Proteolysis targeting chimeric pharmaceutical

FIELD OF THE INVENTION

[0002] This invention relates generally to the field of ubiquitin-dependent proteolysis, and more specifically to targeted ubiquitination by chimeric constructs.

BACKGROUND OF THE INVENTION

[0003] Degradation of cellular proteins is required for normal maintenance of cellular function, including proliferation, differentiation, and cell death. The irreversible nature of proteolysis makes it well-suited to serve as a regulatory switch for controlling unidirectional processes. This principle is evident in the control of the cell cycle, where initiation of DNA replication, chromosome segregation, and exit from mitosis are triggered by the destruction of key regulatory proteins.

[0004] One of the major pathways to regulate proteins posttranslationally is ubiquitin-dependent proteolysis. The first step in selective degradation is the ligation of one or more ubiquitin molecules to a protein substrate. Ubiquitination occurs through the activity of ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin-protein ligases (E3), which act sequentially to catalyze the attachment of ubiquitin to lysine residues of substrate proteins (See Ciechanover A., et al., BioEssays, 22:442-451 (2000)). The E3s confer specificity to ubiquitination reactions by binding directly to substrate.

[0005] There is a need in the art to provide methods and compositions useful for regulating protein activities, especially in vivo. There is also a need in the art to provide methods for identifying proteins involved in a predetermined function or phenotype.

SUMMARY OF THE INVENTION

[0006] The present invention is based on the discovery that a ubiquitin pathway protein ubiquitinates any target protein once the ubiquitin pathway protein and the target protein are placed in proximity by a chimeric construct that binds the ubiquitin pathway protein and the target protein. Accordingly the present invention provides a composition that ubiquitinates a target protein. The present invention also provides a library of compositions and the use thereof.

[0007] In one embodiment, the present invention provides a composition useful for regulating protein activity. The composition comprises a ubiquitin pathway protein binding moiety and a targeting moiety, wherein the ubiquitin pathway protein binding moiety recognizes a ubiquitin pathway protein and the targeting moiety recognizes a target protein and wherein the ubiquitin pathway protein binding moiety is coupled to the targeting moiety.

[0008] In another embodiment, the present invention provides a library. The library comprises more than one composition wherein each composition has a formula of A-B, wherein A is a ubiquitin pathway protein binding moiety and B is a member of a molecular library, wherein A is coupled to B, and wherein the ubiquitin pathway protein binding moiety recognizes a ubiquitin pathway protein. In a particular embodiment, the library contains a specific ubiquitination recognition peptide (ubiquitin pathway protein binding moiety) with random target protein binding elements (e.g., a chemical compound library). As such, the target protein does not have to be determined in advance.

[0009] In yet another embodiment, the present invention provides a method of ubiquitinating a target protein in a cell. The method comprises administering a composition comprising a ubiquitin pathway protein binding moiety and a targeting moiety, wherein the ubiquitin pathway protein binding moiety is coupled to the targeting moiety and wherein the ubiquitin pathway protein binding moiety recognizes a ubiquitin pathway protein and the targeting moiety recognizes the target protein.

[0010] In still another embodiment, the present invention provides a method of screening a library of the present invention to identify a composition containing a targeting moiety which recognizes a target protein associated with a predetermined function of a cell. The method comprises incubating a cell with a pool of entities from the library; monitoring the predetermined function of the cell; identifying a pool of entities that change the predetermined function of the cell; incubating the cell with a composition from the identified pool of entities; monitoring the predetermined function of the cell; and identifying a composition that changes the predetermined function of the cell, wherein the identified composition contains a targeting moiety which recognizes a target protein associated with the predetermined function.

[0011] In another embodiment, the present invention provides a method of screening a library of the present invention to identify a composition containing a targeting moiety which recognizes a target protein associated with a predetermnined function of a cell. The method comprises incubating a cell with each composition from the library; monitoring the predetermined function of the cell; identifying a composition that changes the predetermined function of the cell; wherein the identified composition contains a targeting moiety which recognizes a target protein associated with the predetermined function.

[0012] In another embodiment, the present invention provides a method of identifying a target protein associated with a predetermined function of a cell. The method comprises incubating a cell with a composition from the library of the present invention; monitoring the predetermined function of the cell; identifying a composition that changes the predetermined function of the cell; identifying a target protein that binds to the identified composition, wherein the target protein is associated with the predetermined function of the cell.

[0013] In yet another embodiment, the present invention provides a method of identifying a target protein associated with a predetermined function of a cell. The method comprises incubating a cell with a pool of entities from the library of the present invention; monitoring the predetermined function of the cell; identifying a pool of entities that change the predetermined function of the cell; incubating the cell with a composition from the identified pool of entities; monitoring the predetermined function of the cell; identifying a composition that changes the predetermined function of the cell; and identifying a target protein that binds to the identified composition, wherein the target protein is associated with the predetermined function of the cell.

BRIEF DESCRIPTION OF THE FIGURES

[0014]
FIGS. 1A and 1B show a schematic representation of a general scheme for Protac-1. FIG. 1A shows that Protac-1 targets MetAP-2 to SCF. Protac-1 is a chimeric molecule that consists of a phosphopeptide moiety and a small molecule moiety that interacts with the protein target. Ub, ubiquitin; H, Hrt1. FIG. 1B shows the synthesis scheme for Protac-1. Fmoc, fluorenylmethoxy-carbonyl; DMF, dimethylformamide; DMAP, dimethylaminopyridine; DSS, dis-uccinimidyl suberate.

