino acid.

3. The isolated peptide of claim 1, wherein X₁ is independently any amino acid or no amino acid, X₂ is independently any amino acid or no amino acid, X₃ is phenylalanine, X₄ is aspartic acid, X₅ is proline, X₆ is alanine, X₇ isoluecine, X₈ is independently any amino acid or no amino acid, X₉ is independently any amino acid or no amino acid, X₁₀ is independently any amino acid or no amino acid, X₁₁ is independently any amino acid or no amino acid, and X₁₂ is independently any amino acid or no amino acid.

4. The isolated peptide of claim 1, wherein X₁ is tyrosine, X₂ is glutamic acid, X₃ is phenylalanine, X₄ is aspartic acid, X₅ is proline, X₆ is alanine, X₇ is isoluecine, X₈ is tyrosine, X₉ is alanine, X₁₀ is aspartic acid, X₁₁ is glutamic acid, and X₁₂ is glutamic acid.

5. The isolated peptide of claim 1, wherein X₁ is phenylalanine, X₂ is glutamic acid, X₃ is phenylalanine, X₄ is aspartic acid, X₅ is proline, X₆ is alanine, X₇ is isoluecine, X₈ is phenylalanine, X₉ is alanine, X₁₀ is aspartic acid, X₁₁ is glutamic acid, and X₁₂ is glutamic acid.

9. The isolated peptide of claim 1, wherein the peptide linker comprises the amino acid sequence set forth as GGPGG (SEQ ID NO:

12).

11. The isolated peptide of claim 1, wherein the peptide is detectably labeled.

12. An isolated nucleic acid comprising a nucleotide sequence encoding the peptide of claim 1.

13. A method for detecting Plk1 kinase activity in a biological sample, comprising:

contacting a biological sample with the peptide of claim 1 in the presence of adenosine triphosphate, or an analog thereof, for a period of time sufficient for Plk1 to phosphorylate the peptide; and

detecting phosphorylation of the peptide, wherein the peptide is phosphorylated on one or more threonine residues,

thereby detecting the kinase activity of Plk1 in a biological sample.

14. The method of claim 13, further comprising contacting the biological sample with a specific binding agent that specifically binds to the peptide when the peptide is phosphorylated on one or more threonine residues, wherein the specific binding agent does not specifically bind to the peptide when the peptide is not phosphorylated on one or more threonine residues, and wherein detecting a complex formed between the specific binding agent and the peptide detects the phosphorylated peptide.

15. The method of claim 13, further comprising comparing the amount of the phosphorylated peptide detected with a control.

16. The method of claim 15, wherein the control is a value indicative of the amount of phosphorylated peptide formed from basal phosphorylation of the peptide, or a value indicative of the amount of phosphorylated peptide formed in the presence of a known amount of isolated Plk1.

17. The method of claim 14, wherein the specific binding agent is isolated Plk1 or an antibody that specifically binds to peptide when the peptide is phosphorylated on a threonine residue.

19. The method of claim 14, wherein the specific binding agent is detectably labeled.

20. The method of claim 13, wherein the peptide is detectably labeled.

21. The method of claim 14, further comprising contacting the biological sample with an antibody that specifically binds to the specific binding agent.

22. The method of claim 21, wherein the antibody is detectably labeled.

23. The method of claim 13, wherein the peptide is immobilized on a solid support.

25. A method for detecting a cancer or determining a predisposition for developing a cancer a subject, the method comprising:

obtaining a biological sample from the subject;

contacting the sample with the peptide according to claim 1 in the presence of adenosine triphosphate, or an analog thereof, for a period of time sufficient for Plk1 to phosphorylate the peptide;

detecting an amount of phosphorylated peptide;

comparing the amount of phosphorylated peptide formed with a control, wherein an increase in the amount of phosphorylated peptide formed relative to the control indicates that the subject has cancer or a predisposition for developing cancer.

26. The method of claim 25, wherein the control is a value indicative of the amount of complex formed from basal phosphorylation of the peptide, or a value indicative of the amount of complex formed in the presence of a known amount of isolated Plk1 or the amount of complex formed in a sample not contacted with the test agent.

