BIFUNCTIONAL MOLECULES AND THEIR USE IN THE DISRUPTION OF PROTEIN-PROTEIN INTERACTIONS

Abstract: Bifunctional inhibitor molecules and methods for their use in the inhibition of protein-protein interactions are provided. The subject bifunctional inhibitor molecules are conjugates of a target protein ligand and a blocking protein ligand, where these two moieties are optionally joined by a linking group. In the subject methods, an effective amount of the bifunctional inhibitor molecule is administered to a host in which the inhibition of a protein-protein interaction is desired. The bifunctional inhibitor molecule simultaneously binds to its corresponding target and blocking proteins to produce a tripartite complex that inhibits the target protein-protein interaction. The subject methods and compositions find use in a variety of applications, including therapeutic applications.

Description: BIFUNCTIONAL MOLECULES AND THEIR USE IN THE DISRUPTION OF PROTEIN-PROTEIN INTERACTIONS ACKNOWLEDGMENT This invention was made with United States Government support under Grant No CA39612 awarded by National Institutes of Health The United States Government has certain rights in this invention CROSS REFERENCE TO RELATED APPLICATIONS Pursuant to 35 U S C § 119 (e), this application claims priority to the filing date of the United States Provisional Patent Application Serial No 60/166,675 filed November 19, 1999, the disclosure of which is herein incorporated by reference INTRODUCTION Technical Field The field of this invention is pharmacology Background of the Invention The inhibition or activation of biological processes with drugs that act as antagonists or agonists is the basis of the chemotherapy of diseases For a small molecule to interact with high afiBnity and specificity with a given protein target requires that the molecule is able to establish a sufficient number of molecular interactions which will provide the required free energy of binding If the active site of a protein is a deep pocket, small molecule drugs can take advantage of interactions that can be established on all sides of the molecule However, if the active site of a protein target is relatively flat, the contacts a small molecule can establish may be limited to one face As a result, the affinity of the small molecule for the target may not be very high The active site or the target site of many potential drug targets is relatively flat Usually, these sites are not catalytically active but instead they are used to bind to other proteins The interface of such protein-protein interactions can be rather large and may involve contacts between 20 or 30 amino acids on both proteins. Due to the flat surface small molecules often cannot establish the number of contacts required for a high affinity binding event that can compete with the binding of the large protein ligand to the target. It is considered an intractable problem to develop small molecules that can disrupt such protein-protein interactions. As such, the development of a technique that would allow the disruption of protein-protein interactions, particularly through use of a small molecule therapeutic agent, is of particular interest. Relevant Literature Patent publications of interest include: WO 91/01743; WO 94/18317; WO 95/02684; WO 95/10302; WO 96/06111; WO 96/12796; WO 96/13613; WO 97/25074; WO 97/29372; WO 98/11437; WO 98/47916; U.S. Patent No. 5,830,462; U.S. Patent No. 5,843,440; and U.S. Patent No. 5,871,753. References of interest include: Briesewitz et al., Proc. Nat'l Acad. Sci. USA (March 1999) 96: 1953-1958; Clardy, Proc. Nat'l Acad. Sci. USA (March 1999) 1826-1827; Crabtree & Schreiber, Elsevier Trends Journal (November 1996) 418-422; Spencer et al., Curr. Biol. (July 1996) 6:839-847; Spencer et al., Science (1993) 262: 1019; Chakraborty et al., Chem & Biol. (March 1995) 2:157-161; Ho et al., Nature (1996) 382: 822; Riviera et al., Nature Medicine (1996) 2: 1028; Klemm et al., Current Biology (1997) 7: 638; Belshaw et al., Proc. Nat'l. Acad. Sci. USA (1996) 93: 4604; Livnah et al., Science (1996) 273: 464; Johnson et al., Chemistry and Biology, (1997) 4: 939; Garboczi et al., Nature (1996) 384: 134; Kissenger et al., Nature (1995) 378:641; Griffith et al., Cell (1995) 82: 507; Choi et al., Science (1996) 273:239. Also of interest are Kramer et al., J. Biol. Chem (1992) 267:18598-18604; and Varshavsky, Proc. Nat'l Acad. Sci. USA (March 1998) 95: 2094-2099; Varshavsky, Proc. Nat'l Acad. Sci. USA (April 1995) 92:3663-3667; and Mu et al., Biochem. Biophys. Res. Comrn. (1999)255:75-79. SUMMARY OF THE INVENTION Bifunctional inhibitor molecules and methods for their use in the inhibition of protein-protein interactions are provided. The subject bifunctional inhibitor molecules are conjugates of a target protein ligand and a blocking protein ligand, where these two moieties are optionally joined by a linking group. In the subject methods, an effective amount of the bifunctional inhibitor molecule is administered to a host in which the inhibition of a protein-protein interaction is desired. The bifunctional inhibitor molecule simultaneously binds to its corresponding target and blocking proteins to produce a tripartite complex that inhibits the target protein-protein interaction between the target protein and a second binding protein The subject methods and compositions find use in a variety of applications, including therapeutic applications BRIEF DESCRIPTION OF THE FIGURES Fig 1 provides a schematic representation of how the subject bifunctional molecules inhibit protein-protein interactions in a manner analogous to the way antibodies block such interactions Fig 2 provides a representation of how NFAT translocates into the nucleus after activation of calcineurin with lonomycin Fig 3 provides a graphical representation of assay results which demonstrate that binding of FRB to FKBP-NFAT with rapamycin abolishes translocation into the nucleus Fig 4 provides a schematic representation of how steπc blockage prevents access of regulatory proteins to NFAT DESCRIPTION OF THE SPECIFIC EMBODIMENTS Bifunctional inhibitor molecules and methods for their use in the inhibition of protein-protein interactions between a target protein and a second binding protein are provided The subject bifunctional inhibitor