Molecular scaffolds for kinase ligand development

• US 20050164300A1
• Filed: 09/15/2004
• Published: 07/28/2005
• Est. Priority Date: 09/15/2003
• Status: Abandoned Application

• Current Assignee: Plexxikon Inc (Daiichi Sankyo Company Limited)
• Original Assignee: Plexxikon Inc (Daiichi Sankyo Company Limited)
• Inventors: Ibrahim, Prabha L., Bremer, Ryan E., Zuckerman, Rebecca L., Artis, Dean R., Gillette, Samuel J., Hurt, Clarence R.
• Application Number:
  US10/941,635
• Publication Number:
  US 20050164300A1
• Time in Patent Office: Days
• US Class Current: 435/7.100
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 40/04 Libraries containing only organic compounds
• 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: G16B 15/00 ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
• CPC Class Codes: G16B 15/30 Drug targeting using structural data; Docking or binding prediction
• CPC Class Codes: G16B 35/00 ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
• CPC Class Codes: G16B 35/20 Screening of libraries
• CPC Class Codes: G16C 20/50 Molecular design, e.g. of drugs
• CPC Class Codes: G16C 20/60 In silico combinatorial chemistry

Abstract

Molecular scaffolds for compounds active on protein kinases are described, along with methods for using such scaffolds for kinase ligand development. The use of kinase structural information, exemplified with PIM-1 crystals and structural information can, for example, be used for identifying molecular scaffolds and for developing ligands that bind to and modulate particular kinases.

Claims

1. A kinase scaffold library comprising at least one set of compounds, each set consisting essentially of a plurality of compounds of a chemical structure selected from the group consisting of Formula I, II, III, IV, V, VI, and VII.

2. The library of claim 1, wherein said set comprises at least 50 different compounds.

3. The library of claim 1, wherein said library comprises at least 3 said sets.

4. The library of claim 1, wherein a majority of compounds in said set have been demonstrated to bind to one or more kinases.

5. The library of claim 4, wherein said kinases comprise a plurality of kinases selected from the group consisting of PIM-1, Pyk2, c-Abl, Her2, cMet, VEGFR, EGFR, cKit, Pkcβ

, p38, Cdk2, Akt, Gsk3β

.

6. The library of claim 1, wherein each said compound is in a separate well in a plate or plurality of plates.

7. A system for fitting compounds in binding sites of one or more protein kinases, comprising

an electronic kinase scaffold library comprising at least one collection of electronic representations of compounds of a chemical structure selected from the group consisting of Formula I, II, III, IV, V, VI, and VII, wherein said kinase scaffold library is embedded in a computer memory device, wherein said electronic representations of said compounds can be selectively retrieved and functionally connected with computer software adapted to fit electronic representations of compounds in an electronic representation of a binding site of a kinase.

8. The system of claim 7, further comprising at least one electronic representation of a kinase binding site embedded in computer memory such that said electronic representation of a kinase binding site can be functionally connected with said computer software.

9. The system of claim 8, where said electronic representation of kinase binding sites comprises electronic representations of binding sites of a plurality of kinases selected from the group consisting of PIM-1, Pyk2, c-Abl, Her2, cMet, VEGFR, EGFR, cKit, Pkcβ

, p38, Cdk2, Akt, Gsk3β

.

10. A method for obtaining improved ligands binding to a protein kinase, comprising

determining whether a derivative of a compound of Formula I, II, III, IV, V, VI, or VII binds to said kinase with greater affinity or greater specificity or both than said compound, wherein binding with greater affinity or greater specificity or both indicates that said derivative is an improved ligand.

11. The method of claim 10, wherein said derivative has at least 10-fold greater affinity or specificity or both than said compound.

12. The method of claim 10, wherein said derivative has at least 100-fold greater affinity or specificity or both.

13. The method of claim 10, wherein said kinase is selected from the group consisting of PIM-1, Pyk2, c-Abl, Her2, cMet, VEGFR, EGFR, cKit, Pkcβ

, p38, Cdk2, Akt, Gsk3β

.

14. A method for developing ligands specific for a particular kinase, comprising

determining whether a derivative of a compound of Formula I, II, III, IV, V, VI, or VII that binds to a plurality of kinases has greater specificity for said particular kinase than said compound.

15. The method of claim 14, wherein said compound binds to said kinase with an affinity at least 10-fold greater than for binding to any of said plurality of kinases.

16. The method of claim 15, wherein said compound interacts with at least one of PIM-1 residues 49, 52, 65, 67, 121, 128, and 186.

17. The method of claim 14, wherein said compound binds weakly to said plurality of kinases.

18. The method of claim 14, wherein said kinase is selected from the group consisting of PIM-1, Pyk2, c-Abl, Her2, cMet, VEGFR, EGFR, cKit, Pkcβ

, p38, Cdk2, Akt, Gsk3β

.

