Title:  Synthetic bifunctional molecules containing a drug moiety and presenter protein ligand

United States Patent:  6,372,712

Assignee:  The Board of Trustees of the Leland Stanford Jr. University (Palo Alto, CA); The Howard Hughes Medical Institute (Chevy Chase, MD)

Filed:  May 21, 1999

Bifunctional molecules and methods for their use in the production of binary complexes in a host are provided. The bifunctional molecule is a conjugate of a drug moiety and a presenter protein ligand. The molecular weight of the bifunctional molecule is preferably less than about 5000 daltons, and the drug moiety may have a molecular weight of from about 50 to 2000 daltons. The drug moiety and presenter protein ligand may be covalently linked directly or through a linking group. The drug moiety binds to a drug target such as a protein and the presenter protein ligand binds to a presenter protein that is not the drug target such as extracellular or intracellular protein. Presenter proteins include peptidyl prolyl isomerase (FKBP), Heat Shock Protein 90 (Hsp90), steroid hormone receptors, cytoskeletal proteins, albumin and vitamin receptors. When the presenter protein is FKBP, ligands include FK506, rapamycin and cyclosporin A which may have an introduced functional group such as hydroxyl, amino, carboxyl, aldehyde, carbonate, carbamate, azide, thiol or ester for attaching the drug moiety. In the methods of use, an effective amount of the bifunctional molecule is administered to the host. The bifunctional molecule binds to the presenter protein to produce a binary complex such that the drug exhibits at least one of improved affinity, specificity or selectivity as compared to the corresponding free drug. The methods and bifunctional molecules find use in a variety of therapeutic applications.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Bifunctional molecules, synthesis and screening methods, and methods for their use in the production of at least binary complexes in a host are provided. The bifunctional molecules have a drug moiety covalently linked to a presenter protein ligand, either directly or through a linking group. In the subject methods, an effective amount of the bifunctional molecule is administered to the host. Upon administration, the bifunctional molecule binds to the presenter protein to produce the binary complex. The binary complex has an enlarged target binding surface area as compared to the free drug such that at least one of enhanced affinity, specificity or selectivity are observed as compared to the free drug. In a first embodiment in which increased affinity is observed, the binary complex binds to the target to form a tripartite complex characterized by the presence of presenter-target binding interactions as well as drug-target binding interactions. The subject methods and compositions find use in a variety of therapeutic applications. In further describing the subject invention, the bifunctional molecules and methods for their production will be described first, followed by a discussion of applications in which the bifunctional molecules find use.

Before the subject invention is described further, it is to be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.

In this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.

Though not wishing to be bound by any particular theory, the subject invention provides a means for improving at least one of the affinity or specificity or selectivity of a small molecule drug for its desired target by enlarging the target binding surface area of the drug moiety as compared to the free drug. Enhanced affinity, specificity or selectivity of the drug is accomplished by presenting it to its drug target as a binary complex made up of a bifunctional molecule of the drug and presenter protein ligand bound to a presenter protein. Interactions between the presenter protein and the drug target, such as favorable interactions, neutral interactions or repulsive interactions, in combination with interactions between the drug moiety and its target, result in a modulation of the overall binding profile of the drug moiety for its various targets, as compared to a free drug control. As such, by administering a small molecule drug as a bifunctional molecule according to the subject invention, one can achieve improved results as compared to the results obtainable by administration of the small molecule drug by itself. 

Bifunctional Molecule

A critical element of the subject invention is the bifunctional molecule. The bifunctional molecule is a non-naturally occurring or synthetic compound. The bifunctional molecule is further characterized in that the presenter protein ligand and the drug moiety are different, such that the bifunctional molecule may be viewed as a heterodimeric compound produced by the joining of two different moieties. In many embodiments, the presenter protein ligand and the drug moiety are chosen such that the corresponding drug target and presenter protein do not naturally associate with each other to produce a biological effect. In many preferred embodiments, the bifunctional molecules are capable of simultaneously binding two distinct compounds, i.e. a target and a presenter protein, to form a tripartite complex. The bifunctional molecule has a drug moiety bonded to a ligand for a presenter protein, either directly or through a linking group. The molecular weight of the bifunctional molecule is generally at least about 100 D, usually at least about 400 D and more usually at least about 500 D, and may be as great as 2000 D or greater, but usually does not exceed about 5000 D.