molecules are conjugates of a target protein ligand and a blocking protein ligand, where these two moieties are optionally joined by a linking group In the subject methods, an effective amount of the bifunctional inhibitor molecule is administered to a host in which the inhibition of a protein-protein interaction is desired The bifunctional inhibitor molecule simultaneously binds to its corresponding target and blocking proteins to produce a tripartite complex that inhibits the target protein-protein interaction The subject methods and compositions find use in a varietv of applications, including therapeutic applications 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. BIFUNCTIONAL MOLECULE The bifunctional inhibitor molecule of the present invention is a non-naturally occurring or synthetic compound that includes a target protein ligand and blocking protein ligand, where these two moieties are optionally joined by a linking group. The bifunctional inhibitor molecule is further characterized in that the target protein ligand and the blocking protein ligand 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 blocking protein ligand and the target protein ligand are chosen such that the corresponding target protein and blocking protein do not naturally associate with each other to produce a biological effect. The bifunctional inhibitor molecules are also capable of simultaneously binding their corresponding target and blocking proteins to form a tripartite complex, where the tripartite complex inhibits the binding of the target protein to a second binding protein. See Fig. 1. In forming the tripartite complex, the target protein ligand may bind to a site of the target protein that is the same as the site that is bound by the second binding protein. Alternatively, the target protein ligand may bind to a site of the target protein that is different from the site that is bound by the second binding protein, i.e. the second binding protein binding site of the target protein. An important feature of the subject molecules is that they are small. As such, the molecular weight of the subject bifunctional inhibitor molecules 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. Bifunctional molecules are generally described by the formula: Z-L-X wherein X is a target protein ligand; L is bond or linking group; and Z is a blocking protein ligand; with the proviso that X and Z are different. Target Protein Ligand: X The target protein ligand X may be any molecule, as well as a binding portion or fragment thereof, that is capable of specifically binding to the target protein of the protein pair of the protein- protein interaction whose inhibition is desired. Generally, X is a small organic molecule that is capable of binding to the target of interest As X has a low molecular weight, 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 target protein ligand X is capable of interacting with a target protein in the host into which the bifunctional inhibitor molecule is administered during practice of the subject methods The target protein may be a number of different types of naturally occurring proteins, where targets of interest include both intracellular and extracellular targets proteins As mentioned above, the target protein is a first member of the pair of proteins whose interaction is to be inhibited in the subject methods 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, lmmunoglobulins, e g IgE, cell adhesion receptors, such as integrins, etc, ion channels, transmembrane pumps, transcription factors, signaling proteins, and the like The X moiety of the subject bifunctional inhibitor molecules are therefore chosen in view of the target protein of the target protein-protein interaction or binding event As such, the X moiety may be a number of different ligands, depending on the particular endogenous blocking protein to which it is intended to bind In many preferred embodiments, the X moiety has an affinity for its target 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 X moiety has an affinity for its target protein of between about 10 " and 10 " M In certain embodiments, the X moiety portion of the bifunctional inhibitor molecule is also specific for the target protein in the context of its binding activity when present in the bifunctional inhibitor molecule, in that it does not significantly bind or substantially affect non-target proteins when it is present m the bifunctional inhibitor molecule The target protein ligand of the bifunctional inhibitor compounds includes 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 target protein ligand and its intended target Specifically, the target protein ligand includes 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 target protein ligand will also comprise a region that may be modified and/or participate in covalent linkage to the other components of the bifunctional inhibitor molecule, such as the blocking protein ligand or linker, without substantially adversely affecting the target protein ligand's ability to bind to its target The target protein ligands often compπse 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 target protein ligands are structures found among biomolecules, including peptides, saccharides, fatty acids, steroids, puπnes, pyπmidines, derivatives, structural analogs or combinations thereof Such compounds may be screened to identify those of interest, where a vaπety of different screening protocols are known in the art The target protein ligand of the bifunctional molecule may be derived from a naturally occurring or synthetic compound that may be obtained from a wide vaπety of sources, including libraπes 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 ohgonucleo tides and o gopeptides 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 libraπes and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraπes Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, estenfication, amidification, etc to produce structural analogs As such, the target protein ligand may be obtained from a library of naturally occurring or synthetic molecules, including a library of compounds produced through combinatoπal means, l e a compound diversity combinatorial library When obtained from such libraπes, the target protein ligand employed will