19. A method for developing ligands binding to a kinase, comprising

determining the orientation of at least one molecular scaffold of Formula I, II, III, IV, V, VI, or VII in co-crystals with said kinase;

and

identifying chemical structures of said molecular scaffolds, that, when modified, alter the binding affinity or binding specificity or both between the molecular scaffold and said kinase; and

synthesizing a ligand wherein one or more of the chemical structures of the molecular scaffold is modified to provide a ligand that binds to said kinase with altered binding affinity or binding specificity or both.

20. The method of claim 19, wherein said molecular scaffold is a weak binding compound.

21. The method of claim 19, wherein said molecular scaffold binds to a plurality of kinases.

22. The method of claim 19, wherein said molecular scaffold interacts with at least 3 kinases selected from the group consisting of PIM-1, Pyk2, c-Abl, Her2, cMet, VEGFR, EGFR, cKit, Pkcβ

, p38, Cdk2, Akt, and Gsk3β

.

23. The method of claim 19, wherein said kinase is selected from the group consisting of PIM-1, Pyk2, c-Abl, Her2, cMet, cKit, Pkcβ

, Cdk2, and Akt.

24. A method for developing ligands with increased specificity on a kinase, comprising

testing a derivative of a kinase binding compound of Formula I, II, III, IV, V, VI, or VII for increased specificity on said kinase, wherein increased specificity is indicative that said derivative is a ligand with increased specificity.

25. The method of claim 24, wherein said kinase binding compound binds to at least 5 different human kinases.

26. The method of claim 24, wherein said kinase binding compound binds to at least 10 different human kinases.

27. The method of claim 24, wherein said kinase is selected from the group consisting of PIM-1, Pyk2, c-Abl, Her2, cMet, VEGFR, EGFR, cKit, Pkcβ

, p38, Cdk2, Akt, Gsk3β

.

28. A method for identifying a ligand binding to a kinase, comprising

determining whether a derivative compound that includes a core structure selected from the group consisting of Formula I, II, III, IV, V, VI, and VII binds to said kinase with altered binding affinity or specificity or both as compared to the parent compound.

29. A co-crystal of a kinase and a binding compound of Formula I, II, III, IV, V, VI, or VII.

30. The co-crystal of claim 29, wherein said binding compound interacts with at least one of PIM-1 residues 49, 52, 65, 67, 121, 128, and 186.

31. The co-crystal of claim 29, wherein said kinase is selected from the group consisting of PIM-1, Pyk2, c-Abl, Her2, cMet, VEGFR, EGFR, cKit, Pkcβ

, p38, Cdk2, Akt, Gsk3β

.

32. The co-crystal of claim 29, wherein said co-crystal is in an X-ray beam.

33. A method for obtaining co-crystals of PIM-1 with a binding compound of Formula I, II, III, IV, V, VI, or VII, comprising subjecting PIM-1 protein at 5-20 mg/ml to crystallization conditions substantially equivalent to Hampton Screen 1 conditions 2, 7, 14, 17, 23, 25, 29, 36, 44, or 49 in the presence of binding compound for a time sufficient for crystal development.

34. The method of claim 33, wherein said binding compound is added to said protein to a final concentration of 0.5 to 1.0 mM.

35. The method of claim 34, wherein said binding compound is in a dimethyl sulfoxide solution.

36. The method of claim 33, wherein said crystallization condition is 0.4-0.9 M sodium acetate trihydrate pH 6.5, 0.1 M imidazole;

or 0.2-0.7 M. sodium potassium tartrate, 00.1 M MES buffer pH 6.5.

37. A method for modulating kinase activity, comprising

contacting said kinase with a compound of Formula I, II, III, IV, V, VI, or VII.

38. The method of claim 37, wherein said kinase is selected from the group consisting of PIM-1, Pyk2, c-Abl, Her2, cMet, VEGFR, EGFR, cKit, Pkcβ

, p38, Cdk2, Akt, Gsk3β

.

39. The method of claim 37, wherein said compound is at a concentration of 200 μ

M or less.

40. A method for treating a patient suffering from a disease or condition characterized by abnormal kinase activity, comprising

administering to said patient a compound of Formula I, II, III, IV, V, VI, or VII active on said kinase.

41. The method of claim 40, wherein said kinase is selected from the group consisting of PIM-1, Pyk2, c-Abl, Her2, cMet, VEGFR, EGFR, cKit, Pkcβ

, p38, Cdk2, Akt, Gsk3β

.

42. The method of claim 40 wherein said disease or condition is a cancer.

43. The method of claim 40, wherein said disease or condition is an inflammatory disease or condition.

44. An electronic representation of a binding site of a kinase with a compound of a chemical structure selected from the group consisting of Formula I, II, III, IV, V, VI, and VII bound therein.

