COMPOSITIONS AND METHODS FOR MODULATION OF PLK1 KINASE ACTIVITY

• US 20080279874A1
• Filed: 05/06/2008
• Published: 11/13/2008
• Est. Priority Date: 05/07/2007
• Status: Abandoned Application

• Current Assignee: Wyeth (Pfizer Inc.)
• Original Assignee: Wyeth (Pfizer Inc.)
• Inventors: Krishnamurthy, Girija, Loganzo, Frank JR., Tan, Xingzhi Cindy, Ding, Weidong Warren, Patel, Jagruti Hasmukh
• Application Number:
  US12/115,750
• Publication Number:
  US 20080279874A1
• Time in Patent Office: Days
• US Class Current: 424/185.1
• 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: A61P 37/04 Immunostimulants
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 16/44 against material not provided for elsewhere , e.g. haptens, metals, DNA, RNA, amino acids
• CPC Class Codes: G01N 2333/9121 with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases

Abstract

Described are compositions and methods for activating a Plk1 protein as well as phospho-specific anti-Myt1 antibodies that can be used to detect phosphorylation of Myt1. Activated Plk1 protein, phospho-specific anti-Myt1 antibodies, and/or Plk1 substrates can be used in screening assays to identify compounds that modulate the ability of Plk1 to phosphorylate and/or bind to a Plk1 substrate.

Claims

1. A method of activating a polo-like kinase-1 (Plk1) protein, the method comprising incubating a Plk1 protein in a buffer comprising (i) a divalent cation selected from the group consisting of manganese, calcium, nickel, and zinc and (ii) adenosine triphosphate (ATP), wherein the divalent cation and ATP are present in amounts sufficient to increase the kinase activity of the Plk1 protein.

2. The method of claim 1, wherein the divalent cation is manganese.

3. The method of claim 1, wherein the buffer comprises manganese chloride (MnCl₂).

4. The method of claim 3, wherein the buffer comprises at least 10 mM MnCl₂.

5. The method of claim 1, wherein the buffer comprises a detergent.

6. The method of claim 5, wherein the detergent is 3-[(3-Cholamidopropyl)dimethyl ammonio]-1-propanesulfonate (CHAPS).

7. The method of claim 6, wherein the buffer comprises at least 0.05% CHAPS.

8. The method of claim 1, wherein at least 100 μ

g/ml of the Plk1 protein is incubated in the buffer.

9. The method of claim 8, wherein at least 165 μ

g/ml of the Plk1 protein is incubated in the buffer.

10. The method of claim 1, wherein the Plk1 protein is incubated in the buffer for a period of at least one hour.

11. The method of claim 1, wherein the PLK1 protein is activated in the absence of a Plk1 substrate.

12. The method of claim 1, wherein the PLK1 protein is activated in the presence of a Plk1 substrate.

13. A method of detecting the kinase activity of a Plk1 protein, the method comprising:

providing a Plk1 protein activated by the method of claim 1;

contacting the activated Plk1 protein with a Plk1 substrate under conditions effective to permit phosphorylation of the Plk1 substrate; and

measuring phosphorylation of the Plk1 substrate,

wherein phosphorylation of the Plk1 substrate indicates kinase activity of the Plk1 protein.

14. The method of claim 13, wherein the Plk1 substrate is a polypeptide comprising full length membrane-associated tyrosine- and threonine-specific cdc-2 inhibitory kinase (Myt1) or a fragment thereof that is subject to phosphorylation by Plk1.

15. The method of claim 13, wherein the Plk1 substrate is a polypeptide comprising a fragment of Myt1 that is subject to phosphorylation by Plk1 on a serine residue that corresponds to position 426 of Myt1, on a serine residue that corresponds to position 435 of Myt1, on a serine residue that corresponds to position 469 of Myt1, or on a threonine residue that corresponds to position 495 of Myt1.

16. The method of claim 15, wherein the Plk1 substrate comprises SEQ ID NO:

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, or SEQ ID NO;

14.

