Tandem analyses of noncovalently driven effectors for modulatory mapping of activities of protein sites

• US 20040176575A1
• Filed: 01/16/2004
• Published: 09/09/2004
• Est. Priority Date: 01/17/2003
• Status: Active Grant

• Current Assignee: Advanced Proteome Therapeutics Inc. (Advanced Proteome Therapeutics Corp.)
• Original Assignee: Advanced Proteome Therapeutics Inc. (Advanced Proteome Therapeutics Corp.)
• Inventors: Krantz, Alexander
• Granted Patent: US 7,927,579 B2
• Time in Patent Office: Days
• US Class Current: 530/351
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

The present invention provides methods of discovering and mapping secondary binding sites on biological molecules (e.g., proteins), the effects, if any, of site occupancy on the primary function of the molecule, and the screening of small molecules against the secondary binding sites. The invention further provides novel complexes for modification of secondary binding sites and the resulting modified biological molecules.

Claims

1. A method for modification of a target group in or near a secondary ligand binding site on a biological molecule comprising:

contacting a biological molecule with at least one site-specific activated polymer complex comprising;

a functional group reactive with the target group;

a polymer linked to the functional group; and

a ligand moiety that specifically binds to a secondary ligand binding site,

thereby effecting site-specific covalent bonding of the target group on the biological molecule via the functional group.

2. The method of claim 1, wherein the polymer is selected from the group consisting of a polyalkylene oxide polymer, a polynucleotide, a polysaccharide, a protected polypeptide, a polyethylene glycol polymer, a biotinylated polyethylene glycol polymer, and a fluorescent polyethylene glycol polymer.

3. The method of claim 1, wherein the ligand moiety preferentially binds with the secondary ligand binding site.

4. The method of claim 1, wherein the biological molecule is selected from the group consisting of:

a peptide, a polypeptide, a protein, an enzyme, a receptor, an antibody, a hormone, a cytokine, and functional fragments thereof.

5. The method of claim 1, wherein the biological molecule comprises a plurality of secondary ligand binding sites, and the ligand moiety binds to only one of the plurality of secondary ligand binding target sites.

6. The method of claim 1, wherein the biological molecule is contacted with the activated polymer complex in the presence of at least one other molecule comprising at least one other reactive group that covalently bonds to the functional group, and the ligand moiety is selected to avoid binding that would effect covalent bonding of the other reactive group to the functional group linked to said polymer.

7. The method of claim 1, wherein the ligand moiety of the activated polymer complex is a peptidyl ligand moiety.

8. The method of claim 7, wherein the functional group of the activated polymer complex is directly linked to the polymer so that the polymer becomes linked via the functional group directly to the target group and the peptidyl ligand moiety does not become linked to the biological molecule.

9. The method of claim 8, wherein said activated polymer complex is selected from the group consisting of activated polymer complexes shown in Eqns. 5-7 of FIG. 1B and Eqns. and 9-11 of FIG. 1C.

10. The method of claim 7, wherein the functional group is linked to the polymer via an intervening linker so that the polymer becomes linked to the target group via the intervening linker and the ligand moiety also becomes linked to the biological molecule.

11. The method of claim 10, wherein the intervening linker comprises at least one peptidyl bond of the peptidyl ligand moiety that becomes linked to the biological molecule.

12. The method of claim 11, wherein the activated polymer complex is selected from the group consisting of activated polymer complexes shown in Equations 13-15 of FIG. 1D.

13. The method of claim 10, wherein the intervening linker is linked to the peptidyl ligand moiety that becomes linked to the biological molecule such that the intervening linker comprises no peptidyl bond of the peptidyl ligand moiety.

14. The method of claim 13, wherein the activated polymer complex is selected from the group consisting of activated polymer complexes in Equation 8 of FIG. 1B and Equation 12 of FIG. 1C.

15. The method of claim 7, wherein the peptidyl ligand moiety is further linked reversibly to a solid support, either directly or via the polymer.

16. The method of claim 1, wherein the functional group is selected from the group consisting of:

a dithioester;

a thioloester;

thionoester;

a selenoester;

a selenoloester;

a selenonooester;

a phosphonoester;

a phosphoric ester;

a phosphinic ester;

a sulfonate ester;

an isocyanate;

a diazoester;

a diazoketone;

a diazoamide;

an acylphosphate;

an imine;

a thioimin;

an o-salicylate;

a p-salicylate;

a m-salicylate;

a disulfide;

an acetylene dicarboxylate dimethyl ester and/or a half ester or half amide thereof;

a fumaric acid or maleic acid and their esters and derivatives thereof;

an α

-nitroacrylic acid ester;

a phenylglyoxal;

a glyoxal;

a 2,3-butanedione;

a cyclohexanedione;

an α

-keto- or β

-keto acid or ester;

a di- or tri-fluoromethylketone;

a trinitrobenzenesulfonic acid;

a tetranitromethane;

a diethylpyrocarbonate;

a N-bromosuccinimide;

a dithiothreitol;

a sodium tetrathionate;

a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide;

a N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline;

a fluoro-2,4-dinitrobenzene; and

a (4-aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole.