[0015]
FIGS. 2A and 2B shows that MetAP-2 binds Protac specifically and in a concentration-dependent manner. FIG. 2A shows that MetAP-2 (9 μM) was incubated with increasing concentrations of Protac-1 at room temperature for 45 min. The last two lanes depict MetAP-2 that was incubated with the individual components of protac-1: either free IPP (50 μM) or free OVA (50 μM), as indicated (IPP stands for IkB phosphopeptide, and OVA stands for ovalicin). After incubation, samples were supplemented with SDS/PAGE loading buffer, separated by SDS/PAGE, and immunoblotted with MetAP-2 antiserum. FIG. 2B shows the same as 2A, except MetAP-2 (9 μM) plus Protac-1 (10 μM) were supplemented with either IPP (50 μM) or OVA (50 μM), as indicated. Protac binding to MetAP-2 was inhibited by the addition of OVA, but not by the addition of IPP.

[0016]
FIG. 3 shows that Protac-1 recruits MetAP-2 to SCFβ-TRCP. Extracts from 293T cells transiently transfected with either control vector or plasmids expressing FLAG epitope-tagged Cul-1 and β-TRCP were subject to affinity purification on anti-FLAG resin to yield either control beads or SCFβ-TRCP beads. The matrices then were mixed with the preformed MetAP-2-Protac-1 complex (input), incubated, and separated into pellet (bound) and unbound (sup) fractions. Proteins were fractionated on an SDS/10% polyacrylamide gel, and immuno-blotted with anti-MetAP-2 antiserum. MetAP-2 and MetAP-2-Protac-1 refer to free MetAP-2 and MetAP-2 complexed with Protac-1, respectively.

[0017]
FIGS. 4A, 4B, 4C, and 4D show that Protac mediates MetAP-2 ubiquitination by SCF. FIG. 4A shows ubiquitination of the 46-kDa fragment of MetAP-2. MetAP-2-Protac-1 mixture was added to either control (Mock) or SCFβ-TRCP beads (1) supplemented with ATP plus purified E1, E2 (Cdc34), and ubiquitin. The E2, UbcH5c (500 ng), was also tested in the reaction, which resulted in the same degree of ubiquitination as observed with Cdc34 (data not shown). Reactions were incubated for 1 hr at 30° C. and were evaluated by SDS/PAGE followed by Western blotting with anti-MetAP-2 antiserum. FIG. 4B shows ubiquitination of full-length (67-kDa) MetAP-2. Same as FIG. 4A, except that the 67-kDa preparation of MetAP-2 was used, and E1, E2, plus ubiquitin were either added at normal (1×) or 2-fold higher (2×) levels, as indicated. FIG. 4C shows that ubiquitination of MetAP-2 by SCFβ-TRCP depends on Protac-1. Same as A, except that methylubiquitin (Me) was substituted for ubiquitin, as indicated, and the reactions depicted in lanes 5-7 lacked Protac-1. In lane 7, unlinked IPP and OVA were added at 100 μM in place of Protac-1. FIG. 4D shows that Protac-1-dependent ubiquitination of MetAP-2 is competitively inhibited by IPP. Same as FIG. 4A, except that reactions in lanes 3 and 4 were supplemented with 100 μM each IPP and OVA, respectively.

[0018]
FIG. 5 shows that MetAP-2-Protac is degraded in Xenopus extracts. The MetAP-2-Protac-1 mixture or MetAP-2 alone was added to Xenopus egg extract fortified with OVA (100 μM), IKK-EE (0.4 μg), and OA (10 μM). Where indicated, reactions were either deprived of IKK-EE or OA, or were further supplemented with 50 μM LLnL or 10 μM epoxomicin (Epox). Reactions were incubated for the indicated times at room temperature, terminated by adding SDS/PAGE loading buffer, and evaluated by SDS/PAGE followed by Western blotting with anti-MetAP-2 antiserum.

[0019]
FIG. 6 depicts the general schematic of how different disease-promoting proteins might be recruited to different ubiquitin ligases for ubiquitination and degradation by unique Protacs.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present invention relates in general to targeted ubiquitination using a composition, e.g., a chimeric construct containing a ubiquitin pathway protein binding moiety and a targeting moiety. The present invention also provides a library of compositions useful for identifying proteins associated or involved with a predetermined function of cells.

[0021] According to the present invention, a ubiquitin pathway protein binding moiety is any suitable structure that recognizes and binds to a ubiquitin pathway protein. In general, a ubiquitin pathway protein is any entity or complex that is capable of catalyzing or causing to catalyze the transfer of a ubiquitin or ubiquitin-like modifying polypeptide, e.g., Nedd8, APG12 or ISG15/UCRP to another protein. In one embodiment, a ubiquitin pathway protein is a ubiquitin protein ligase or E3 protein. There are at least 100 distinct E3 proteins encoded by the human genome (See Winston, J., et al., Curr. Bio., 9:1180-82 (1999)). For example, one particular class of E3 proteins are Skp1-Cullin-F box (SCF) complexes, each complex containing Skp1, a member of Cullin family, e.g., the RING-H2 protein Hrt1, also known as Roc1 or Rbx1, and an F box protein (See Deshaies, R. J., Annu. Rev. Cell Dev. Biol., 15:435-67 (1999)). These components are conserved from yeast to mammals.

[0022] In another embodiment, a ubiquitin pathway protein is a protein that is involved in or a component of a ubiquitin-like pathway, which transfers ubiquitin-like modifying polypeptides, e.g., SUMO, Nedd8, APG12 or ISG15/UCRP. Components of a ubiquitin-like pathway are usually homologues of a ubiquitin pathway. For example, the ubiquitin-like pathway for SUMO includes a homologue of a ubiquitin protein activating enzyme or E1 protein, ubiquitin protein conjugating enzyme or E2 protein and ubiquitin ligase or E3 protein.