17. The method of claim 13, wherein the Plk1 substrate is less than 50 amino acids in length.

18. The method of claim 13, wherein the Plk1 substrate is Myt1, cell cycle phosphatase Cdc25C, Cyclin B1, early mitotic inhibitor 1 (Emi1), anaphase-promoting complex/cyclosome 1 (APC1), anaphase-promoting complex/cyclosome subunit 3 (APC3), anaphase-promoting complex subunit 8 (APC8), nucleolar phosphoprotein B23 (B23/Nucleophosmin), breast cancer type 2 susceptibility protein homolog (BRCA2), centrosomal protein of 55 kDa (Cep55), kinesin family member 23 (KIF23/CHO1/Mklp1), Cohesin, Cohesin, golgi reassembly stacking protein 1 (GRASP65), heat shock transcription factor 1 (HSF1), Kizuna, kinesin family member 20A (KIF20A/MkIp2/Rabkinesin6), ninein-like protein (Nlp), nuclear migration protein nudC (NudC), p53, Plk1-interacting checkpoint helicase (PICH), peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (Pin1), stathmin 1/oncoprotein 18 (Stathmin/Op18), translationally-controlled tumor protein homolog (TCTP), Vimentin, Weel, tumor protein p73 (p73), Bora, DNA topoisomerase II alpha, origin recognition complex 1 (Hbo1), Aurora B, Mitotic centromere-associated kinesin (MCAK), Rho-associated, coiled-coil containing protein kinase 2, MLF1 interacting protein (PBIP1), budding uninhibited by benzimidazoles 1 homolog beta (BubR1), cytoplasmic polyadenylation element-binding protein (CPEB), human phosphatase HsCdc14A, or small GTP/GDP-binding protein Ran.

21. A method of identifying a compound that inhibits phosphorylation of a Plk1 substrate, the method comprising:

providing a Plk1 protein activated by the method of claim 1;

contacting, in the presence of a candidate compound, the activated Plk1 protein with a Plk1 substrate; and

measuring phosphorylation of the Plk1 substrate;

wherein decreased phosphorylation of the Plk1 substrate in the presence of the candidate compound as compared to phosphorylation of the Plk1 substrate that occurs in the absence of the candidate compound indicates that the candidate compound inhibits phosphorylation of the Plk1 substrate by the Plk1 protein.

24. The method of claim 13, wherein measuring phosphorylation of the Plk1 substrate comprises:

contacting the Plk1 substrate with an antibody that (i) is conjugated to a first fluorescent agent and (ii) specifically binds to the Plk1 substrate when the Plk1 substrate is phosphorylated on a serine or threonine residue, wherein the Plk1 substrate is conjugated to a second fluorescent agent; and

detecting the occurrence of fluorescence resonance energy transfer between the first fluorescent agent and the second fluorescent agent as an indicator of phosphorylation of the Plk1 substrate.

25. The method of claim 13, wherein measuring phosphorylation of the Plk1 substrate comprises:

contacting the Plk1 substrate with an antibody that (i) is conjugated to a detection moiety and (ii) specifically binds to the Plk1 substrate when the Plk1 substrate is phosphorylated on a serine or threonine residue;

removing antibody that is not bound to the Plk1 substrate; and

detecting the detection moiety associated with the Plk1 substrate as an indicator of phosphorylation of the Plk1 substrate.

26. The method of claim 13, wherein measuring phosphorylation of the Plk1 substrate comprises passaging the Plk1 substrate through a stationary phase, wherein increased or decreased retardation of the Plk1 substrate during passage through the stationary phase indicates the phosphorylation status of the Plk1 substrate.

29. A method of assessing the ability of a compound to inhibit phosphorylation of a Plk1 substrate by a Plk1 protein in a cell, the method comprising:

providing a cell expressing a Plk1 protein and a Plk1 substrate;

incubating the cell in the presence of the compound identified by the method of claim 21; and

measuring the amount of the Plk1 substrate in the cell after incubating the cell in the presence of the compound;

wherein a difference in the amount of the Plk1 substrate in the cell after incubation with the compound as compared to the amount of the Plk1 substrate in the cell in the absence of incubation with the compound indicates that the compound inhibits phosphorylation of the Plk1 substrate by the Plk1 protein.