17. The method of claim 1, wherein reactivity of the functional group is photochemically induced.

18. The method of claim 7, wherein the peptidyl ligand moiety comprises at least one synthetic amino acid or one surrogate amino acid.

19. The method of claim 18, wherein the synthetic amino acid is selected from the group consisting of:

β

-alanine, γ

-aminobutyrate, O-methyl-substituted threonine, O-methyl-substituted serine, and O-methyl-substituted tyrosine.

20. The method of claim 18, wherein the surrogate amino acid is selected from the group consisting of β

-thiazolealanine, β

-thiadiazole-alanine, β

-isothiazole-alanine, β

-isoxazole-alanine, oxazole-alanine, β

-benzoxazole-alanine, β

-benzisoxazole-alanine, β

-benzisothiazoles, β

-benzthiazoles, and 2-acylimino-3H-thiazoline derivatives.

21. An activated polymer complex for secondary site-specific modification of a target group in or near a secondary ligand binding site, the activated polymer complex comprising:

a functional group potentially reactive with the target group;

a polymer linked to the functional group; and

a secondary site-specific ligand moiety.

22. The activated polymer complex of claim 21, wherein the polymer is selected from the group consisting of a polyalkylene oxide polymer, a polynucleotide, a polysaccharide, a protected polypeptide, a polyethylene glycol polymer, a biotinylated polyethylene glycol polymer, and a fluorescent polyethylene glycol polymer.

23. The activated polymer complex of claim 21, wherein the secondary-site specific ligand moiety is a peptidyl ligand moiety.

24. The activated polymer complex of claim 23, wherein the functional group is directly linked to the polymer so that when the functional group becomes covalently linked to the reactive group on the target molecule, the polymer becomes linked via the functional group directly to the reactive group and the peptidyl ligand moiety does not become linked to the target molecule.

25. The activated polymer complex of claim 24, wherein the activated polymer complex is selected from the group consisting of activated polymer complexes shown in Eqns. 5-7 of FIG. 1B and Eqns. and 9-11 of FIG. 1C.

26. The activated polymer complex of claim 23, wherein the functional group is linked to the polymer via an intervening linker so that when the functional group becomes covalently linked to the reactive group on the target molecule, the polymer becomes covalently linked via the intervening linker and the ligand moiety also becomes linked to the target molecule.

27. The activated polymer complex of claim 26, wherein the intervening linker comprises at least one peptidyl bond of the peptidyl ligand moiety that becomes linked to the target molecule.

28. The activated polymer complex of claim 27, wherein the activated polymer complex is selected from the group consisting of activated polymer complexes shown in Equations 13-15 of FIG. 1D.

29. The activated polymer complex of claim 26, wherein said intervening linker that links the functional group to the polymer is linked to the peptidyl ligand moiety that becomes linked to the target molecule such that the intervening linker comprises no peptidyl bond of the peptidyl ligand moiety.

30. The activated polymer complex of claim 29, wherein the activated polymer complex is selected from the group consisting of activated polymer complexes in Equation 8 of FIG. 1B and Equation 12 of FIG. 1C.

31. The activated polymer complex of claim 23, wherein the peptidyl ligand moiety is further linked reversibly to a solid support, either directly or via the polymer.

32. The activated polymer complex of claim 23, wherein the functional group is selected from the group consisting of:

a dithioester;

a thioloester;

a thionoester;

a selenoester;

a selenoloester;

a selenonooester;

a phosphonoester;

a phosphoric ester;

a phosphinic ester;

a sulfonate ester;

an isocyanate;

a diazoester;

a diazoketone;

a diazoamide;

an acylphosphate;

an imine;

a thioimin;

an o-salicylate;

a p-salicylate;

a m-salicylate;

a disulfide;

an acetylene dicarboxylate dimethyl ester and/or a half ester or half amide thereof;

a fumaric acid or maleic acid and their esters and derivatives thereof;

an α

-nitroacrylic acid ester;

a phenylglyoxal;

a glyoxal;

a 2,3-butanedione;

a cyclohexanedione;

an α

-keto- or α

-keto acid or ester;

a di- or tri-fluoromethylketone;

a trinitrobenzenesulfonic acid;

a tetranitromethane;

a diethylpyrocarbonate;

a N-bromosuccinimide;

a dithiothreitol;

sodium tetrathionate;

a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide;

a N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline;

a fluoro-2,4-dinitrobenzene; and

a (4-aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole.