[0023] A ubiquitin pathway protein can be expressed in a tissue specific or regulated manner. For example, VACM-1 receptor (aka CUL-5), and F-box protein, NFB42 are expressed in a tissue specific manner. In one embodiment, a ubiquitin pathway protein is an RING-based or HECT-based ubiquitin ligase.

[0024] According to one embodiment of the present invention, a ubiquitin pathway protein binding moiety of the present invention is any suitable ligand to a ubiquitin pathway protein, e.g., ubiquitin protein ligase or E3 protein or homologues thereof. In another embodiment, a ubiquitin pathway protein binding moiety of the present invention is any ubiquitin pathway protein binding peptide, domain or region of an ligand to a ubiquitin pathway protein. In still another embodiment, a ubiquitin pathway protein binding moiety of the present invention recognizes and binds to a ubiquitin pathway protein in a regulated manner. For example, IkBa is a well defined ligand for mammalian SCFβ-TRCP complex. The mammalian F box protein, β-TRCP/E3RS, has been shown to bind IkBa, a negative regulator of NFkB (See Yaron, A., et al., Nature (London), 396:590-94 (1998)). The SCFβ-TRCP complex promotes the ubiquitination and subsequent degradation of IkBa, which results in activation of NFkB during the inflammatory response (See Deshaies, R. J., Annu. Rev. Cell Dev. Biol., 15:435-67 (1999)). The recruitment of IkBa to SCFβ-TRCP is mediated by a ubiquitin pathway protein binding domain, which is a 10-aa peptide within IkBa, DRHDSGLDSM (See Yaron, A., et al., Nature (London), 396:590-94 (1998); and Yaron, A., et al., EMBO J., 16: 6486-94 (1997)). In response to diverse inflammatory signals, IkBa kinase (IKK) phosphorylates this motif on both serines, which triggers the binding of IkBa to β-TRCP.

[0025] According to the present invention, a targeting moiety is any structure that recognizes and binds to a target protein. A target protein can be any protein that one desires to regulate its level or activity, e.g., to alter the activity through ubiquitin-dependent proteolysis or through attachment of ubiquitin or ubiquitin-like modifying polypeptide to lysine residues that are important for the protein's activity or structure. For example, a target protein can be a protein involved in cell cycle, signal transduction, cell differentiation, cell dedifferentiation, cell growth, production of cytokines or other biological modifiers, production of regulatory or functional proteins, pro-inflammatory signaling, or the glucose regulation pathway. In one embodiment, a target protein can be a protein that is not known to be ubiquitinated or not known to be a substrate for any ubiquitin pathway protein.

[0026] In another embodiment, a target protein is a disease related protein, e.g., a protein for which changes in its function or activity cause disease, or whose function is considered important to the propagation of the disease state. The target protein may be either stable or unstable, e.g., androgen receptor, estrogen receptor, myc, cyclin B, Ras, or cyclin E.

[0027] In still another embodiment, a target protein is methionine aminopeptidase-2 (MetAP-2). MetAP-2 catalyzes the cleavage of N-terminal methionine from nascent polypeptides (See Li, X., et al. Proc. Natl. Acad. Sci. USA, 92:12357-61 (1995)) and is the primary target of the potent angiogenesis inhibitors fumagillin and ovalicin (OVA; See Sin, N., et al., Proc. Natl. Acad. Sci. USA, 94:6099-103 (1997); and Griffith, E., et al., Chem. Biol., 4: 461-71(1997)). Both of these compounds inhibit MetAP-2 by covalently binding His-231 in the active site. The consequent reduction in MetAP-2 activity blocks endothelial cell proliferation by causing p53-dependent arrest in the G1 phase of the cell cycle (See Yeh, J., et al., Proc. Natl. Acad. Sci. USA, 97: 12782-87 (2000)).

[0028] Structures that recognize and bind to a target protein are well known or readily available to one skilled in the art. For example, a targeting moiety for MetAP-2 can be an angiogenesis inhibitor including, without limitation, fumagillin and ovalicin. MetAP-2 is a naturally occurring receptor for the anti-angiogenic drug fumagillin, and forms a covalent linkage with both fumagillin and the related compound ovalicin. For example, as shown in EXAMPLE 6, a chimeric compound was prepared including IkBa phosphopeptide linked to ovalicin. MetAP-2 is ubiquitinated in the presence of this chimeric compound and is then degraded by proteosomes.

[0029] According to the present invention, the composition of the present invention may further comprise a cell delivering moiety. A cell delivering moiety is any structure that facilitates the delivery of the composition or promotes transduction of the composition into cells. In one embodiment, a cell delivering moiety is derived from virus protein or peptide, e.g., a tat peptide. In another embodiment, a cell delivering moiety is a hydrophobic compound capable of penetrating cell membranes. Alternatively, a ubiquitin pathway protein binding moiety that is more susceptible for cell membrane penetration is used to enhance the cell membrane transduction of the composition. For example, a ubiquitin pathway protein binding peptide derived from the HIV Vpu protein.

[0030] According to the present invention, the ubiquitin pathway protein binding moiety can be coupled, e.g., covalently by any suitable means to the targeting moiety. For example, a ubiquitin pathway protein binding moiety, IkBa peptide is coupled to a targeting moiety, OVA through Fmoc-Gly. In one embodiment, the composition of the present invention is a chimeric fusion protein and is encoded by a polynucleotide in an expression system. In another embodiment, the ubiquitin pathway protein binding moiety is covalently coupled to the targeting moiety through a linker, e.g., a linker which has a binding domain for the ubiquitin pathway protein binding moiety as well as targeting moiety.