have demonstrated an affinity for its protein target in an appropπate screening assay for the activity Combinatorial libraπes, as well as methods for the production and screening, are known in the art and described in 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 Blocking Protein Ligand Z Z is a ligand for a blocking protein present in the host into which the bifunctional inhibitor molecule is to be administered The blocking protein ligand of the subject bifunctional inhibitor molecules binds to a specific blocking protein present in the host In many embodiments, the binding interaction between the blocking protein and the blocking protein ligand is non-covalent, such that no covalent bonds are produced between the bifunctional molecule and the blocking protein upon binding of the two entities The blocking protein ligand (Z) has a low molecular weight - 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 In certain embodiments, the blocking protein ligand, in the context of the bifunctional molecule, has substantially no pharmacological activity at its effective concentration beyond binding to the blockmg protein, l e it does not directly cause a blocking protein-mediated pharmacological event to occur upon binding at its effective concentration to the blocking protein, where a blocking protein- mediated pharmacological event is a pharmacologically relevant event which is directly modulated by the blocking protein in the absence of the subject bifunctional molecules By "pharmacologic activity" is meant an activity that modulates or alters a biological process so as to result in a phenotypic change, e g cell death, cell proliferation etc 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) In other certain embodiments, the blocking protein ligand may have some pharmacological activity, where this pharmacological activity does not adversely effect the host to the extent that the therapy in which the bifunctional molecule is employed places the host in a worst condition than prior to the therapy In other words, pharmacological activity in the blocking protein ligand may be tolerated in these embodiments to the extent that any consequences of such activity, if any, are outweighed by the benefits provided by the bifunctional molecule The blocking protein to which the ligand of the bifunctional inhibitor molecule binds may be any protein that is present in the host at the time the bifunctional molecule is introduced to the host, l e the blocking protein is endogenous to the host The blocking protein may or may not have one or more modified residues, e g residues that are glycosylated, such that the blocking protein may or may not be a glycoprotem Furthermore, the blocking 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 hpids, where such complexes or structures may include hpoproteins, hpid bilayers, and the like However, in many embodiments, the blocking protein that is recruited by the blocking protein ligand of the bifunctional molecule will be by itself, l e it will not be part of a larger structure of a plurality of biological molecules Though the blocking 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 pπor administration of the protein or a nucleic acid composition encoding the same, such as through gene therapy, the blocking 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 blocking protein is a protein that is present in the region of host occupied by the target protein. As such, where the target protein is an intracellular protein, the blocking protein will be an intracellular protein present in the cell comprising the target, typically expressed in the cell comprising the target, i.e. the blocking protein and the target protein are co-expressed in the same cell. Likewise, where the target protein is an extracellular protein, the blocking 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 blocking protein is one that is present in the host in sufficient quantities such that, upon binding of at least a portion of blocking protein present in the host to the bifunctional molecule, adverse pharmacological effects do not occur. In other words, the blocking 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 blocking protein population to the bifunctional molecule. The amount of diminished activity of the blocking 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 blocking protein activity, if any. In certain situations, a large decrease in overall blocking protein activity may be acceptable, e.g. where the blocking protein activity aggravates the condition being treated. The size of the blocking protein should be sufficient to sterically inhibit the target protein- protein interaction between the target protein and the second binding protein when a tripartite complex is produced between the target protein, the bifunctional inhibitor molecule and the blocking protein. In many embodiments, the blocking protein is at least about 5 to 10 kD, and may be as high as 1000 to 2000 kD or higher, where the upper limit for the blocking protein is about 300 kD in certain embodiments. Specific blocking 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 blocking 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. (Oct. 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 mclude FKBP 12, FKBP 52, FKBP 14 6 (described m U S Patent No 5,525,523, the disclosure of which is herein incorporated by reference). FKBP 12 6 (described m U S Patent No 5,457,182 the disclosure of which is herein mcorporated by reference). FKBP 13 (described m U S Patent No 5,498,597, the disclosure of which is herein incorporated by reference), and HCB (described m U S Patent No 5,196,352 the disclosure of which is herein incorporated by reference), where FKBP 12 and FKBP 52 are of particular interest in certain embodiments as intracellular blocking protems Also of specific interest m certain embodiments as blocking protems are cyclophilins A number of cyclophilins are known in the art and are described m Trandinh et al , FASEB J (December 1992) 6 3410-3420, Harding et al . Transplantation (August 1988) 46 29S-35S Specific cyclophilins of mterest as intracellular blocking protems mclude cyclophilm A, B, C, D, E, and the like, where cyclophilm A is of particular mterest Instead of being an intracellular protein, the