30. The method of claim 29, wherein the Plk1 substrate is Emi1.

31. The method of claim 30 wherein an increase in the amount of Emi1 in the cell after incubation with the compound as compared to the amount of Emi1 in the cell in the absence of incubation with the compound indicates that the compound inhibits phosphorylation of Emi1 by the Plk1 protein.

41. The method of claim 13, wherein the Plk1 substrate is centromere protein B (CENPB).

19. A method of detecting the kinase activity of a Plk1 protein, the method comprising:

contacting a Plk1 protein with a Plk1 substrate under conditions effective to permit phosphorylation of the Plk1 substrate, wherein the Plk1 substrate is a polypeptide comprising full length Myt1 or a fragment of Myt1 that is subject to phosphorylation by Plk1 on a threonine residue that corresponds to position 495 of Myt1;

contacting the Plk1 substrate with a phospho-specific anti-pT495 Myt1 antibody; and

measuring binding of the antibody to the Plk1 substrate to thereby detect phosphorylation of the Plk1 substrate;

wherein phosphorylation of the Plk1 substrate indicates kinase activity of the Plk1 protein.

20. The method of claim 19, wherein, prior to contacting the Plk1 protein with the Plk1 substrate, the Plk1 protein is incubated in a buffer comprising an amount of manganese and ATP sufficient to increase the kinase activity of the Plk1 protein.

23. The method of claim 19, wherein the measuring occurs in a cell.

27. The method of claim 19, wherein the contacting occurs in a cell.

28. The method of claim 27, wherein the cell is a mammalian cell.

22. A method of identifying a compound that inhibits phosphorylation of a Plk1 substrate, the method comprising:

contacting, in the presence of a candidate compound, a Plk1 protein with a Plk1 substrate, wherein the Plk1 substrate is a polypeptide comprising full length Myt1 or a fragment of Myt1 that is subject to phosphorylation by Plk1 on a threonine residue that corresponds to position 495 of Myt1;

contacting the Plk1 substrate with a phospho-specific anti-pT495 Myt1 antibody; and

measuring binding of the antibody to the Plk1 substrate to thereby detect phosphorylation of the Plk1 substrate;

wherein decreased phosphorylation of the Plk1 substrate in the presence of the candidate compound as compared to phosphorylation of the Plk1 substrate that occurs in the absence of the candidate compound indicates that the candidate compound inhibits phosphorylation of the Plk1 substrate by the Plk1 protein.

32. An isolated peptide that is less than 50 amino acids in length and comprises SEQ ID NO:

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, or a variant thereof, wherein the variant is a phosphorylation substrate of Plk1.

33. The peptide of claim 32, wherein the peptide comprises SEQ ID NO:

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, or SEQ ID NO;

12.

34. The peptide of claim 33, wherein the peptide comprises a variant of SEQ ID NO:

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, or SEQ ID NO;

12 in which at least one but not more than five amino acid residues are substituted, deleted, or inserted.

35. The peptide of claim 34, wherein the peptide consists of SEQ ID NO:

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, or SEQ ID NO;

12.

36. The peptide of claim 32, wherein the peptide is phosphorylated on a threonine residue that corresponds to position 495 of Myt1.

37. The peptide of claim 36, wherein the peptide has the amino acid sequence depicted in SEQ ID NO:

13.

38. An isolated antibody that specifically binds to a peptide whose amino acid sequence consists of SEQ ID NO:

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, or SEQ ID NO;

13.

39. The antibody of claim 38, wherein the antibody preferentially binds the peptide when phosphorylated on a threonine amino acid residue that corresponds to position 495 of Myt1.

40. A method of generating an immune response in a mammal, the method comprising administering to the mammal an effective amount of the peptide of claim to 31.

42. A method of detecting the kinase activity of a Plk1 protein, the method comprising:

contacting a Plk1 protein with a Plk1 substrate under conditions effective to permit phosphorylation of the Plk1 substrate, wherein the Plk1 substrate is a polypeptide comprising a CENPB protein or a fragment of a CENPB protein that is subject to phosphorylation by Plk1; and

measuring phosphorylation of the Plk1 substrate;

wherein phosphorylation of the Plk1 substrate indicates kinase activity of the Plk1 protein.