[0031] In yet another embodiment, the ubiquitin pathway protein binding moiety can be coupled noncovalently to the targeting moiety upon the presence of a signal factor, e.g., the presence or the level of an intracellular metabolite, regulatory protein, etc. For example, the ubiquitin pathway protein binding moiety and the targeting moiety can be coupled when they simultaneously chelate an intracellular metabolite.

[0032] In still another embodiment, the ubiquitin pathway protein binding moiety can include a first coupling moiety and the targeting moiety can include a second coupling moiety such that the first and the second coupling moiety are coupled or bind to each other in the presence of a signal factor or enzymatic activity in vitro or in vivo (e.g., phosphorylation of the first coupling moiety by a kinase that is produced by cancer cells enables it to bind to the second coupling moiety.

[0033] Another feature of the present invention provides a library of the compositions of the present invention. In one embodiment, the targeting moiety of the composition of the library is a member of a molecular library. A molecular library can be any collection of molecules, including without limitation, a combinatorial library, a small molecule library, a receptor library, and a ligand library. In one embodiment, a member of a molecular library is covalently coupled to the ubiquitin pathway protein binding moiety, e.g., through a linker.

[0034] The compositions and the libraries of compositions provided by the present invention can be used individually or collectively as a group to perform target ubiquintination. Such target ubiquitination is useful for regulating protein levels or activities, thus providing therapeutic treatment for disease conditions. One or more compositions of the present invention can be administered to ubiquitinate a target protein either in vitro or in vivo. In one embodiment, two or more compositions of the present invention with the same targeting moiety, but different ubiquitin pathway protein binding moieties are administered to cells to ubiquitinate a target protein, e.g., ubiquitinate a target protein with a desired rate or degree. In another embodiment two or more compositions of the present invention with the same ubiquitin pathway protein binding moiety, but different targeting moiety are administered to cells to ubiquitinate different target proteins. In still another embodiment, one or more compositions with a ubiquitin pathway protein binding moiety that recognizes a ubiquitin pathway protein expressed in a tissue specific manner are administered to cells to ubiquitinate one or more target proteins in a tissue specific manner, e.g., at a specific location.

[0035] In one embodiment, the invention includes a combinatorial chemical library in which every member of the library is of the following structure: ubiquitin pathway protein binding moiety-X, wherein ubiquitin pathway protein binding moiety is a ubiquitin recognition element that is functionally defined as a moiety that binds to any part of the surface of an E2 or E3, such that it promotes the ubiquitination of a protein attached directly or indirectly to the moiety, and X is a moiety that binds to a target protein. In the library contemplated here, every member of the library shares a common, but contains a distinct X moiety. In one aspect, screening of such a combinatorial chemical library of the composition described above, is achieved by contacting the library with cells, after which a specific property of the cells is monitored (e.g., rate of cell proliferation, DNA synthesis, gene expression, etc). Specific compounds within the library would bind to a protein (protein Z) that plays a critical role in some aspect of cell physiology (e.g., gene expression). By binding to protein Z, the compound would direct its ubiquitination and degradation via the ubiquitin pathway protein binding moiety of the compound, thereby leading to a change in gene expression which could be detected by a suitable assay. It should be understood that such a library may contain a specific ubiquitin recognition element moiety coupled to a random library of target protein binding element moieties (e.g., a chemical library). Such a library is useful for identifying a target responsible or related to a cell function (e.g., cell proliferation).

[0036] In another aspect, a combinatorial chemical library of the composition described above is screened by contacting every member of the library with cells engineered in such a way that the activity or concentration of the desired target (protein Z) can be readily monitored (e.g., target is expressed as a fusion protein to GFP). For example, specific compounds within the library would bind to the protein Z-GFP chimera. By binding to protein Z, the compound would direct its ubiquitination and degradation via the ubiquitin pathway protein binding moiety of the compound, thereby leading to a decrease in GFP fluorescence. Similarly, such a screen could be performed with the exception that the screen is performed with protein Z-GFP added to a cell lysate that reconstitutes ubiquitin-dependent degradation by the proteasome.

[0037] In another embodiment, a combinatorial chemical library is constructed in which every member of the library has the following structure: X-TRE, wherein TRE is a target recognition element specific for a particular target that is functionally defined as a moiety that binds to any part of the surface of the target, such that TRE promotes the ubiquitination or degradation of the target if it enables the target to be linked, directly or indirectly, to a component of the ubiquitin/proteasome pathway. In the library contemplated here, every member of the library shares a common TRE moiety, but contains a distinct X moiety.

[0038] In another aspect, a combinatorial chemical library of the composition described above is screened by contacting every member of the library with cells that are engineered to report the activity or concentration of the protein to which the TRE binds. One would then screen for compounds that alter the activity or concentration of the protein to which TRE binds. For example, if the TRE binds GFP, one would use cells (or a cell lysate that reconstitutes ubiquitin-dependent degradation by the proteasome) that are engineered to express GFP, and then search for compounds in the library that bring about a reduction in GFP fluorescence. It is contemplated that some of these compounds will contain ‘X’ moities that bind to components of the ubiquitin/proteasome pathway, and thereby enable the recruitment of degradation machinery to GFP via the X-TRE compound. While not wanting to be bound by a particular theory, it is believed that it may be possible that some active compounds might bypass the ubiquitin pathway entirely, and directly tether the target to the proteasome.

[0039] The compositions of the present invention useful for therapeutic treatment can be administered alone, in a composition with a suitable pharmaceutical carrier, or in combination with other therapeutic agents. An effective amount of the compositions to be administered can be determined on a case-by-case basis. Factors should be considered usually include age, body weight, stage of the condition, other disease conditions, duration of the treatment, and the response to the initial treatment.