33. The activated polymer complex of claim 23, wherein reactivity of the functional group with the reactive group on the biological molecule is photochemically induced.

34. The activated polymer complex of claim 23, wherein the peptidyl ligand moiety comprises at least one synthetic amino acid or one surrogate amino acid.

35. The activated polymer complex of claim 34, wherein the synthetic amino acid is selected from the group consisting of:

β

-alanine, γ

-aminobutyrate, O-methyl-substituted threonine, O-methyl-substituted serine, and O-methyl-substituted tyrosine.

36. The activated polymer complex of claim 34, wherein the surrogate amino acid is selected from the group consisting of β

-thiazolealanine, β

-thiadiazole-alanine, (3-isothiazole-alanine, β

-isoxazole-alanine, oxazole-alanine, β

-benzoxazole-alanine, β

-benzisoxazole-alanine, β

-benzisothiazoles, β

-benzthiazoles, and 2-acylimino-3H-thiazoline derivatives.

37. A composition comprising the activated polymer complex of any of claims 21-36 and a pharmaceutically acceptable carrier.

38. A method for identifying an activated polymer complex for secondary site-specific modification of a target group in or near a secondary ligand binding site, the method comprising the steps of:

(a) contacting a biological molecule with a library of activated polymer complexes, each activated polymer complex comprising;

a functional group potentially reactive with the target group;

a polymer linked to the functional group, and

a secondary site-specific ligand moiety; and

(b) identifying an activated polymer complex that and binds with specificity to a secondary ligand binding site.

39. The method of claim 38, comprising the step of determining whether the activated polymer complex affects the primary activity of the biological molecule.

40. The method of claim 38, comprising the step of determining whether the activated polymer complex inhibits or enhances the primary activity of the biological molecule.

41. The method of claim 38, comprising the step of determining the rate of modification by quenching the reaction at a predetermined time and measuring the amount of modification.

42. The method of claim 38, comprising the step of at least partially digesting the biological molecule to determine the position of attachment of an activated polymer complex.

43. A method for identifying a competitive site-specific activated polymer complex comprising the step of:

identifying a second activated polymer complex for secondary site-specific modification of the target group in or near the secondary ligand binding site using the method of claim 38.

44. A method for selecting a secondary-site specific effector molecule comprising the steps of:

(a) identifying one or more secondary-site specific molecules that modulate stable covalent bonding of a reactivity probe with a target group in or near a secondary ligand binding site on a biological molecule; and

(b) selecting a secondary-site specific molecule that affects a primary binding site activity of the biological molecule, wherein said secondary-site specific molecule is a secondary-site specific effector molecule.

45. The method of claim 44, wherein step (a) comprises determining the extent of specific binding of one or more test molecules.

46. The method of claim 45, wherein the extent of specific binding is determined using NMR spectroscopy or X-ray crystallography.

47. The method of claim 45, wherein step (a) further comprises determining the difference in the extent of stable covalent bonding in the absence and presence of said test molecule.

48. The method of claim 47, wherein the reactivity probe comprises a leaving group that is displaced upon covalent bonding with the biological molecule, and the difference in the extent of stable covalent in the absence and presence of said test molecule is the difference in the amount of leaving group displaced in the present and the absence of said test molecule.

49. The method of claim 47, wherein a correlation between the extent of specific binding of a test molecule to the biological molecule, and a difference in the extent of stable covalent in the absence and presence of said test molecule, identifies a molecule as a secondary-site specific molecule.

50. The method of claim 44, wherein the primary binding site activity is an enzymatic activity, antibody binding activity, or a receptor binding activity.

51. The method of claim 44, wherein the secondary site-specific molecule blocks primary binding site activity.

52. A method of selecting a secondary-site specific inhibitor molecule comprising:

selecting one or more secondary-site specific effector molecules according to claim 44;

and

selecting a secondary-site specific effector molecule that inhibits the primary binding site activity, wherein said secondary-site specific effector molecule is a secondary-site specific inhibitor molecule.

53. The method of claim 52, further comprising determining whether inhibitor is a competitive inhibitor or a noncompetitive or uncompetitive inhibitor.

54. The method of claim 44, wherein the biological molecule is a peptide, a polypeptide, a protein, an enzyme, a receptor, an antibody, a hormone, a cytokine and a functional fragments thereof.

55. The method of claim 44, wherein the effector molecule further comprises a second functional group.

56. The method of claim 55, wherein the second functional group effects stable covalent bonding with an anchor group on the biological molecule.

