Affinity labeling libraries and applications thereof

Details Affinity labeling libraries and applications thereof Affinity labeling libraries and applications thereof

1996-02-07

2001-08-21

Ponnaluri, Padmashri (Department: 1627)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C530S300000, C530S344000, C530S380000, C436S501000, C436S536000, C435S091500, C435S091500, C435S091500, C435S091500, C435S091500

Reexamination Certificate

active

06277583

ABSTRACT:

INTRODUCTION
1.Technical Field
The field of this invention is the specific covalent affinity labeling of target macromolecules, affinity labeling libraries and various applications thereof.
2. BACKGROUND
It is frequently desirable to have the ability to covalently link a particular compound of interest to a specific target, e.g. one of a mixture of components or a site on a macromolecule. For example, covalent linkage of a compound to a specific target site on a catalytic enzyme can result in permanent inhibition of the catalytic activity of that enzyme.
Furthermore, covalent linkage of a compound to a specific target site on a biological receptor can result in either agonism or antagonism at that receptor site. Moreover, covalent linkage of a biologically active compound to a specific target macromolecule present in the human vasculature can result in an effective increase in the half-life of that biologically active molecule. Having the ability to specifically and covalently link a compound of interest to a macromolecular target or target site, therefore, can have profound implications for drug design and pharmaceutical therapy in humans.
Except for molecules that exhibit very high binding affinities, non-covalent interactions between two molecules are generally in equilibrium, where a substantial proportion of the molecules remain separated. Therefore, if one wishes to have stable interactions between a molecule and a target site, one usually resorts to covalent bonding.
Non-covalent interactions between two complementary biological molecules, however, are extremely well known in the art. For example, a double-stranded nucleic acid may be constructed from non-covalent interactions between two complementary single-stranded nucleic acids. The two single-stranded nucleic acids possess a specific affinity for one another because of the complementarity of their respective sequences. In addition, a number of biological proteins specifically and non-covalently interact with complementary sites on other macromolecules. These specific non-covalent interactions include such things as receptor/protein interactions and immunoglobulin/antigen interactions.
Both non-covalent and covalent interactions provide alternative means for attaching labels to macromolecular target sites. The hallmark of an affinity label molecule, however, is that it labels a macromolecular target by using both of the above described types of molecular interaction. For example, an affinity label not only possesses a certain degree of specific complementarity with a target macromolecule, but it also forms a stable covalent bond with that target macromolecule. As a result, affinity labels can selectively attach to a macromolecular target or target site in a complex mixture. Affinity labels also generally exhibit a greater degree of preferential binding to their target macromolecule than do other molecules bearing a chemically reactive group but which lack specific complementarity with the target.
Affinity labels which have both a specific complementarity to a macromolecular target and the ability to covalently bind to that target are presently known in the art and have previously been constructed by attaching potentially reactive electrophilic groups to the binding determinants of an enzyme substrate or a ligand for a specific biological receptor. (Methods of Enzymology, Vol. XL VI, 1977, eds., Jakoby, W. B. and Wilchek, M., Academic Press, New York). For example, affinity labeling of enzymes has proven to be successful because enzymes often use a wide assortment of nucleophilic side chains to catalyze their reactions. This provides the opportunity to attach potentially reactive electrophilic groups to enzyme substrate analogs so that binding of those substrate analogs by the enzyme brings those potentially reactive electrophilic groups into close spatial proximity to the nucleophilic side chains of the enzyme. Because the potentially reactive electrophilic groups of the substrate analog are brought into close spatial proximity to the nucleophilic side chains of the enzyme, covalent bonds between these reactive groups may readily form. Thus, enzyme substrate analogs possessing potentially reactive electrophilic groups have proven to be prime candidates for the affinity labeling of enzymes.
Affinity labeling of biological receptors and other non-catalytic proteins, however, has proven to be much less successful, but not necessarily because of difficulties related to identifying a tight binding complementary group. For example, medicinal chemists have been able to produce numerous antagonist molecules which exhibit high specific affinities for receptor targets. However, since biological receptors are not catalytic, it is difficult to know how potentially reactive groups on the receptor molecule and on the affinity label are spatially situated relative to one another without specific structural information. As such, forming covalent bonds between affinity label molecules and non-catalytic proteins has proven to be rather difficult.
Thus, one of the difficulties encountered in the affinity labeling of macromolecular targets is that it is often necessary to possess structural information about the macromolecular target and the location of its potentially reactive sites. A general approach for affinity labeling of macromolecules, therefore, regardless of whether those macromolecules possess catalytic activity and without possessing structural information about those macromolecules, would be invaluable because it would have numerous applications which arise from the ability to specifically link a compound of interest to a macromolecular target or target site in a complex mixture.
Having the ability to affinity label macromolecular targets or specific target sites without requiring that the target be catalytic or without possessing detailed structural information about the target gives rise to numerous beneficial applications. These numerous applications include new opportunities for enzyme inhibition, antagonism and agonism of biological receptors, attaching biologically active compounds to specific target sites in vitro and/or in vivo, e.g., for the purpose of increasing the half-life of those biologically active compounds, labeling macromolecules for premature destruction, directing biologically active compounds to specific sites in vivo and exploiting numerous in vivo sites that have been hitherto difficult to target, including tumorigenic sites. These many applications have profound implications for drug design and discovery, including the generation of specific linkages to specific sites on various living cells and tissues for therapeutic and prophylactic purposes. There is, therefore, substantial interest in providing for improved methods of specifically affinity labeling various macromolecular targets and target sites of interest.
SUMMARY OF THE INVENTION
Novel methods for preferentially producing target protein conjugates between oligomeric affinity label molecules and target proteins are provided. These methods represent a complete system for both producing and identifying affinity label molecules which preferentially bind to a macromolecular target or target site and conjugating those preferentially binding affinity label molecules to a target macromolecule, usually in a complex mixture of macromolecules. The methods comprise combining a mixture of proteins comprising the target protein with an affinity label conjugate comprising an oligomer and a biologically active compound wherein the affinity label conjugate has an activated functional group capable of covalently bonding to a functionality on the target protein. The affinity label conjugate is produced by preparing an oligomeric combinatorial library comprising an activatable functional group and activating the functional group of the members of the combinatorial library. The activated affinity label conjugate members of the combinatorial library are combined with the target protein, selecting one or more members of the combinatorial library which preferenti

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