[0040] Typically, the compositions are prepared as an injectable, either as a liquid solution or suspension. However, solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The composition can also be formulated into an enteric-coated tablet or gel capsule according to known methods in the art.

[0041] The compositions of the present invention may be administered in any way which is medically acceptable which may depend on the disease condition or injury being treated. Possible administration routes include injections, by parenteral routes such as intravascular, intravenous, intraepidural or others, as well as oral, nasal, ophthalmic, rectal, topical, or pulmonary, e.g., by inhalation. The compositions may also be directly applied to tissue surfaces, e.g., during surgery. Sustained release administration is also specifically included in the invention, by such means as depot injections or erodible implants.

[0042] According to another feature of the present invention, the libraries of compositions provided by the present invention are useful for identifying target proteins that are involved in a predetermined function. For example, compositions of the libraries of the present invention can be screened either individually or be divided into a first subset of different pools of compositions for screening. Cells are incubated with and without each composition or pool of compositions and a predetermined function of the cells is monitored to determine the effect of each composition or pool of compositions.

[0043] A predetermined function of cells can be any relevant or interested function, including without limitation, cell growth, cell death, cell cycle regulation, cell differentiation, cell dedifferentiation, pro-inflammatory signaling, signal transduction, cytokine production, production of regulatory or functional proteins, and glucose regulation.

[0044] If a pool of compositions is identified as affecting a predetermined function of cells, then the identified pool of compositions is broken down further into a second subset of pools to repeat the same type of screening with the first subset of pools. Such subdivision of pools can be repeated as many times as necessary so that at the end the identified pool has a reasonable number of compositions to be screened individually.

[0045] An individual composition identified as affecting a predetermined function of cells contains a targeting moiety that recognizes a target protein involved in the predetermined function of cells. Such identified composition can be used either therapeutically to affect the predetermined function of cells or to screen cell extracts or expression libraries to identify proteins that bind to the targeting moiety of the identified composition, e.g., target proteins, thus identify proteins involved in the predetermined function of cells. The identified target proteins are useful for developing novel drugs or therapeutic treatments.

EXAMPLES

[0046] The following examples are intended to illustrate but not to limit the invention in any manner, shape, or form, either explicitly or implicitly. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

Example 1

Methodology

[0047] Synthesis of I kBa-OVA Protac

[0048] OVA(1.4 mmol) was dissolved in 10 ml of methanol at 0° C., and NaBH4 (3.0 mmol) was added slowly. After 30 min of stirring, methanol was removed under reduced pressure, and the resulting crude product was purified by flash column chromatography to yield ovalicinol (1.15 mmol, 82%). Fmoc-Gly was coupled to the ovalicinol to give Fmoc-Gly-ovalicinol. Specifically, dimethylformamide (DMF, 28 μl) was added to dichloromethane solution (30 ml) containing Fmoc-Gly-OH (3.56 mmol) and oxalyl chloride (7.12 mmol) at 0° C. After 3 hr of stirring at room temperature, dichloromethane was removed under nitrogen atmosphere. The resulting solid residue was redissolved in dichloromethane (10 ml) and was combined with ovalicinol (0.6 mmol) and dimethylaminopyridine (4.7 mmol) in dichloromethane (30 ml) at 0° C. The reaction mixture was stirred for 2 hr at room temperature. After dichloromethane was removed under reduced pressure, the resulting residue was flash-chromatographed to provide Fmoc-Gly-ovalicinol (0.39 mmol, 65%). Next, Fmoc-Gly-ovalicinol (0.09 mmol) was treated with 20% piperidine in DMF (2 ml) at room temperature for 10 min, and the DMF was removed under high vacuum. The resulting solid was redissolved in 2 ml of DMSO, and disuccin-imidyl suberate (0.9 mmol) was added at room temperature. After overnight stirring, DMSO was removed under high vacuum, and the resulting crude product was flash-chromato-graphed to give monosuccinimidyl suberate-Gly-ovalicinol (0.06 mmol, 68%).

[0049] Monosuccinimidyl suberate-Gly-ovalicinol (12 μmol) in DMSO (0.6 ml) was added to DMSO solution (1 ml) containing I kBa peptide (3.67 μmol) and dimethylaminopyridine (11 μmol). After 20 min stirring at room temperature, the coupling reaction was completed, which was confirmed by a Kaiser test. DMSO was removed under high vacuum, and the resulting crude product was repeatedly washed with dichloromethane and methanol to remove excess monosuccinimidyl suberate-Gly-ovalicinol to give the final product, IkBa peptide-suberate-Gly-ovalicinol (5.8 mg, 2.59 μmol, 70%). The final product was characterized by electrospray (ES) mass spectrometry. ES-MS (M 1 H)1 for ovalicinol-Gly-suberate-IkBa peptideydimethylaminopyridine was 2,231.56 Da. All other intermediates were characterized by 500-MHz 1H NMR spectroscopy.

[0050] MetAP-2-Protac-1 Coupling Assay

[0051] MetAP-2 (9 μM) was incubated with increasing concentrations of Protac-1 (dissolved in water) at room temperature for 45 min. Reactions were supplemented with SDS loading dye, fractionated on an SDS/10% polyacryl-amide gel, transferred onto a nitrocellulose membrane, and immunoblotted with rabbit polyclonal anti-MetAP-2 antiserum (Zymed). Enhanced chemiluminescence was performed by using Amersham Pharmacia detection reagents.