The bifunctional molecule is further characterized in that the drug moiety has improved activity as compared to free drug. By improved activity is meant that the drug moiety has a more desirable effect with respect to the condition being treated, as compared to the corresponding free drug from which the drug moiety of the bifunctional molecule is derived. In many embodiments, the bifunctional molecule is characterized by having improved affinity for its target as compared to its corresponding drug, i.e. a control. The magnitude of enhanced affinity and/or specificity will be at least about 2 fold, usually at least about 5 fold and in many embodiments at least 10 fold. In many embodiments, the affinity of the bifunctional molecule for its target will be at least about 10^-4 M, usually at least about 10^-6 M. Additionally and/or alternatively, the bifunctional molecule exhibits improved specificity for its target as compared to a free drug control. Additionally and/or alternatively, the bifunctional molecule exhibits improved selectively for its target as compared to a free drug control.

Bifunctional molecules are generally described by the formula:

Z--L--X

wherein

X is a drug moiety;

L is bond or linking group; and

Z is a ligand for an endogenous presenter protein; with the proviso that X and Z are different.

Drug Moiety: X

The drug moiety X may be any molecule, as well as binding portion or fragment thereof, that is capable of modulating a biological process in a living host, either by itself or in the context of the presenter protein/bifunctional molecule binary complex. Generally, X is a small organic molecule that is capable of binding to the target of interest. As the drug moiety of the bifunctional molecule is a small molecule, it generally has a molecular weight of at least about 50 D, usually at least about 100 D, where the molecular weight may be as high as 500 D or higher, but will usually not exceed about 2000 D.

The drug moiety is capable of interacting with a target in the host into which the bifunctional molecule is administered during practice of the subject methods. The target may be a number of different types of naturally occurring structures, where targets of interest include both intracellular and extracellular targets, where such targets may be proteins, phospholipids, nucleic acids and the like, where proteins are of particular interest. Specific proteinaceous targets of interest include, without limitation, enzymes, e.g. kinases, phosphatases, reductases, cyclooxygenases, proteases and the like, targets comprising domains involved in protein-protein interactions, such as the SH2, SH3, PTB and PDZ domains, structural proteins, e.g. actin, tubulin, etc., membrane receptors, immunoglobulins, e.g. IgE, cell adhesion receptors, such as integrins, etc, ion channels, transmembrane pumps, transcription factors, signaling proteins, and the like.

The drug moiety of the bifunctional compound will include one or more functional groups necessary for structural interaction with the target, e.g. groups necessary for hydrophobic, hydrophilic, electrostatic or even covalent interactions, depending on the particular drug and its intended target. Where the target is a protein, the drug moiety will include functional groups necessary for structural interaction with proteins, such as hydrogen bonding, hydrophobic-hydrophobic interactions, electrostatic interactions, etc., and will typically include at least an amine, amide, sulfhydryl, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. As described in greater detail below, the drug moiety will also comprise a region that may be modified and/or participate in covalent linkage to the other components of the bifunctional molecule, such as the presenter protein ligand or linker, without substantially adversely affecting the moiety's ability to bind to its target.

The drug moieties often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Also of interest as drug moieties are structures found among biomolecules, including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Such compounds may be screened to identify those of interest, where a variety of different screening protocols are known in the art.

The drug moiety of the bifunctional molecule may be derived from a naturally occurring or synthetic compound that may be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including the preparation of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

As such, the drug moiety may be obtained from a library of naturally occurring or synthetic molecules, including a library of compounds produced through combinatorial means, i.e. a compound diversity combinatorial library. When obtained from such libraries, the drug moiety employed will have demonstrated some desirable activity in an appropriate screening assay for the activity. Combinatorial libraries, as well as methods for the production and screening, are known in the art and described in: U.S. Pat. Nos. 5,741,713; 5,734,018; 5,731,423; 5,721,099; 5,708,153; 5,698,673; 5,688,997; 5,688,696; 5,684,711; 5,641,862; 5,639,603; 5,593,853; 5,574,656; 5,571,698; 5,565,324; 5,549,974; 5,545,568; 5,541,061; 5,525,735; 5,463,564; 5,440,016; 5,438,119; 5,223,409, the disclosures of which are herein incorporated by reference.