43. The method of claim 42, wherein the CENPB protein comprises the sequence of SEQ ID NO:

19.

44. The method of claim 42, wherein the Plk1 substrate is a polypeptide comprising a fragment of a CENPB protein that is subject to phosphorylation by Plk1 on a serine residue that corresponds to position 43 of CENPB, on a serine residue that corresponds to position 156 of CENPB, on a threonine residue that corresponds to position 169 of CENPB, on a serine residue that corresponds to position 307 of CENPB, or on a threonine residue that corresponds to position 396 of CENPB.

45. The method of claim 44, wherein the Plk1 substrate comprises SEQ ID NO:

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, or SEQ ID NO;

25.

46. The method of claim 42, wherein the Plk1 substrate is less than 50 amino acids in length.

48. The method of claim 42, wherein measuring phosphorylation of the Plk1 substrate comprises:

contacting the Plk1 substrate with a phospho-specific anti-CENPB antibody; and

measuring binding of the antibody to the Plk1 substrate to thereby detect phosphorylation of the Plk1 substrate.

49. The method of claim 48, wherein the phospho-specific anti-CENPB protein antibody specifically recognizes CENPB at an epitope comprising a phosphorylated serine residue that corresponds to position 43 of CENPB, a phosphorylated serine residue that corresponds to position 156 of CENPB, a phosphorylated threonine residue that corresponds to position 169 of CENPB, a phosphorylated serine residue that corresponds to position 307 of CENPB, or a phosphorylated threonine residue that corresponds to position 396 of CENPB.

50. The method of claim 42, wherein measuring phosphorylation of the Plk1 substrate comprises:

contacting the Plk1 substrate with an antibody that (i) is conjugated to a detection moiety and (ii) specifically binds to the Plk1 substrate when the CENPB protein is phosphorylated on a serine or threonine residue; and

detecting the detection moiety associated with the Plk1 substrate as an indicator of phosphorylation of the Plk1 substrate.

51. The method of claim 42, wherein measuring phosphorylation of the Plk1 substrate comprises passaging the Plk1 substrate through a stationary phase, wherein an increased or decreased retardation of the Plk1 substrate during passage through the stationary phase indicates the phosphorylation status of the Plk1 substrate.

53. The method of claim 42, wherein the measuring occurs in a cell.

47. A method of identifying a compound that inhibits phosphorylation of a Plk1 substrate, the method comprising:

contacting, in the presence of a candidate compound, a Plk1 protein with a Plk1 substrate, wherein the Plk1 substrate is a polypeptide comprising a CENPB protein or a fragment of a CENPB protein that is subject to phosphorylation by Plk1; and

measuring phosphorylation of the Plk1 substrate;

wherein decreased phosphorylation of the Plk1 substrate in the presence of the candidate compound as compared to phosphorylation of the Plk1 substrate that occurs in the absence of the candidate compound indicates that the candidate compound inhibits phosphorylation of the Plk1 substrate by the Plk1 protein.

52. A method for identifying a compound that inhibits an interaction between a Plk1 protein and a Plk1 substrate, the method comprising:

contacting, in the presence of a candidate compound, a Plk1 protein with a Plk1 substrate, wherein the Plk1 substrate is a polypeptide comprising a CENPB protein or a fragment of a CENPB protein that binds to Plk1; and

measuring binding of the Plk1 protein to the Plk1 substrate;

wherein decreased binding of the Plk1 protein to the Plk1 substrate in the presence of the candidate compound as compared to binding of the Plk1 protein to the Plk1 substrate that occurs in the absence of the candidate compound indicates that the candidate compound inhibits an interaction between the Plk1 protein and the Plk1 substrate.

54. A method of inhibiting phosphorylation of a CENPB protein by a Plk1 protein, the method comprising administering to a subject an effective amount of a compound that inhibits phosphorylation of a CENPB protein by a Plk1 protein.