57. The method of claim 44, wherein the effector molecule is a small organic compound.

58. The method of claim 44, wherein the effector molecule comprises a peptidyl ligand moiety.

59. The method of claim 44, wherein the effector molecule comprises a carbohydrate ligand moiety.

60. A secondary-site specific effector molecule identified by a method according to claim 44.

61. A library of secondary-site specific effector molecules identified by a method according to claim 44.

62. A method for modulating secondary site-specific modification of a biological molecule, comprising:

contacting the biological molecule with the secondary-site specific effector molecule identified in claim 44.

63. The method of claim 62, wherein the primary binding site activity is an enzymatic activity, antibody binding activity, or a receptor binding activity.

64. The method of claim 62, wherein the secondary site-specific molecule inhibits or blocks primary binding site activity.

65. A method for modulatory mapping of a biological molecule comprising:

contacting a biological molecule with a reactivity probe comprising;

a ligand moiety that specifically binds to a secondary ligand binding site on a biological molecule, and

a functional group reactive with a target group in or near the secondary site, thereby effecting site-specific covalent bonding of the target group on the biological molecule via the functional group;

mapping the secondary ligand binding site and the target group in or near the secondary ligand binding site on the biological molecule.

66. The method of claim 65, comprising determining whether the reactivity probe effects a primary binding site activity of the biological molecule and mapping the effect, if any, of the secondary ligand binding site on the primary binding site activity.

67. A method for obtaining a map of secondary ligand binding sites and target group sites in or near the secondary ligand binding sites on a biological molecule, comprising mapping a plurality of secondary ligand binding sites and target groups on a molecule according to the method of claim 65.

68. The method of claim 67, further comprising mapping whether the reactivity probe affects a primary binding site activity of the biological molecule and mapping the effect, if any, of the secondary ligand binding site on the primary binding site activity.

69. The method of claim 67, comprising mapping primary binding site activity.

70. The method of claim 69, wherein the primary binding site activity is an enzymatic activity, a receptor binding activity, or an antibody binding site.

71. A modified biological molecule comprising:

a biological molecule having a target group covalently bonded via a functional group to a polymer, wherein the target group is in or near a secondary ligand binding site.

72. The biological molecule of claim 71, further comprising a ligand moiety bound to the secondary ligand binding site.

73. The biological molecule of claim 72, wherein the ligand moiety is linked to the polymer.

74. The biological molecule of claim 71, wherein the polymer is selected from the group consisting of a polyalkylene oxide polymer, a polynucleotide, a polysaccharide, a protected polypeptide, a polyethylene glycol polymer, a biotinylated polyethylene glycol polymer and a fluorescent polyethylene glycol polymer.

75. The biological molecule of claim 72, wherein the ligand moiety is a peptidyl ligand moiety.

76. The biological molecule of claim 75, wherein the peptidyl ligand moiety comprises at least one synthetic amino acid or one surrogate amino acid.

77. The biological molecule of claim 76, wherein the synthetic amino acid is selected from the group consisting of:

β

-alanine, γ

-aminobutyrate, O-methyl-substituted threonine, O-methyl-substituted serine, and O-methyl-substituted tyrosine.

78. The biological molecule of claim 76, wherein the surrogate amino acid is selected from the group consisting of β

-thiazolealanine, β

-thiadiazole-alanine, (3-isothiazole-alanine, β

-isoxazole-alanine, oxazole-alanine, β

-benzoxazole-alanine, β

-benzisoxazole-alanine, β

-benzisothiazoles, β

-benzthiazoles, and 2-acylimino-3H-thiazoline derivatives.

79. The biological molecule of claim 71, wherein the biological molecule comprises a peptide, a polypeptide or a protein.

80. The biological molecule of claim 79, wherein the biological molecule is selected from the group consisting of:

an enzyme, a receptor, an antibody, a hormone, a cytokine and a functional fragment of an enzyme, a receptor, an antibody, a hormone or a cytokine.

81. The biological molecule of claim 71, wherein the site-specific modification increases the safety or the efficacy of the biological molecule as compared to an unmodified biological molecule.

82. The biological molecule of claim 71, wherein the site-specific modification increases the dosing period of the biological molecule as compared to an unmodified biological molecule.

83. The biological molecule of claim 82, wherein the dosing period is at least doubled as compared to an unmodified biological molecule.

84. The biological molecule of claim 71, wherein the site-specific modification reduces an immune response in an organism as compared to an unmodified biological molecule.

85. The biological molecule of claim 71, wherein the site-specific modification masks the biological molecule from the immune system.

86. A composition comprising the biological molecule of any of claims 71-85 and a pharmaceutically acceptable carrier.

87. A method of treating a subject comprising administering the biological molecule or composition of any one of claims 71-86 such that the subject is benefited.

88. The method of claim 87, wherein the method is employed to treat HIV, hepatitis, endocrine disorders, proliferative disorders, infectious disease, diabetes, emphysema, or cancer.