The drug moiety of the bifunctional molecule may be the whole compound or a binding fragment or portion thereof that retains its affinity and specificity for the target of interest while having a linkage site for covalent bonding to the presenter protein ligand or linker.

Presenter Protein Ligand: Z

Z is a ligand for a presenter protein present in the host into which the bifunctional molecule is to be administered. The presenter protein ligand of the subject bifunctional molecules binds to a specific presenter protein present in the host. The binding interaction between the presenter protein and the presenter protein ligand is non-covalent, such that no covalent bonds are produced between the bifunctional molecule and the presenter protein upon binding of the two entities. The presenter protein ligand is small, where the size of the presenter protein ligand does not exceed about 4950 daltons, usually does not exceed about 4925 daltons and more usually does not exceed about 4900 daltons, where the size of the presenter protein ligand is generally at least about 50 daltons and more usually at least about 100 daltons. The presenter protein ligand, in the context of the bifunctional molecule, has substantially no pharmacological activity at its effective concentration beyond binding to the presenter protein, i.e. it does not directly cause a presenter protein-mediated pharmacological event to occur upon binding at its effective concentration to the presenter protein, where a presenter protein-mediated pharmacological event is a pharmacologically relevant event which is directly modulated by the presenter protein in the absence of the subject bifunctional molecules. As used herein, pharmacological event is an event that is distinct from a biochemical event (e.g. inhibition a prolyl isomerase activity) or a biological event (e.g. inducement of a cell to express new genes).

The presenter protein to which the ligand of the bifunctional molecule binds may be any protein that is present in the host at the time the bifunctional molecule is introduced to the host, i.e. the presenter protein will be endogenous to the host. The presenter protein may or may not have one or more modified residues, e.g. residues that are glycosylated, such that the presenter protein may or may not be a glycoprotein. Furthermore, the presenter protein that is recruited by the bifunctional molecule may or may not be part of a complex or structure of a plurality of biological molecules, e.g. lipids, where such complexes or structures may include lipoproteins, lipid bilayers, and the like. However, in many embodiments, the presenter protein that is recruited by the presenter protein ligand of the bifunctional molecule will be by itself, i.e. will not be part of a larger structure of a plurality of biological molecules. Though the presenter protein may be a protein that is not native to the host but has been introduced at some time prior to introduction of the bifunctional molecule, e.g. through prior administration of the protein or a nucleic acid composition encoding the same, such as through gene therapy, the presenter protein will, in many embodiments, be a protein that is native to and naturally expressed by at least some of the host's cells, i.e. a naturally occurring protein in the host. The presenter protein is a protein that is present in the region of host occupied by the drug target. As such, where the drug target is an intracellular drug target, the presenter protein will be an intracellular protein present in the cell comprising the target, typically expressed in the cell comprising the target, i.e. the presenter protein and target are co-expressed in the same cell. Likewise, where the drug target is an extracellular drug target, the presenter protein will be an extracellular protein that is found in the vicinity of the target.

Although not a requirement in certain embodiments, in many preferred embodiments the presenter protein is one that is present in the host in sufficient quantities such that, upon binding of at least a portion of presenter protein present in the host to the bifunctional molecule, adverse pharmacological effects do not occur. In other words, the presenter protein in these preferred embodiments is one in which its native and desirable biological activity, if any, is not diminished by an unacceptable amount following binding of the portion of the presenter protein population to the bifunctional molecule. The amount of diminished activity of the presenter protein that is acceptable in a given situation is determined with respect to the condition being treated in view of the benefits of treatment versus the reduction of overall presenter protein activity, if any. In certain situations, a large decrease in overall presenter protein activity may be acceptable, e.g. where the presenter protein activity aggravates the condition being treated.

Specific presenter proteins of interest include intracellular and extracellular proteins. Intracellular proteins of interest include: peptidyl-prolyl isomerases, e.g. FKBPs and cyclophilins; ubiquitously expressed molecular chaperones, e.g. Heat Shock Protein 90 (Hsp90); steroid hormone receptors, e.g. estrogen receptors, glucocorticoid receptors, androgen receptors; retinoic acid binding protein, cytoskeletal proteins, such as tubulin and actin; etc.