[0052] Tissue Culture and Transfections

[0053] 293T cells were cultured in DMEM with 10% (vol/vol) FBS (GIBCO), penicillin (100 units/ml), strep-tomycin (100 mg/ml), and L-glutamine (2 mM). Cells were split 1:5 before the day of transfection and transiently transfected with 40 μg of plasmid. Cells were 60% confluent in 100-mm dishes on the day of transfection. DNA [20 μg of pFLAG-Cul1 (RDB 1347) and 20 μg of pFLAG-β-TRCP(RDB1189)] was added and the cells were transfected using calcium phosphate precipitation, as described (See Lyapina, S. A., et al., Proc. Natl. Acad. Sci. USA, 95:7451-56 (1998)). Cells were harvested 30 hr after transfection. Five micrograms of pGL-1, a plasmid containing the cytomegalovirus (CMV) promoter linked to the green fluorescent protein (GFP) cDNA, was cotransfected into cells at the same time to assess transfection efficiency. In all experiments, greater than 80% of the cells were GFP-positive at the time of harvesting.

[0054] Immunoprecipitation and Ubiquitination Assays

[0055] 293T cell pellets were lysed with 200 μl of lysis buffer (25 mMTris/Cl, pH 7.5/150 mM NaCl/0.1% Triton X-100/5 mM NaF/0.05 mM EGTA/1 mM PMSF). Pellets from cells transfected with vector, pFLAG-β-TRCP, or pFLAG-Cul-1 were vortexed for 10 sec, then incubated on ice for 15 min. After centrifugation at 13,000 rpm in an Eppendorf microfuge (Germany) for 5 min at 4° C., the supernatant was added to 20 μl of FLAG M2 beads (Sigma), which were washed with lysis buffer three times before immunoprecipitation. Lysates were incubated with the beads on a rotator for 2 hr at 4° C., followed by one wash with buffer A (25 mM Hepes buffer, pH 7.4/0.01% Triton X-100/150 mM NaCl) and one wash with buffer B (the same buffer without the Triton X-100). For binding assays (FIG. 3), 50% (10 μl) of the 9 μM MetAP-2/50 μM Protac-1 mixture was loaded as input; the other 50% was added to the beads. After the addition of ligand, the beads were rotated at room temperature for 1 hr. The beads were washed once each with buffers A and B. After centrifugation at 13,000 rpm in an Eppendorf microfuge (Germany) for 1 min, half of the bead and supernatant fractions, representing bound and unbound, respectively, were evaluated by Western blotting as described above. For ubiquitination reactions, 4 μl of 18 μM MetAP-2, 4 μl of 100 μM Protac-1, 0.5 ml of 0.1 μg/μl purified mouse E1, 1 μl of 0.5 μg/μl human Cdc34 E2, and 1 μl of 25 mM ATP were added to 20 μl (packed volume) of washed FLAG-M2 beads. For competition experiments, the IPP (100 μM final) or OVA(100 μM final) was added simultaneously with the Protac-1. Reactions were incubated for 1 hr at 30° C. in a thermomixer (Eppendorf) with constant mixing. SDS/PAGE loading buffer was added to terminate reactions, which were evaluated by Western blotting as described above.

[0056] The experiments performed herein used two different preparations of MetAP-2. One preparation consisted primarily of a 47-kDa fragment that was generated either by a contaminating protease or by slow autoproteolysis. The second preparation consisted almost entirely of full-length 67-kDa MetAP-2. Essentially identical results were obtained with both preparations.

[0057] Degradation Experiments with Xenopus Extracts

[0058] Extracts from unfertilized Xenopus laevis eggs were prepared the day of the experiment, as described (See Murray, A. W., Methods Cell Biol., 3:581-605 (1991)). The MetAP-2-Protac-1 mixture (4 μl of 9 μM MetAP-2 plus 50 μMProtac-1) or MetAP-2 alone was added to 10 ml of extract in addition to OVA (10 μM final), constitutively active IKK (IKK-EE, 0.4 μg), and okadaic acid (OA; 10 μM final). N-acetyl-leu-leu-norleucinal (LLnL, 50 μM final), epoxomicin (10 μM final), or DMSO vehicle were added to the indicated concentrations to inhibit degradation by the proteasome. The protease inhibitors chymostatin, pepstatin, and leupeptin (15 μg/μl final concentration) also were added to the extracts. Reactions were incubated for the indicated times at room temperature and terminated by adding 50 μl of SDS/PAGE loading buffer. Samples were evaluated by Western blotting as described above.

Example 2

[0059] MetAP-2 Specifically Binds Protac-1 in Vitro

[0060] The IkBa-OVA chimera, Protac-1 (FIG. 1B), was synthesized as described above. To demonstrate that purified MetAP-2 bound Protac-1, we incubated MetAP-2 (18 mM) with increasing concentrations of Protac-1 (FIG. 2A). A Western blot analysis was performed with anti-MetAP-2 antiserum. At Protac-1 concentrations of 10 μM and 10 mM, two bands were observed; the lower band represents unbound MetAP-2, and the upper band represents a complex of MetAP-2 bound to Protac-1. The addition of Protac-1 at higher concentrations did not increase the yield of MetAP-2-Protac-1 complexes, suggesting that only a fraction of the MetAP-2 molecules were active and able to bind Protac-1. Combining MetAP-2 with either free IPP or free OVA did not yield the doublet observed with Protac-1. The specificity of MetAP-2 interaction with Protac-1 in vitro was also tested. Free OVA, but not free IPP, inhibited the formation of the MetAP-2-Protac-1 complex (FIG. 2B). Therefore, the results demonstrate that MetAP-2 was specifically conjugated to the OVA moiety of Protac-1 in a concentration-dependent manner.