endogenous blocking protem may be an extracellular or serum protem Serum blocking protems of particular interest are those that are relatively abundant m the serum of the host and meet the above criteria for suitable endogenous blocking protems By relatively abundant is meant that the concentration of the serum blocking protem is at least about 1 ng/ml, usually at least about 10 μg/ml and more usually at least about 15 μg/ml Specific serum protems of interest as blocking protems mclude albumin, Vitamin A bmding protems and Vitamin D bmdmg proteins, β-2 macroglobulm, with albumin being a particularly preferred blocking protem The Z moiety of the subject bifunctional inhibitor molecules will therefore be chosen m view of the endogenous blocking protem that is to be recruited to produce the tripartite complex that inhibits the target protein-protem mteraction or bmdmg event As such, the Z moiety may be a number of different ligands. dependmg on the particular endogenous blocking protem to which it is mtended to bind In many preferred embodiments, the Z moiety has an affinity for its blocking protem of at least about 10 "4 M. usually at least about 10 6 molar and more usually at least about 10 M, where m many embodiments the Z moiety has an affinity for its blocking protem of between about 10 "9 and 10 "12 M The Z moiety portion of the bifunctional molecule should also be specific for the blocking protem m the context of its bmdmg activity when present in the bifunctional molecule, m that it does not significantly bmd or substantially affect non-blocking protems when it is present m the bifunctional molecule Representative ligands capable of serving as the Z moiety of the bifunctional molecule mclude ligands for intracellular protems. 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. anti itotic 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 prefeπed intracellular blocking proteins in many embodiments of the subject invention are members of the peptidyl-prolyl isomerase family, particularly the FKBP and cyclophilin subsets of this family. Where peptidyl-prolyl isomerase blocking 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 and 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. Patent 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 as e U as synthetic derivatives and mimetics thereof, including those described in U.S. Patent 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; 4,220,61 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 blocking proteins. Such ligands should specifically bind to their respective blocking protein with an affinity of at least about 10 "4 M. Ligands of interest for use in binding to extracellular blocking 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 jomed together through linking moiety L, where L may be either a bond or a linking group Where hnkmg groups are employed, such groups are chosen to provide for covalent attachment of the two ligand moieties through the linking group, as well as the desired structural relationship of the bifunctional molecule with respect to its mtended blocking protem Linking groups of mterest may vary widely dependmg on the nature of the target and blockmg ligand moieties The linking group, when present, should preferably be biologically inert Appropπate linkers can be readily identified by those of skill in the art using screening assays which evaluate the ability of the candidate bifunctional inhibitor to form a tripartite complex that inhibits the target protem-protem mteraction or bmdmg event A vaπety of hnkmg groups are known to those of skill m 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 descπbed above, the size of the lmker 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 lmkers will comprise a spacer group terminated at either end with a reactive functionality capable of covalently bondmg to the target protem ligand or blocking protem ligand moieties Spacer groups of interest possibly mclude 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 bmd to metals such that the presence of a metal ion coordmates two or more ligands to form a complex Specific spacer elements mclude 1,4-dιamιnohexane, xylylenediamme. terephthalic acid, 3,6-dιoxaoctanedιoιc acid, ethylenediamine-N.N-diacetic acid, l,l '-ethylenebιs(5-oxo-3-pyrrolιdmecarboxylιc acid), 4,4'- ethylenedipipeπdine Potential reactive functionalities mclude nucleophihc functional groups (amines, alcohols, thiols, hydrazides), electrophilic functional groups (aldehydes, esters, vmyl ketones, epoxides. lsocyanates, maleimides), functional groups capable of cycloaddition reactions, forming disulfide bonds, or bmdmg to metals Specific examples include primary and secondary amines, hydroxamic acids, N-hydroxysuccmimidyl esters, N-hydroxysuccinimidyl carbonates, oxycarbonyhmidazoles, mtrophenylesters, trifluoroethyl esters, glycidyl ethers, vmylsulfones, and maleimides Specific linker groups that may find use in the subject bifunctional molecules mclude heterofunctional compounds, such as azidobenzoyl hydrazide, N-[4-(p-azιdosalιcylammo)butyl]- 3'-[2'-pyπdyldιthιo]propιonamιd), bis-sulfosuccmimidyl suberate, dimethyladipimidate, disuccinimidyltartrate, N- -maleimidobutyryloxysuccmimide ester, N-hydroxy sulfosuccmιmιdyl-4- azidobenzoate, N-succinimidyl [4-azιdophenyl]-l,3'-dιthιopropιonate, N-succinimidyl [4- ιodoacetyl]amιnobenzoate, glutaraldehyde, and succinimidyl 4-[N-maleιmιdomethyl]cyclohexane-l- carboxylate, 3-(2-pyπdyldιthιo)propιonιc acid N-hydroxysuccmimide ester (SPDP), 4-(N- maleιmιdomethyl)-cyclohexane-l-carboxylιc acid N-hydroxysuccmimide ester (SMCC), and the like METHODS OF MAKING BIFUNCTIONAL MOLECULES The bifunctional molecules of the subject invention may be prepared usmg any convenient methodology As mdicated above, the bifunctional inhibitor molecule is one that inhibits a target protem-protem mteraction or bmdmg event between a target protem and a second bmdmg protein Generally, a ligand that specifically bmds to the target protem is first identified The ligand may be a previously identified molecule or compound having the desired target bmdmg activity, or one that has been newly discovered usmg one or more compound discovery techniques The bifunctional inhibitor molecule is then generally produced from the target protem ligand usmg