Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1998-06-26
2002-01-01
Ponnaluri, Padmashri (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S005000, C435S007100, C435S007500, C435S091500, C435S091500, C435S091500, C435S091500, C436S173000
Reexamination Certificate
active
06335155
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to novel molecular methods useful for quickly and unambiguously identifying small organic molecule ligands for binding to specific sites on target biological molecules. Small organic molecule ligands identified according to the methods of the present invention find use, for example, as novel therapeutic drug lead compounds, enzyme inhibitors, labeling compounds, diagnostic reagents, affinity reagents for protein purification, and the like.
BACKGROUND OF THE INVENTION
The primary task in the initial phase of generating novel biological effector molecules is to identify and characterize one or more tightly binding ligand(s) for a given biological target molecule. In this regard, many molecular techniques have been developed and are currently being employed for identifying novel ligands that bind to specific sites on biomolecular targets, such as proteins, nucleic acids, carbohydrates, nucleoproteins, glycoproteins and glycolipids. Many of these techniques exploit the inherent advantages of molecular diversity by employing combinatorial libraries of potential ligand compounds in an effort to speed up the identification of functional ligands. For example, combinatorial synthesis of both oligomeric and non-oligomeric libraries of diverse compounds combined with high-throughput screening assays has already provided a useful format for the identification of new lead compounds for binding to chosen molecular targets.
While combinatorial approaches for identifying biological effector molecules have proven useful in certain applications, these approaches also have some significant disadvantages. For example, current synthetic technology is limited in that it allows one to synthesize only a relatively small fraction of the total number of possible library members for any given molecule type. As such, even when appropriate high-throughput screening assays are available for screening a library, only a small fraction of the total number of possible members of any molecule type will be represented in the library and, therefore, screened for the ability to bind to the chosen target. Thus, combinatorial approaches often do not allow one to identify the “best” ligand for a target molecule of interest.
Additionally, even when appropriate screening assays are available, in many cases techniques which allow identification of the actual library member(s) which bind most effectively to the target are not available or provide ambiguous results, making the actual identification and characterization of functional ligand molecules difficult or impossible. Furthermore, many approaches currently employed to identify novel ligands are dependent upon only a single specific chemistry, thereby limiting the usefulness of such approaches to only a narrow range of applications. Finally, many of the approaches currently employed are expensive and extremely time-consuming. Thus, there is a significant interest in developing new methods which allow rapid, efficient and unambiguous identification of small organic molecule ligands for selected biomolecular targets. It is also desired that such techniques are adaptable to a variety of different chemistries, thereby being useful for a wide range of applications.
Schiff base adduct formation involves the reaction of an available aldehyde or ketone functionality with a primary amine to form an imine-bonded complex. While the Schiff base adduct is relatively unstable, numerous groups have employed aldehyde or ketone compounds for bonding to primary amine functionalities on proteins of interest for a variety of purposes (see, e.g., Pollack et al.,
Science
242:1038-1040 (1988), Abraham et al.,
Biochemistry
34:15006-15020 (1995) and Boyiri et al.,
Biochemistry
34:15021-15036 (1995)). We herein describe novel techniques useful for rapidly and efficiently identifying organic molecule ligands that bind to specific sites on biomolecular targets, wherein those techniques are adaptable to a variety of different chemistries, preferably Schiff base adduct formation between a target polypeptide and one or more members of a library of potential organic molecule ligands. These methods allow one to unambiguously identify and characterize the organic molecule ligand that binds most efficiently to the chosen target. Additionally, the herein described methods are quick, easy to perform and inexpensive as compared to other currently employed methods.
SUMMARY OF THE INVENTION
Applicants herein describe a molecular approach for rapidly and efficiently identifying small organic molecule ligands that are capable of interacting with and binding to specific sites on biological target molecules, wherein ligand compounds identified by the subject methods may find use, for example, as new small molecule drug leads, enzyme inhibitors, labeling compounds, diagnostic reagents, affinity reagents for protein purification, and the like. The herein described approaches allow one to quickly screen a library of small organic compounds to unambiguously identify those that have affinity for a particular site on a biomolecular target. Those exhibiting affinity for interacting with a particular site are capable of forming a covalent bond with a chemically reactive group at that site, whereby small organic compounds capable of covalent bond formation may be readily identified and characterized. Such methods may be performed quickly, easily and inexpensively and provide for unambiguous results. The small organic molecule ligands identified by the methods described herein may themselves be employed for numerous applications, or may be coupled together in a variety of different combinations using one or more linker elements to provide novel binding molecules.
With regard to the above, one embodiment of the present invention is directed to a method for identifying an organic molecule ligand that binds to a site of interest on a biological target molecule, wherein the method comprises:
(a) obtaining a biological target molecule that comprises or has been modified to comprise a chemically reactive group, wherein the site of interest on the target molecule comprises the chemically reactive group;
(b) combining the target molecule with one or more members of a library of organic compounds that are capable of covalently bonding to the chemically reactive group, wherein at least one member of the library binds to the site of interest to form a covalent bond with the chemically reactive group to form a target molecule/organic compound conjugate; and
(c) identifying the organic compound that forms a covalent bond with the chemically reactive group.
In particular embodiments, the biological target molecule is a polypeptide, a nucleic acid, a carbohydrate, a nucleoprotein, a glycopeptide or a glycolipid, preferably a polypeptide, which may be, for example, an enzyme, a hormone, a transcription factor, a receptor, a ligand for a receptor, a growth factor, an immunoglobulin, a steroid receptor, a nuclear protein, a signal transduction component, an allosteric enzyme regulator, and the like. The target molecule may comprise the chemically reactive group without prior modification of the target molecule or may be modified to comprise the chemically reactive group, for example, when a compound comprising the chemically reactive group is bound to the target molecule.
Other embodiments of the above described methods employ libraries of organic compounds which comprise aldehydes, ketones, oximes, hydrazones, semicarbazones, carbazides, primary amines, secondary amines, tertiary amines, N-substituted hydrazines, hydrazides, alcohols, ethers, thiols, thioethers, thioesters, disulfides, carboxylic acids, esters, amides, ureas, carbamates, carbonates, ketals, thioketals, acetals, thioacetals, aryl halides, aryl sulfonates, alkyl halides, alkyl sulfonates, aromatic compounds, heterocyclic compounds, anilines, alkenes, alkynes, diols, amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolines, enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonyl chlorid
Braisted Andrew C.
Erlanson Dan
Wells Jim
Ponnaluri Padmashri
Sunesis Pharmaceuticals, Inc.
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