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

• US 7,927,579 B2
• Filed: 01/16/2004
• Issued: 04/19/2011
• 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
• Primary Examiner(s): Yang; N. C.
• Application Number:
  US10/759,513
• Publication Number:
  US 20040176575A1
• Time in Patent Office: 2,650 Days
• Field of Search: 435/174, 435/177, 435/178, 435/180, 435/181, 435/183, 435/184, 435/188, 435/964, 436/518, 436/528, 436/529, 436/531, 436/532, 436/174, 530/345, 530402-411, 544/224, 544/232, 544/239
• US Class Current: 424/1.69
• 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 of a protein comprising:

contacting the protein 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 the secondary ligand binding site, thereby effecting site-specific covalent bonding of the target group on the protein via the functional group,

and eliminating the ligand moiety from the protein-polymer conjugate after the specific covalent bonding of the target group on the protein,

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;

a diazoester;

an acylphosphate;

an o-salicylate;

a p-salicylate;

a m-salicylate;

a disulfide; and

an α

-keto- or β

-keto acid or ester;

wherein the polymer is a polyethylene glycol polymer;

wherein the secondary site-specific ligand moiety is a peptidyl ligand moiety or carbohydrate ligand moiety; and

wherein the secondary ligand binding site is a site differing from a primary binding site; and

wherein the secondary ligand binding site has no effect on the primary function of the protein.

2. The method of claim 1, wherein the protein 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.

3. The method of claim 1, wherein the protein 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.

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

5. The method of claim 4, 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 protein.

6. The method of claim 5, wherein said activated polymer complex is selected from the group consisting of

7. The method of claim 1, wherein a intervening linker is linked to the peptidyl ligand moiety that becomes linked to the protein such that the intervening linker comprises no peptidyl bond of the peptidyl ligand moiety.

8. The method of claim 7, wherein the activated polymer complex is selected from the group consisting of

9. The method of claim 4, wherein the peptidyl ligand moiety is further linked reversibly to a solid support, either directly or via the polymer.

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

11. The method of claim 4, wherein the peptidyl ligand moiety comprises at least one synthetic amino acid or one surrogate amino acid.

12. The method of claim 11, 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.

13. The method of claim 11, 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.