a rational or combinatorial approach In a rational approach, the bifunctional inhibitor molecules are constructed from their individual components, e g target hgand, protem ligand and optional lmker The components can be covalently bonded to one another through functional groups, as is known m the art, where such functional groups may be present on the components or introduced onto the components usmg one or more steps, e g oxidation reactions, reduction reactions, cleavage reactions and the like Functional groups that may be used m covalently bondmg the components together to produce the bifunctional molecule mclude hydroxy, sulfhydryl, ammo, and the like The particular portion of the different components that are modified to provide for covalent lmkage will be chosen so as not to substantially adversely mterfere with that components desired bmdmg activity, e g for the target protem ligand, a region that does not affect the target bmdmg activity will be modified, such that a sufficient amount of the desired target protem bmdmg activity is preserved Where necessary and/or desired, certain moieties on the components may be protected usmg blockmg groups, as is known m the art, see, e g Green & Wuts, Protective Groups m Organic Synthesis (John Wiley & Sons) (1991) The above component approach to production of the bifunctional inhibitor molecule is best suited for situations where the crystal structures of the blockmg protem, blockmg protem ligand, target protem ligand and target protem are known, such that molecular modeling can be used to determine the optimal linker size, if any, to be employed to jom the different components Alternatively, the bifunctional inhibitor molecule can be produced usmg combinatonal methods to produce large libraπes of potential bifunctional molecules which may then be screened for identification of a bifunctional molecule with the desired bmdmg affinity and/or specificity Methods for producmg and screenmg combinatorial libraπes of molecules mclude 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 herem mcorporated by reference Alternatively, the bifunctional molecule may be produced usmg medicinal chemistry and known structure- activity relationships for the blocking protem ligand and the target protem hgand In particular, this approach will provide msight as to where to jom the two moieties to the lmker SCREENING BIFUNCTIONAL COMPOUNDS The resultant bifunctional inhibitor molecules are then screened for those molecules exhibit the desired inhibitory activity with respect to the target protem-protem mteraction or bmdmg event between the target protem and a second bmdmg protem Screenmg of such activity may be accomplished usmg any convenient protocol For example, in vitro bmdmg assays can be employed m which the occurrence of the target bmdmg event in the presence and absence of blocking protem and the candidate bifunctional inhibitor compound is evaluated In such assays, the occurrence of the bmdmg event in the presence of both the blocking protem and the inhibitor is indicative of a lack of inhibitory activity in the candidate bifunctional molecule, while the absence of a bmdmg event m the presence of both blockmg protem and candidate bifunctional molecule mdicates that the candidate bifunctional molecule possesses the desired inhibitory effect By comparison to appropriate controls, e g evaluation m the absence of candidate bifunctional inhibitor and/or blockmg protein, bifunctional inhibitor molecules that exhibit the desired inhibitory activity are identified In the above described screening assays, one or of the components may be bound to solid support, labeled reagents for the detection of bmdmg events may be employed, etc , as may be required by the particular format employed and is known by those of skill m the art A candidate bifunctional inhibitor molecule will generally be considered to have inhibitory activity with respect to a given target protem-protem mteraction if the amount of target protem-protem bindmg under physiological conditions in the presence of the bifunctional inhibitor is decreased by at least about 2-fold, usually at least about 4- fold and more usually at least about 10-fold as compared to a control m which the bifunctional inhibitor molecule is not present METHODS OF MAKING BIFUNCTIONAL MOLECULES FOR PEPTIDYL-PROLYL ISOMERASE BLOCKING PROTEINS As mentioned above, one class of preferred embodiments of the subject invention are those embodiments in which the bifunctional molecules specifically bind to endogenous peptidyl-prolyl isomerase blocking proteins present in the host into which the bifunctional molecule is introduced. Thus, bifunctional inhibitor molecules of interest include those in which the endogenous blocking protein is either an FKBP or a cyclophilin. In preparing bifunctional molecules from FK506, a suitable attachment site on the FK506 structure is identified, modified as necessary, and then covalently attached to the linker or target protein ligand moiety. The structure of FK506 (also known as tacrolimus) is: The site to which the linker/target ligand moiety is covalently attached is one that, upon covalent attachment, does not ablate the affinity and or specificity of FK506 for its FKBP blocking protein, e.g. FKBP 12 or FKBP 52. As such, positions suitable for use as covalent linkage sites include atoms located between carbon 15 and carbon 25 and the substituents attached to these atoms. For example, oxidation of the allyl group or oxidation of the carbon 18 methylene group; modification of the carbon 22 ketone or the carbon 24 hydroxyl group or alkylation at carbon 21 or carbon 23; as well as the secondary hydroxyl group located on the cyclohexyl ring (carbon 32); are potential specific covalent linkage sites. With FK506, depending on the target protein ligand and/or linker to be attached, it may be desirable to introduce one or more functional moieties onto the FK506 structure. Functional moieties of interest that may be introduced include: hydroxyl groups, amino groups, carboxyl groups, aldehydes, carbonates, carbamates, azides, thiols, and esters, etc. Such groups may be introduced using known protocols, such as oxidation reactions, reduction reactions, cleavage reactions and the like, with or without the use of one or more blocking