27. A method for monitoring a subjects response to treatment for cancer, the method comprising:

obtaining a first biological sample at a first time point and a second biological sample at second later time point from a subject being treated for cancer;

contacting the first biological sample with the peptide according to claim 1 in the presence of adenosine triphosphate, or an analog thereof, for a period of time sufficient for Plk1 to phosphorylate the peptide

detecting a first amount of phosphorylated peptide formed from contacting the peptide with the first biological sample;

contacting the second biological sample with the peptide according to claim 1 in the presence of adenosine triphosphate, or an analog thereof, for a period of time sufficient for Plk1 to phosphorylate the peptide;

detecting a second amount of phosphorylated peptide formed from contacting the peptide with the second biological sample; and

comparing the first amount of phosphorylated peptide formed with the second amount of phosphorylated peptide formed, wherein an increase in the second amount of phosphorylated peptide formed relative to the first amount of phosphorylated peptide formed indicates that the subject is not responding to the treatment for cancer and wherein a decrease in the second amount of phosphorylated peptide formed relative to the first amount of phosphorylated peptide formed indicates that the subject is responding to the treatment for cancer.

28. The method of claim 25, further comprising contacting the biological sample(s) with a specific binding agent that specifically binds to the peptide when the peptide is phosphorylated on one or more threonine residues or a specific binding agent that specifically binds to the peptide when the peptide is not phosphorylated on a threonine residue;

and

detecting a complex formed between the peptide and the specific binding agent that specifically binds to the peptide when the peptide is phosphorylated on one or more threonine residues, wherein detecting a complex formed between the peptide and the specific binding agent that specifically binds to the peptide when the peptide is phosphorylated identifies the test agent as one that does not inhibit Plk1 kinase activity;

or

detecting a complex formed between the peptide and the specific binding agent that specifically binds to the peptide when the peptide is not phosphorylated, wherein detecting a complex formed between the peptide and the specific binding agent that specifically binds to the peptide when the peptide is not phosphorylated identifies the test agent as one that inhibits Plk1 kinase activity.

29. The method of claim 28, wherein the specific binding agent is an antibody that specifically binds to the peptide when the peptide is phosphorylated on a threonine residue, isolated Plk1 or an antibody that specifically binds to the peptide when the peptide is not phosphorylated.

32. The method of claim 28, wherein the specific binding agent is detectably labeled.

33. The method of claim 28, further comprising contacting the sample with an antibody that specifically binds to the specific binding agent.

34. The method of claim 33, wherein the antibody that specifically binds to the specific binding agent is detectably labeled.

35. The method of claim 25, wherein the peptide is immobilized on a solid support.

46. A kit for detecting the activity of Plk1, the kit comprising one or more peptides according to claim 1.

47. The method of claim 27, further comprising contacting the biological sample(s) with a specific binding agent that specifically binds to the peptide when the peptide is phosphorylated on one or more threonine residues or a specific binding agent that specifically binds to the peptide when the peptide is not phosphorylated on a threonine residue;

and

detecting a complex formed between the peptide and the specific binding agent that specifically binds to the peptide when the peptide is phosphorylated on one or more threonine residues, wherein detecting a complex formed between the peptide and the specific binding agent that specifically binds to the peptide when the peptide is phosphorylated and identifies the test agent as one that does not inhibit Plk1 kinase activity;

or

detecting a complex formed between the peptide and the specific binding agent that specifically binds to the peptide when the peptide is not phosphorylated, wherein detecting a complex formed between the peptide and the specific binding agent that specifically binds to the peptide when the peptide is not phosphorylated identifies the test agent as one that inhibits Plk1 kinase activity.

48. The method of claim 47, wherein the specific binding agent is an antibody that specifically binds to the peptide when the peptide is phosphorylated on a threonine residue, isolated Plk1, or an antibody that specifically binds to the peptide when the peptide is not phosphorylated.

49. The method of claim 47, wherein the specific binding agent is detectably labeled.

50. The method of claim 47, further comprising contacting the sample with an antibody that specifically binds to the specific binding agent.

51. The method of claim 52, wherein the antibody that specifically binds to the specific binding agent is detectably labeled.

52. The method of claim 47, wherein the peptide is immobilized on solid support.

6-8. -8. (canceled)

10. (canceled)

18. (canceled)

24. (canceled)

30-31. -31. (canceled)

36-45. -45. (canceled)