Example 3

[0061] Protac-1 Recruits MetAP-2 to SCF

[0062] Before testing the activity of Protac-1, an approach to isolate and assay SCFβ-TRCP complexes in vitro as described (See Tan, P., et al., Mol. Cell, 3:327-33 (1999)) was first adapted. Lysates from 293T cells transfected with plasmids that encoded β-TRCP and Cul-1 proteins tagged with the FLAG epitope at the N terminus were immunoprecipitated with anti-FLAG antibody-conjugated resin. Immunoblot analysis confirmed that all components of SCFβ-TRCP were present in the anti-FLAG immunoprecipitate, including Skp1, Hrt1, and the transfected FLAG Cul-1 and FLAG β-TRCP (data not shown). Furthermore, control experiments confirmed that these immunoprecipitates promoted ubiquitination of IKK-phosphorylated glutathione S-transferase-IkBa in a manner that was inhibited by the IPP and Protac-1 (data not shown; ref. 7). To determine whether Protac-1 could recruit MetAP-2 to SCFβ-TRCP, MetAP-2 (18 μM) was first incubated with Protac-1 (100 μM) for 45 min at room temperature. After isolation of SCFβ-TRCP complexes, the anti-FLAG beads were supplemented with the MetAP-2-Protac-1 mixture and rotated at room temperature for 1 hr. The beads and supernatant then were evaluated by Western blot analysis for the presence of MetAP-2. Anti-FLAG beads coated with SCFβ-TRCP, but not control beads preincubated with untransfected 293T cell lysates, specifically retained a fraction of the MetAP-2-Protac-1 complex and not the unliganded MetAP-2 (FIG. 3). These results demonstrate that Protac-1 specifically recruited MetAP-2 to SCFβ-TRCP.

Example 4

[0063] Protac-1 Mediates the Ubiquitination of MetAP-2

[0064] Since Protac-1 was able to tether MetAP-2 to SCFβ-TRcP, the question became whether MetAP-2 could be ubiquitinated. To answer this question, anti-FLAG beads coated with SCFβ-TRCP was supplemented with ATP, MetAP-2-Protac-1, plus purified E1, E2 (human Cdc34), and ubiquitin. After incubation, Western blot analysis was performed with anti-MetAP-2 antiserum. This experiment was repeated with two different preparations of purified MetAP-2: one contained primarily a 47-kDa autocatalyzed cleavage product (FIG. 4A), and the other contained full-length 67-kDa protein (FIG. 4B). In both cases, MetAP-2-Protac-1 was extensively modified in the presence of SCFβ-TRCP-coated beads but not control beads (Mock). Substitution of the methyl ubiquitin, which acts as a chain-terminator of polyubiquitination, collapsed the high molecular weight forms of modified MetAP-2 to a series of 2-3 bands migrating directly above unmodified MetAP-2-Protac-1 complex (FIG. 4C, compare lanes 2 and 3 with lane 4), confirming that MetAP-2 was ubiquitinated by SCFβ-TRCP. The next test was whether MetAP-2 ubiquitination depended on Protac-1. As shown in FIG. 4C, MetAP-2 was not ubiquitinated in the absence of either SCFβ-TRCP (lane 1) or Protac-1 (lanes 5 or 6; for some experiments, methyl ubiquitin was used in place of ubiquitin to simplify detection of ubiquitin conjugates). Moreover, unlinked OVA (50 mM) plus IPP (50 mM) were not able to substitute for the OVA-IPP conjugate (lane 7). Protac-1-dependent ubiquitination of MetAP-2 was specific; it was readily competed for by free IPP (FIG. 4D, lane 3 vs. lane 2). In contrast, free OVA did not compete (FIG. 4D, lane 4), presumably because it was unable to displace the Protac-1 that previously was linked covalently to MetAP-2. Taken together, these observations indicate that Protac-1 specifically elicited ubiquitination of MetAP-2 by SCFβ-TRCP, and that successful targeting required that the two components of Protac-1 be bound together as a chimeric molecule.

Example 5

[0065] MetAP-2-Protac-1 is Degraded in Xenopus Egg Extracts

[0066] The experiments described above demonstrated that MetAP-2 was ubiquitinated in a Protac-1-dependent manner by highly purified SCFβ-TRCP. However, the key issues are whether Protac-1 can specifically activate MetAP-2 degradation, and whether targeted degradation can be achieved by endogenous ubiquitin/proteasome pathway components at typical intracellular concentrations. To address these questions, MetAP-2 was preincubated with Protac-1 to allow the complexes to form, and then added the mixture to Xenopus egg extract supplemented with IKK-EE (See Mercurio, F., Zhu, et al., Science, 278:860-66. (1997)), OA, and OVA. The addition of IKK-EE and OA was intended to sustain phosphorylation of the IkBa peptide moiety of Protac-1, whereas OVA was added to prevent the further linkage of Protac-1 to MetAP-2. Remarkably, MetAP-2-Protac-1 complex (top band) but not MetAP-2 alone (bottom band) was mostly degraded after 30 min (FIG. 5). Degradation of MetAP-2-Protac-1 was attenuated in extracts supplemented with the proteasome inhibitors LLnL or epoxomicin (See Meng, L., et al., Proc. Natl. Acad. Sci. USA, 96:10403-08 (1999)), but not by other protease inhibitors (chymotrypsin, pepstatin, and leu-peptin) added to the reaction. Moreover, addition of both IKK-EE and OA was required for optimal degradation of MetAP-2-Protac-1. Similarly, we have seen specific turnover of the MetAP-2-Protac-1 complex, but not free MetAP-2, in three independent experiments. Because the IPP does not have lysines and the OVA does not have free amino groups, it is unlikely that Protac itself serves as a target for ubiquitin-dependent proteolysis. Taken together, these results demonstrate that Protac-1 targeted MetAP-2 for degradation by means of the proteasome. MetAP-2 turnover was very specific, in that maximal degradation required agents predicted to sustain phosphorylation of the IkBa peptide.