Of particular interest as intracellular presenter proteins are cis-trans peptidyl-prolyl isomerases which interact with many proteins because of their chaperonin/isomerase activity, e.g. FKBPs and cyclophilins. Peptidyl-prolyl isomerases of interest include FKBPs. A number of different FKBPs are known in the art, and include those described in: Sabatini et al., Mol. Neurobiol. (October 1997) 15:223-239; Marks, Physiol. Rev. (July 1996) 76:631-649; Kay, Biochem J. (March, 1996) 314: 361-385; Braun et al., FASEB J. (January 1995) 9:63-72; Fruman et al, FASEB J. (April 1994) 8:391-400; and Hacker et al., Mol. Microbiol. (November 1993) 10: 445-456. FKBPs of interest include FKBP 12, FKBP 52, FKBP 14.6 (described in U.S. Pat. No. 5,525,523, the disclosure of which is herein incorporated by reference); FKBP 12.6 (described in U.S. Pat. No. 5,457,182 the disclosure of which is herein incorporated by reference); FKBP 13 (described in U.S. Pat. No. 5,498,597, the disclosure of which is herein incorporated by reference); and HCB (described in U.S. Pat. No. 5,196,352 the disclosure of which is herein incorporated by reference); where FKBP 12 and FKBP 52 are of particular interest as intracellular presenter proteins.

Also of specific interest as presenter proteins are cyclophilins. A number of cyclophilins are known in the art and are described in Trandinh et al., FASEB J. (December 1992) 6: 3410-3420; Harding et al., Transplantation (August 1988) 46: 29S-35S. Specific cyclophilins of interest as intracellular presenter proteins include cyclophilin A, B, C, D, E, and the like, where cyclophilin A is of particular interest.

Instead of being an intracellular protein, the endogenous presenter protein may be an extracellular or serum protein. Serum presenter proteins of particular interest are those that are relatively abundant in the serum of the host and meet the above criteria for suitable endogenous presenter proteins. By relatively abundant is meant that the concentration of the serum presenter protein is at least about 1 ng/ml, usually at least about 10 .mu.g/ml and more usually at least about 15 .mu.g/ml. Specific serum proteins of interest as presenter proteins include: albumin, Vitamin A binding proteins and Vitamin D binding proteins, .beta.-2 macroglobulin, with albumin being a particularly preferred presenter protein.

The Z moiety of the subject bifunctional molecules will therefore be chosen in view of the endogenous presenter protein that is to be recruited to produce the at least binary and, in some embodiments, tripartite complex. As such, the Z moiety may be a number of different ligands, depending on the particular endogenous presenter protein to which it is intended to bind. In many preferred embodiments, the Z moiety has an affinity for its presenter protein of at least about 10^-4 M, usually at least about 10^-6 molar and more usually at least about 10^-8 M, where in many embodiments the Z moiety has an affinity for its presenter protein of between about 10^-9 and 10^-12 M. The Z moiety portion of the bifunctional molecule should also be specific for the presenter protein in the context of its binding activity when present in the bifunctional molecule, in that it does not significantly bind or substantially affect non-presenter proteins when it is present in the bifunctional molecule.

Representative ligands capable of serving as the Z moiety of the bifunctional molecule include ligands for intracellular proteins, such as: peptidyl-prolyl isomerase ligands, e.g. FK506, rapamycin, cyclosporin A and the like; Hsp90 ligands, e.g. geldanamycin; steroid hormone receptor ligands, e.g. naturally occurring steroid hormones, such as estrogen, progestin, testosterone, and the like, as well as synthetic derivatives and mimetics thereof, particularly those which bind with high specificity and affinity but do not activate their respective receptors; small molecules that bind to cytoskeletal proteins, e.g. antimitotic agents, such as taxanes, colchicine, colcemid, nocadozole, vinblastine, and vincristine, actin binding agents, such as cytochalasin, latrunculin, phalloidin, and the like.