45. The electronic representation of claim 44, comprising a binding site surface contour.

46. The electronic representation of claim 44, comprising representations of the binding character of a plurality of conserved amino acid residues.

47. A method for identifying potential kinase binding compounds, comprising

fitting at least one electronic representations of a compound of Formula I, II, III, IV, V, VI, or VII in an electronic representation of a kinase binding site.

48. The method of claim 47, wherein said kinase is selected from the group consisting of PIM-1, Pyk2, c-Abl, Her2, cMet, VEGFR, EGFR, cKit, Pkcβ

, p38, Cdk2, Akt, Gsk3β

.

49. The method of claim 48, comprising

removing a computer representation of a compound complexed with said kinase and fitting a computer representation of a compound from a computer database with a computer representation of the active site of said kinase;

and

identifying compounds that best fit said active site based on favorable geometric fit and energetically favorable complementary interactions as potential binding compounds.

50. The method of claim 48, comprising

modifying a computer representation of a compound complexed with said kinase by the deletion or addition or both of one or more chemical groups;

fitting a computer representation of a compound from a computer database with a computer representation of the active site of said kinase; and

identifying compounds that best fit said active site based on favorable geometric fit and energetically favorable complementary interactions as potential binding compounds.

51. The method of claim 48, comprising

removing a computer representation of a compound complexed with said kinase;

and

searching a database for compounds having structural similarity to said compound using a compound searching computer program or replacing portions of said compound with similar chemical structures using a compound construction computer program.

52. A method for attaching a kinase binding compound to an attachment component, comprising

identifying energetically allowed sites for attachment of a said attachment component on a kinase binding compound of Formula I, II, III, IV, V, VI, or VII;

and

attaching said compound or derivative thereof to said attachment component at said energetically allowed site.

53. The method of claim 52, wherein said attachment component is a linker for attachment to a solid phase medium, and said method further comprises attaching said compound or derivative to a solid phase medium through a linker attached at a said energetically allowed site.

54. The method of claim 52, wherein said kinase is selected from the group consisting of PIM-1, Pyk2, c-Abl, Her2, cMet, VEGFR, EGFR, cKit, Pkcβ

, p38, Cdk2, Akt, Gsk3β

.

55. The method of claim 52, wherein said kinase comprises conserved residues matching at least one of PIM-1 residues 49, 52, 65, 67, 121, 128, and 186.

56. The method of claim 53, wherein said linker is a traceless linker.

57. The method of claim 53, wherein said kinase binding compound or derivative thereof is synthesized on a said linker attached to said solid phase medium.

58. The method of claim 57, wherein a plurality of said compounds or derivatives are synthesized in combinatorial synthesis.

59. The method of claim 53, wherein attachment of said compound to said solid phase medium provides an affinity medium.

60. The method of claim 52, wherein said attachment component comprises a label.

61. The method of claim 60, wherein said label comprises a fluorophore.

62. A modified compound, comprising

a compound of Formula I, II, III, IV, V, VI, or VII, with a linker moiety attached thereto.

63. The compound of claim 62, wherein said linker is attached to an energetically allowed site for binding of said modified compound to a kinase selected from the group consisting of PIM-1, Pyk2, c-Abl, Her2, cMet, VEGFR, EGFR, cKit, Pkcβ

, p38, Cdk2, Akt, Gsk3β

.

64. The compound of claim 62, wherein said linker is attached to a solid phase.

65. The compound of claim 62, wherein said linker comprises or is attached to a label.

66. The compound of claim 62, wherein said linker is a traceless linker.

67. A method for developing a ligand for a kinase comprising conserved residues matching one or more of PIM-1 residues 49, 52, 65, 67, 121, 128, and 186, comprising

determining whether a compound of Formula I, II, III, IV, V, VI, or VII binds to said kinase.

68. The method of claim 67, wherein said kinase is selected from the group consisting of PIM-1, Pyk2, c-Abl, Her2, cMet, VEGFR, EGFR, cKit, Pkcβ

, p38, Cdk2, Akt, Gsk3β

.

69. The method of claim 67, wherein said kinase comprises conserved residues matching at least 2 of PIM-1 residues 49, 52, 65, 67, 121, 128, and 186.

70. The method of claim 67, wherein said kinase comprises conserved residues matching PIM-1 residues 49, 52, 65, 67, 121, 128, and 186.

71. The method of claim 67, further comprising determining whether said compound modulates said kinase.

72. The method of claim 67, wherein said determining comprises computer fitting said compound in a binding site of said kinase.

73. The method of claim 67, further comprising forming a co-crystal of said kinase and said compound.

74. The method of claim 73, further comprising determining the binding orientation of said compound with said kinase.