groups to prevent unwanted side reactions. In some instances, it is desirable to covalently attach the target protein Hgand directly to FK506, often activated FK506. In such instances, the reactive functional group(s) introduced onto the FK506 structure will depend primarily on the nature of the target protein hgand to be attached. Thus, for peptidic target protein ligands, specific pairings of interest include: FK506 carbonates for reacting with amino groups of peptides; FK506 carboxylic acids for reacting with amino groups of peptides; FK506 amines for reacting with carboxylic acid groups of peptides; FK506 maleimide for reacting with thiol groups of peptides; and the like. Alternatively, where the Hgand moiety is a steroid, potential pairings of interest include: FK506 N-hydroxysuccinimidyl carbonate and partner amine; FK506 aldehyde and partner amine; FK506 aldehyde and partner hydrazide; FK506 hydroxy group and partner carboxylic acid OR alkyl haHde; FK506 thiol and partner maleimide and the like. Following introduction of the reactive functional group(s) onto the FK506 structure, the activated FK506 is then combined with the Hgand moiety/Hnker under conditions sufficient for covalent bonding to occur. Another embodiment of particular interest are bifunctional molecules of cyclosporin A or analogs thereof. The structure of cyclosporin A is: As with the FK506 bifunctional molecules, the cyclosporin A will be conjugated to the target protein Hgand in a manner such that cyclosporin A does not substantially lose its affinity for cyclophiHn. Preferred positions on the cyclosporin A structure that may serve as covalent linkage sites include: residues 4, 5, 6, 7, 8; while less preferred but still possible residues include: 1,2, 3, 9, 10 and 11. Where necessary, reactive functionaHties may be introduced onto the cyclosporin structure, where such functionalities include: hydroxyl groups, amino groups, carboxyl groups, aldehydes, carbonates, carbamates, azides, thiols, and esters, etc., with the particular functionality of interest being chosen with respect to the specific linker or target protein Hgand to be attached. METHODS OF USE, PHARMACEUTICAL PREPARATIONS AND KITS The subject bifunctional inhibitor molecules find use in methods of inhibiting protem-protem mteractions between a first target protem and a second bmdmg protem This "second bmdmg protem" can also be called an "effector protein," meaning that bmdmg of the target protem to the effector protem is responsible for a biological/physiological response the blockage or inhibition of which is desired with the bifunctional molecules The target protem-protem mteraction or bmdmg event may be one that occurs in vitro or m vivo, but m many preferred embodiments is one that occurs in vivo, m that it occurs m a living organism or host By inhibiting protem-protem mteractions is meant that the presence of the subject bifunctional inhibitor molecules reduces the amount of bmdmg between the first target protem and the second bmdmg protem, generally by at least about 2-fold , usually by at least about 4-fold and more usually by at least about 10-fold, where m certain cases the target protein-effector protem mteraction between the target protem and the second bmdmg protem is completely inhibited, l e the bindmg event does not occur The subject bifunctional compounds inhibit the target bmdmg event between the target protem and the second bmdmg protem by forming a tπpartite complex that mcludes the target protem, the bifunctional molecule and the blockmg protem, where the blockmg protein member of the tripartite complex inhibits or prevents bmding of the second bmdmg protem to the target protem As such, the subject methods can also be viewed as methods of producing tripartite complexes that inhibit or prevent the bmdmg of a target protem to a second bindmg protem In the methods of the subject mvention, an effective amount of the bifunctional molecule is administered to the host, where "effective amount" means a dosage sufficient to produce the desired result, e g an improvement m a disease condition or the symptoms associated therewith which are mediated at some point by the protein/protein mteraction that is inhibited by the bifunctional condition The bifunctional molecule may be administered to the host usmg any convenient means capable of producing the desired result Thus, the bifunctional molecule can be incorporated into a vaπety of formulations for therapeutic administration More particularly, the bifunctional molecule of the present mvention can be formulated into pharmaceutical compositions by combmation with appropriate, pharmaceutically acceptable earners or diluents, and may be formulated mto preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, omtments, solutions, suppositories, injections, inhalants and aerosols As such, administration of the bifunctional molecule can be achieved m various ways, mcludmg oral, buccal, rectal, parenteral, intraperitoneal, lntradermal, transdermal. mtracheal, etc , administration In pharmaceutical dosage forms, the bifunctional molecule may be administered alone or m combmation with other pharmaceutically active compounds The following methods and excipients are merely exemplary and are in no way krniting For oral preparations, the bifunctional molecules can be used alone or m combmation with appropπate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, com starch or potato starch, with bmders, such as crystalline cellulose, cellulose derivatives, acacia, com starch or gelatins, with disintegrators, such as com starch, potato starch or sodium carboxymethylcellulose, with lubricants, such as talc or magnesium stearate, and if desired, with diluents, buffering agents, moistenmg agents, preservatives and flavoring agents The bifunctional molecules can be formulated mto preparations for injection by dissolving, suspending or emulsifying them m an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glyceπdes, esters of higher aliphatic acids or propylene glycol, and if desired, with conventional additives such as solubilizers, lsotomc agents, suspendmg agents, emulsifying agents, stabilizers and preservatives The bifunctional molecules can be utihzed in aerosol formulation to be administered via inhalation The compounds of the present