As mentioned above, the preferred intracellular presenter proteins are members of the peptidyl-prolyl isomerase family, particularly the FKBP and cyclophilin subsets of this family. Where peptidyl-prolyl isomerase presenter proteins are employed, the bifunctional molecule/peptidyl-prolyl isomerase complex will preferably not substantially bind to the natural peptidyl-prolyl isomerase/ligand target calcineurin so as to result in significant immunosuppression. A variety of ligands are known that bind to FKBPs and may be used in the subject invention. The ligands should specifically bind to an FKBP and have an affinity for the FKBP that is between about 10^-6 and 10^-10 M. Of interest are both naturally occurring FKBP ligands, including FK506 and rapamycin. Also of interest are synthetic FKBP ligands, including those described in U.S. Pat. Nos.: 5,665,774; 5,622,970; 5,516,797; 5,614,547; and 5,403,833, the disclosures of which are herein incorporated by reference.

Also of interest are cyclophilin ligands, where such ligands should specifically bind to cyclophilin with an affinity that is between about 10^-6 and 10^-9 M. A variety of ligands that bind to cyclophilins are also known, where such ligands include the naturally occurring cyclosporins, such as cyclosporin A, as well as synthetic derivatives and mimetics thereof, including those described in U.S. Pat. Nos.: 5,401,649; 5,318,901; 5,236,899; 5,227,467; 5,214,130; 5,122,511; 5,116,816; 5,089,390; 5,079,341; 5,017,597; 4,940,719; 4,914,188; 4,885,276; 4,798,823; 4,771,122; 4,703,033; 4,554,351; 4,396,542; 4,289,851; 4,288,431; 4,220,610 and 4,210,581, the disclosures of which are herein incorporated by reference.

Representative ligands for use as the Z moiety in the bifunctional molecule also include ligands that bind to extracellular presenter proteins. Such ligands should specifically bind to their respective presenter protein with an affinity of at least about 10^-4 M. Ligands of interest for use in binding to extracellular presenter proteins include: albumin ligands, such as arachidonate, bilirubin, hemin, aspirin, ibuprofen, para-amino salicylic acid, myristylate, plamitate, linoleate, warfarin etc.; Vitamin A and derivatives thereof, Vitamin D and derivatives thereof, and the like.

Linking Moiety: L

The Z and X moieties of the bifunctional molecule are joined together through linking moiety L, where L may be either a bond or a linking group. Where linking groups are employed, such groups are chosen to provide for covalent attachment of the drug and ligand moieties through the linking group, as well as the desired structural relationship of the bifunctional molecule with respect to its intended presenter protein. Linking groups of interest may vary widely depending on the nature of the drug and ligand moieties. The linking group, when present, should preferably be biologically inert. Appropriate linkers can readily be identified using the affinity, specificity or selectivity assays described supra. A variety of linking groups are known to those of skill in the art and find use in the subject bifunctional molecules. The linker groups should be sufficiently small so as to provide a bifunctional molecule having the overall size characteristics as described above, the size of the linker group, when present, is generally at least about 50 daltons, usually at least about 100 daltons and may be as large as 1000 daltons or larger, but generally will not exceed about 500 daltons and usually will not exceed about 300 daltons. Generally, such linkers will comprise a spacer group terminated at either end with a reactive functionality capable of covalently bonding to the drug or ligand moieties. Spacer groups of interest possibly include aliphatic and unsaturated hydrocarbon chains, spacers containing heteroatoms such as oxygen (ethers such as polyethylene glycol) or nitrogen (polyamines), peptides, carbohydrates, cyclic or acyclic systems that may possibly contain heteroatoms. Spacer groups may also be comprised of ligands that bind to metals such that the presence of a metal ion coordinates two or more ligands to form a complex. Specific spacer elements include: 1,4-diaminohexane, xylylenediamine, terephthalic acid, 3,6-dioxaoctanedioic acid, ethylenediamine-N,N-diacetic acid, 1,1'-ethylenebis(5-oxo-3-pyrrolidinecarboxylic acid), 4,4'-ethylenedipiperidine. Potential reactive functionalities include nucleophilic functional groups (amines, alcohols, thiols, hydrazides), electrophilic functional groups (aldehydes, esters, vinyl ketones, epoxides, isocyanates, maleimides), functional groups capable of cycloaddition reactions, forming disulfide bonds, or binding to metals. Specific examples include primary and secondary amines, hydroxamic acids, N-hydroxysuccinimidyl esters, N-hydroxysuccinimidyl carbonates, oxycarbonylimidazoles, nitrophenylesters, trifluoroethyl esters, glycidyl ethers, vinylsulfones, and maleimides. Specific linker groups that may find use in the subject bifunctional molecules include heterofunctional compounds, such as azidobenzoyl hydrazide, N-[4-(p-azidosalicylamino)butyl]-3'-[2'-pyridyldithio]propionamid), bis-sulfosuccinimidyl suberate, dimethyladipimidate, disuccinimidyltartrate, N- -maleimidobutyryloxysuccinimide ester, N-hydroxy sulfosuccinimidyl-4-azidobenzoate, N-succinimidyl [4-azidophenyl]-1,3'-dithiopropionate, N-succinimidyl [4-iodoacetyl]aminobenzoate, glutaraldehyde, and succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate, 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP), 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester (SMCC), and the like.

