Tandem analyses of noncovalently driven effectors for modulatory mapping of activities of protein sites
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Publication Number
US-7927579-B2
Publication Date
2011-04-19
Expiration Date
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.
Core Innovation
The invention describes a tandem mapping approach for identifying and mapping secondary ligand binding sites on proteins using covalent bonding that is made site-specific. A method is provided in which a protein is contacted with at least one site-specific activated polymer complex that includes a functional group reactive with a target group and a ligand moiety that specifically binds to a secondary ligand binding site.
The activated polymer complex effecting site-specific covalent bonding is constructed such that covalent attachment occurs via the functional group between the target group and the protein. After covalent bonding of the target group on the protein, the ligand moiety is eliminated from the protein-polymer conjugate, so that the covalent modification is associated with the contacted secondary binding site while the ligand moiety does not remain in the conjugate.
The secondary binding site is a site differing from a primary binding site and has no effect on the primary function of the protein. The invention describes activated polymer complex architectures in which the polymer is a polyethylene glycol polymer and the secondary site-specific ligand moiety is a peptidyl ligand moiety or a carbohydrate ligand moiety, enabling identification of effector/effector binding site relationships and mapping of modulated covalent bonding.
In applications described in the document narrative, the approach is used with tandem analyses and mapping modalities to connect secondary ligand binding with covalent labeling outcomes. A specific narrative describes HIV protease inactivation and mapping associated with cysteine/histidine-mediated inhibition and resultant PEGylated protein mapping.
Claims Coverage
The independent claim is directed to a method for site-specific modification of a target group in or near a secondary ligand binding site using a site-specific activated polymer complex and eliminating the ligand moiety after covalent bonding. The dependent claims refine aspects including photochemical induction of functional-group reactivity and activated polymer complex linkage architectures, as well as constraints on peptidyl ligand composition and structural variables for the activated polymer complex, and narrowing secondary-site binding selection.
Site-specific activated polymer complex for secondary site covalent labeling
Contacting a 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.
Eliminating the ligand moiety after covalent bonding
Eliminating the ligand moiety from the protein-polymer conjugate after the specific covalent bonding of the target group on the protein.
Reactive functional group selection for target-group modification
Selecting the functional group 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.
Polyethylene glycol polymer with secondary ligand moiety
Having the polymer be a polyethylene glycol polymer and having the secondary site-specific ligand moiety be a peptidyl ligand moiety or carbohydrate ligand moiety.
Secondary ligand binding site differing from primary function
Having the secondary ligand binding site be a site differing from a primary binding site, wherein the secondary ligand binding site has no effect on the primary function of the protein.
Photochemically induced functional-group reactivity
Inducing reactivity of a functional group photochemically.
Linkage architecture preventing peptidyl ligand linkage to the protein
Providing that, in an activated polymer complex, a functional group is directly linked to the polymer to connect the polymer directly to the target group while preventing the peptidyl ligand moiety from linking to the protein.
Peptidyl ligand moiety using synthetic or surrogate amino acids
Having the peptidyl ligand moiety comprise at least one synthetic amino acid or one surrogate amino acid.
Selecting a defined synthetic amino-acid set
Selecting the synthetic amino acid from β-alanine, γ-aminobutyrate, O-methyl-substituted threonine, O-methyl-substituted serine, or O-methyl-substituted tyrosine.
Defined activated polymer complex structural variables
Selecting the activated polymer complex from polymer complex structures defined by variables B (hydrogen, biotinyl, or a fluorescent group), Q (O or S), W and Z (each independently N or CH), X (S, O, NH, or the unspecified P-containing substituent shown in the figure), q (0 or 1), R (hydrogen, aryl, alkyl, or aralkyl), Y (from the chemical substituents shown in the figure), R′ (a protecting group, biotinyl, or a fluorescent group), and n (an integer from 0 to 5).
Overall claim coverage centers on using a site-specific activated polymer complex to achieve covalent modification via a reactive functional group targeted to a secondary ligand binding site, followed by eliminating the ligand moiety, with dependent claims refining photochemical induction, linkage architecture that prevents peptidyl-ligand attachment to the protein, constraints on synthetic/surrogate amino acids in peptidyl ligands, and explicit structural-variable definitions for activated polymer complexes.
Stated Advantages
Enables site-specific covalent bonding of a target group on a protein at a secondary ligand binding site.
Allows mapping of secondary ligand binding sites by coordinating ligand binding with covalent bonding and subsequent ligand elimination.
Eliminates the ligand moiety from the protein-polymer conjugate after covalent bonding of the target group.
Uses a secondary ligand binding site that differs from a primary binding site and has no effect on the primary function of the protein.
Documented Applications
Tandem mapping narrative describing HIV protease inactivation and mapping associated with cysteine/histidine-mediated inhibition using PEGylated protein mapping.
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