mvention can be formulated mto pressuπzed acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like Furthermore, the bifunctional molecules can be made mto suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases The compounds of the present mvention can be admmistered rectally via a suppository The suppository can mclude vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherem each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contams a predetermmed amount of the composition containing active agent Similarly, unit dosage forms for injection or intravenous administration may comprise the active agent m a composition as a solution m sterile water, normal saline or another pharmaceutically acceptable carrier The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit contammg a predetermined quantity of compounds of the present invention calculated m an amount sufficient to produce the desired effect m association with a pharmaceutically acceptable diluent, earner or vehicle The specifications for the novel unit dosage forms of the present mvention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound m the host The pharmaceutically acceptable excipients, such as vehicles, adjuvants, earners or diluents, are readily available to the public Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffermg agents, tomcity adjusting agents, stabiHzers, wettmg agents and the like, are readily available to the pubHc Those of skill wiH readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects Preferred dosages for a given compound are readily determinable by those of skill m the art by a variety o f means The subject methods find use m the treatment of a variety of different disease conditions which are mediated, at least m part, by the occmrence of protem-protem mteractions between a target protem and a second bmdmg protem The specific disease conditions treatable by with the subject bifunctional compounds are varied Thus, disease conditions include cellular prohferative diseases, such as neoplastic diseases, autoimmune diseases, cardiovascular diseases, hormonal abnormality diseases, mfectious diseases, and the like By treatment is meant at least an amelioration of at least one of the symptoms associated with the disease condition afflicting the host, where amelioration is used m a broad sense to refer to at least a reduction m the magnitude of a parameter, e g symptom, associated with the pathological condition being treated, such as inflammation and pain associated therewith As such, treatment also mcludes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e g prevented from happening, or stopped, e g terminated, such that the host no longer suffers from the pathological condition, or at least the symptoms that characterize the pathological condition A variety of hosts are treatable accordmg to the subject methods Generally such hosts are "mammals" or "mammaHan," where these terms are used broadly to describe organisms which are withm the class mammaHa, including the orders carnivore (e g , dogs and cats), rodentia (e g , mice, guinea pigs, and rats), and pnmates (e g , humans, chimpanzees, and monkeys) In many embodiments, the hosts will be humans Kits with unit doses of the bifunctional molecule, usually in oral or injectable doses and often m a storage stable formulation, are provided In such kits, m addition to the containers contammg the unit doses will be an informational package insert descnbmg the use and attendant benefits of the bifunctional molecules m treatmg pathological condition of mterest Prefened compounds and unit doses are those described herem above The following examples are offered by way of illustration and not by way of limitation EXPERIMENTAL I Introduction The NFAT transcπption factor plays an important role in the activation of T-lymphocytes A molecule that inhibits the NFAT transcπption factor would be a potent lmmunosuppressant drug The regulation of NFAT mvolves two important events first, NFAT has to be dephosphorylated which unveils a nuclear translocation signal Second, the nuclear translocation machinery has to bmd to the nuclear translocation signal to move the protem from the cytoplasm mto the nucleus The critical phosphorylation sites and the nuclear localization signal are found m the N-termmus of NFAT II Assay Design and Results Usmg the blocker protem approach to mterfere with dephosphorylation or translocation of NFAT requires havmg a ligand m the N-termmus of NFAT that, by itself, does not cause any inactivation of the protem Such a ligand has not been identified, yet, because so far such ligands have been of no biological mterest In order to overcome this problem, a bmdmg site was created for a ligand at the N-termmus of NFAT by fusing it to an FKBP protem which can bmd the small molecule rapamycin The resultmg FKBP-NFAT fusion protem is regulated normally After transfection of the cDNA mto COS cells, the expressed protem is localized m the cytoplas When the cells are treated with lonomycm, FKBP-NFAT translocates mto the nucleus lonomycm is an lonophore which leads to an mcrease in intracellular calciu As a result, the phosphatase calcmeuπn is activated Calcmeurm dephosphorylates FKBP-NFAT and the nuclear translocation machinery translocates the fusion protein into the nucleus as it would NFAT (Figure 2) Like FK 06 and cyclosporin, rapamycin is a bifunctional molecule It bmds FKBP and the protem kmase FRAP simultaneously FRAP is a large protem but withm this large protem the rapamycm bmdmg site maps to a small 87 ammo acid domain This domain can be coexpressed m cells together with FKBP-NFAT In the absence of rapamycm, the FRB domain and FKBP do not mteract and FKBP-NFAT is translocated normally after stimulation with lonomycm However, when rapamycm is added, the molecule bmds FKBP and the FKBP-rapamycin complex recruits and bmds FRB This locaHzes the FRB protem domain at the N-termmus of NFAT When lonomycm is now used to trigger the translocation of FKBP-NFAT, the fusion protem remains m the cytoplasm (Figure 3) The FRB domain bound at the N-termmus of NFAT estabhshes a block that prevents the interaction of NFAT with calcineurin or with the nuclear translocation machinery (Figure 4). These results demonstrate that the mode of action of FK506 and cyclosporin