Methods of Making Bifunctional Molecules

The bifunctional molecules of the subject invention may be prepared using any convenient methodology. In many embodiments of the subject invention, the invention is used to improve one or more aspects of an identified and at least partially characterized small molecule drug. Generally, a small molecule drug of interest but lacking in some of the desired biological activities, such as affinity, specificity or selectivity, is first identified. The drug may be a previously identified biologically active agent or compound having the desired target binding activity, or one that has been newly discovered using one or more drug discovery techniques. The bifunctional molecule is then generally produced from the drug using a rational or combinatorial approach.

In a rational approach, the bifunctional molecules are constructed from their individual components, e.g. ligand, linker and drug. The components can be covalently bonded to one another through functional groups, as is known in the art, where such functional groups may be present on the components or introduced onto the components using one or more steps, e.g. oxidation reactions, reduction reactions, cleavage reactions and the like. Functional groups that may be used in covalently bonding the components together to produce the bifunctional molecule include: hydroxy, sulfhydryl, amino, and the like. The particular portion of the different components that are modified to provide for covalent linkage will be chosen so as not to substantially adversely interfere with that components desired binding activity, e.g. for the drug moiety, a region that does not affect the target binding activity will be modified, such that a sufficient amount of the desired drug activity is preserved. Where necessary and/or desired, certain moieties on the components may be protected using blocking groups, as is known in the art, see, e.g. Green & Wuts, Protective Groups in Organic Synthesis (John Wiley & Sons) (1991).

The above component approach to production of the bifunctional molecule is best suited for situations where the crystal structures of the presenter protein, ligand, drug and target are known, such that molecular modeling can be used to determine the optimal linker size, if any, to be employed to join the different components.

Alternatively, the bifunctional molecule can be produced using combinatorial methods to produce large libraries of potential bifunctional molecules which may then be screened for identification of a bifunctional molecule with the desired binding affinity and/or specificity. Methods for producing and screening combinatorial libraries of molecules include: U.S. Pat. Nos. 5,741,713; 5,734,018; 5,731,423; 5,721,099; 5,708,153; 5,698,673; 5,688,997; 5,688,696; 5,684,711; 5,641,862; 5,639,603; 5,593,853; 5,574,656; 5,571,698; 5,565,324; 5,549,974; 5,545,568; 5,541,061; 5,525,735; 5,463,564; 5,440,016; 5,438,119; 5,223,409, the disclosures of which are herein incorporated by reference.

Alternatively, the bifunctional molecule may be produced using medicinal chemistry and known structure-activity relationships for the presenter protein ligand and the drug. In particular, this approach will provide insight as to where to join the two moieties to the linker.

Claim 1 of 15 Claims

What is claimed is:

1. A synthetic bifunctional molecule of less than about 5000 daltons consisting of a drug moiety and a presenter protein ligand joined thereto, wherein said drug moiety binds to a drug target and said presenter protein ligand binds to a presenter protein that is not said drug target and said drug moiety exhibits at least one of enhanced affinity, specificity or selectivity for its target as compared to a corresponding free drug control.

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