55. The method of claim 54, wherein the compound is a polypeptide comprising a CENPB protein or a fragment of a CENPB protein that is subject to phosphorylation by Plk1.

58. The method of claim 54, wherein the subject is a mammal.

59. The method of claim 58, wherein the mammal is a human.

60. The method of claim 54, wherein the subject has a cancer.

61. The method of claim 60, further comprising determining if one or more cells of the subject'"'"'s cancer express a Plk1 protein, a CENPB protein, or a Plk1 protein and a CENPB protein.

56. A method of inhibiting an interaction between a Plk1 protein and a CENPB protein, the method comprising administering to a subject an effective amount of a compound that inhibits an interaction between a Plk1 protein and a CENPB protein.

57. The method of claim 56, wherein the compound is a polypeptide comprising a CENPB protein or a fragment of a CENPB protein that binds to Plk1.

62. A method for evaluating the efficacy of an anti-Plk1 agent, the method comprising:

providing a biological sample obtained from a subject to whom an anti-Plk1 agent has been administered; and

detecting phosphorylation of a CENPB protein in the biological sample;

wherein a decreased level of phosphorylation of the CENPB protein as compared to the level of phosphorylation in a biological sample taken from another subject or from the subject prior to administration of the anti-Plk1 agent indicates that the anti-Plk1 therapy is effective.

63. The method of claim 62, wherein the anti-Plk1 agent inhibits Plk1 kinase activity.

64. The method of claim 62, wherein the anti-Plk1 agent inhibits Plk1 expression.

65. The method of claim 62, wherein the anti-Plk1 agent is scytonemin, ON01910, or BI 2536.

66. An isolated peptide that is less than 50 amino acids in length and comprises a fragment of a CENPB protein that is subject to phosphorylation by Plk1 on a serine residue that corresponds to position 43 of CENPB, on a serine residue that corresponds to position 156 of CENPB, on a threonine residue that corresponds to position 169 of CENPB, on a serine residue that corresponds to position 307 of CENPB, or on a threonine residue that corresponds to position 396 of CENPB.

71. The peptide of claim 66, wherein the peptide is phosphorylated on a serine residue that corresponds to position 43 of CENPB, a serine residue that corresponds to position 156 of CENPB, a threonine residue that corresponds to position 169 of CENPB, a serine residue that corresponds to position 307 of CENPB, or a threonine residue that corresponds to position 396 of CENPB.

72. An isolated antibody that specifically binds to the peptide of claim 66.

73. The antibody of claim 72, wherein the antibody preferentially binds the peptide when phosphorylated on a serine residue that corresponds to position 43 of CENPB, a serine residue that corresponds to position 156 of CENPB, a threonine residue that corresponds to position 169 of CENPB, a serine residue that corresponds to position 307 of CENPB, or a threonine residue that corresponds to position 396 of CENPB.

74. A method of generating an immune response in a mammal, the method comprising administering to the mammal an effective amount of the peptide of claim 66.

67. An isolated peptide that is less than 50 amino acids in length and comprises SEQ ID NO:

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, or SEQ ID NO;

25, or a variant thereof, wherein the variant is a phosphorylation substrate of Plk1.

68. The peptide of claim 67, wherein the peptide comprises SEQ ID NO:

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, or SEQ ID NO;

25.

69. The peptide of claim 67, wherein the peptide comprises a variant of SEQ ID NO:

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, or SEQ ID NO;

25 in which at least one but not more than five amino acid residues are substituted, deleted, or inserted.

70. The peptide of claim 67, wherein the peptide consists of SEQ ID NO:

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, or SEQ ID NO;

25.

75. A method for generating a compound that inhibits the interaction between a Plk1 protein and a CENPB protein, the method comprising:

providing a three-dimensional structure of a molecule or a molecular complex comprising;

(a) a Plk1 protein or a CENPB-binding fragment thereof;

(b) a CENPB protein or a Plk1-binding fragment thereof;

or (c) a molecular complex comprising (a) and (b);

designing, based on the three-dimensional structure, a compound comprising a region that inhibits the interaction between a Plk1 protein and a CENPB protein; and

producing the compound.