is generalizable: a bifunctional molecule can be used to recruit an endogenous blocking molecule to any given drug target so that a steric block is established which prevents other proteins from interacting with the target. It is evident from the above results and discussion that the subject invention provides a powerful new tool for inhibiting protein-protein interactions. Specifically, the subject invention provides small molecule therapeutics which are capable of mhibiting both extraceUular and intracellular protein-protein interactions. As such, the subject methods and compositions provide for new treatment protocols for a variety of disease and other conditions. Accordingly, the invention represents an important advancement in pharmacological science. All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in Hght of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims: WHAT IS CLAIMED IS: 1. A non-naturally occurring bifunctional inhibitor molecule of less than about 5000 daltons that inhibits a binding event between a first target protein and a second binding protein, said bifunctional inhibitor molecule consisting of: a target protein Hgand and a blocking protein Hgand optionaUy joined by a linking group; wherein said bifunctional inhibitor molecule is capable of simultaneously binding said target protein and said blocking protein in a manner sufficient to inhibit said binding event. 2. The bifunctional inhibitor molecule according to Claim 1, wherein said bifunctional inhibitor molecule comprises a linking group. 3. The bifunctional inhibitor molecule according to Claim 1, wherein said bifunctional inhibitor molecule binds to a site of said target protein that is also bound by said second binding protein. 4. The bifunctional inhibitor molecule according to Claim 1 , wherein said bifunctional inhibitor molecule binds to a site of said target protein that is not bound by said second binding protein. 5. The bifunctional inhibitor molecule according to Claim 1, wherein said blocking protein is an extracellular protein. 6. The bifunctional inhibitor molecule according to Claim 1, wherein said blocking protein is an intracellular protein. 7. The bifunctional molecule according to Claim 6, wherein said blocking protein is a peptidyl prolyl isomerase. 8. A synthetic bifunctional inhibitor molecule of less than about 5000 daltons and capable of inhibiting a binding event between a first target protein and a second binding protein, wherein said bifunctional inhibitor molecule is of the formula: Z-L-X wherein: X is target protein ligand; L is a bond or a linking group; and Z is different from X and is a blocking protein Hgand; wherein said bifunctional inhibitor molecule is capable of simultaneously bindmg to said target protein and said blocking protein in a manner sufficient to inhibit said binding event. 9. The bifunctional inhibitor molecule according to Claim 8, wherein X binds to a site of said target protein that is also bound by said second binding protein. 10. The bifunctional inhibitor molecule according to Claim 8, wherein X binds to a site of said target protein that is not bound by said second binding protein. 11. The bifunctional inhibitor molecule according to Claim 8, wherein X has a molecular weight of from about 50 to 2000 D. 12. The bifunctional inhibitor molecule according to Claim 8, wherein said target protein is an extracellular protein. 13. The bifunctional inhibitor molecule according to Claim 8, wherein said target protein is an intracellular protein. 14. The bifunctional inhibitor molecule according to Claim 13, wherein said blocking protein is a peptidyl prolyl isomerase. 15. The bifunctional inhibitor molecule according to Claim 8, wherein Z has substantially no pharmacologic activity apart from binding to a blocking protein. 16. A method for inhibiting a binding event between a first target protein and a second binding protein in a host, said method comprising: administering to said host an effective amount of a bifunctional inhibitor molecule of less than about 5000 daltons consisting of a target protein Hgand and a blocking protein ligand optionally joined by a linking group, wherein said bifunctional inhibitor molecule is capable of simultaneously binding said target protein and said blocking protein in a manner sufficient to inhibit said binding event; whereby a tripartite complex comprising said bifunctional inhibitor molecule, said target protein and said blocking protein is produced that inhibits said binding event. 17. The method according to Claim 16, wherein said bifunctional inhibitor molecule comprises a linking group. 18. The method according to Claim 16, wherein said bifunctional inhibitor molecule binds to a site of said target protein that is also bound by said second binding protein. 19. The method according to Claim 16, wherein said bifunctional inhibitor molecule binds to a site of said target protein that is not bound by said second binding protein. 20. The method according to Claim 16, wherein said tripartite complex is produced intraceUularly. 21. The method according to Claim 16, wherein said tripartite complex is produced extracellularly. 22. The method according to Claim 16, wherein said blocking protein is endogenous to said host. 23. The method according to Claim 22, wherein said blocking protein is selected from the group consisting of: peptidyl-prolyl isomerases, Hsp90, steroid hormone receptors, cytoskeletal proteins, albumin and vitamin receptors. 24. The method according to Claim 16, wherein said bifunctional inhibitor molecule is administered as a pharmaceutical preparation. 25. A pharmaceutical preparation comprising a bifunctional inhibitor molecule according to Claim 1. 26. A kit comprising the pharmaceutical preparation according to Claim 25 and instructions for use in a therapeutic method.

Inventors: BRIESEWITZ ROGER, CRABTREE GERALD R, STANKUNAS KRYN, WANDLESS THOMAS, BRIESEWITZ, ROGER, CRABTREE, GERALD, R., STANKUNAS, KRYN, WANDLESS, THOMAS
Applicants: BRIESEWITZ ROGER, CRABTREE GERALD R, STANKUNAS KRYN, UNIV LELAND STANFORD JUNIOR, WANDLESS THOMAS, BRIESEWITZ, ROGER, CRABTREE, GERALD, R., STANKUNAS, KRYN, THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY, WANDLESS, THOMAS
Application year: 2000
Main classification: C12N9
IPC codes: A61K47