[0067] The present invention provides a method to target proteins, at will, to the ubiquitin/proteasome pathway. The linchpin of the strategy described here is the development of chimeric molecules—referred to as Protacs—that link a desired target protein to a ubiquitin ligase. A related method for inducing selective heterodimerization of proteins to regulate activation and termination of signaling pathways has been described (See Belshaw, P. J., et al., Proc. Natl. Acad. Sci. USA, 93:4604-07 (1996)).

[0068] As a target protein for a proof of principle experiment, MetAP-2, which covalently binds the angiogenesis inhibitor OVA was chosen. For the ubiquitin ligase, the SCFβ-TRCP complex was chosen, for reasons described in more detail below. Although MetAP-2 has 36 lysines, it has not been reported to be an unstable protein in vivo, and it was not clear whether it would serve as a substrate for SCFβ-TRCP, or whether it would be degraded by the ubiquitin/proteasome pathway (See Liu, S., et al., Science, 282:324-27 (1998)). The data presented in the present invention demonstrate that Protac-1 can recruit MetAP-2 to SCFβ-TRCP for ubiquitination. In addition, the data presented in the present invention demonstrate that Protac-1 specifies degradation of MetAP-2 by the endogenous ubiquitin/proteasome pathway in Xenopus egg extracts.

[0069] As one embodiment of the present invention, SCFβ-TRCP was chosen as a candidate ubiquitin ligase for the development of Protac technology for two reasons. First, the apparent constitutive activity of SCF complexes (See Deshaies, R. J., Annu. Rev. Cell Dev. Biol., 15:435-67 (1999)) enables a general strategy to manipulate normal or diseased cells. Second, the mechanism underlying substrate selection is well understood for SCFβ-TRCP. Pioneering work by Ben-Neriah and coworkers (See Yaron, A., et al., Nature (London), 396:590-94 (1998); and Yaron, A., et al., EMBO J., 16:6486-94 (1997)) demonstrated that a 10-aa internal phosphopeptide mediates ubiquitination and degradation of IkBa. Subsequently, it was established that β-TRCP is the receptor that links this phosphopeptide to the ubiquitin/proteasome pathway (See Yaron, A., et al., Nature (London), 396:590-94 (1998); and Karin, M., et al., Annu. Rev. Immunol., 18: 621-63 (2000)). Furthermore, the experimental data provided by Zhou, Howley, and colleagues (See Zhou, P., et al., Mol. Cell, 6:751-56 (2000)) support our discovery. Zhou et al have demonstrated that engineered SCF complexes can be used to target heterologous proteins for destruction. Specifically Zhou et al. fused the human papillomavirus E7 protein to the F-box proteins Cdc4 and b-TRCP to create chimeras, which assemble to form hybrid SCF Cdc4-E7 and SCFβ-TRCP-E7 complexes. E7 binds tightly to retinoblastoma protein (Rb), and the F-box protein/E7 chimeras stimulate turnover of Rb by means of the SCF pathway in both yeast and mammalian cells. The F-box fusion approach depends on gene transfer of the chimeric F-box protein.

[0070] Protacs are useful research tools for manipulating the phenotype of cells by means of the targeted elimination of specific proteins, or as useful therapeutic agents for targeting the elimination of disease-promoting proteins. Furthermore, Protacs may be modified to enhance delivery to cells. For example, attachment of the tat peptide (See Nagahara, H., et al., Nat. Med., 4:1449-52 (1998)) may promote transduction of Protac-1 into cells, or a β-TRCP-binding peptide that is derived from the HIV Vpu protein (See Margottin, F., et al., Mol. Cell., 1:565-74 (1998); and Schubert, U., et al., J. Virol., 72:2280-88 (1998)), and that is phosphorylated by constitutively active casein kinase II may allow delivery of an unphosphorylated Protac to cells. Alternatively one could identify small molecules that can substitute for the E3 targeting activity of the IPP.

[0071] The present invention also provides methods to develop an entire suite of Protac compounds (FIG. 6). Several approaches have been developed for identifying small molecules that bind to any target protein of interest (See Borchardt, A., et al., Chem. Biol., 4:961-68(1997); MacBeath, G., et al., J. Am. Chem. Soc., 121:7967-68 (1999); and You, A. J., et al., Chem. Biol., 4:969-75 (1997)). Candidates emerging from such screens would serve as platforms for the production of new Protacs, regardless of the topology of their interaction with the target protein. Because the human genome encodes a large number of RING-and HECT-based ubiquitin ligases, the present invention could be used to develop multiple Protacs that target a single protein to allow for fine-tuning its rate of degradation. Moreover, because some ubiquitin ligases are likely to exhibit restricted patterns of expression, the present invention can be used to develop Protac-based drugs whose effects are limited to specific tissues.

Example 6

[0072] Targeting the Estrogen Receptor for Ubiquitination and Degradation in Breast Cancer Cells

[0073] Breast cancer is the most common cancer among women and the second largest cause of death as a result of malignancy in women. During their lifetime, 12% of all women in the U.S. will be diagnosed as having breast cancer, and 3.5% will die of this disease. Breast cancer cells often require activation of the estrogen-specific steroid hormone receptor by estrogen to proliferate, and it is well known that steroid hormone receptor signaling plays a pivotal role in progression of breast cancer disease. In estrogen receptor (ER) positive metastatic breast cancer, hormonal therapy either alone or as adjuvant chemotherapy can help to restrain disease progression. In ER-positive breast cancer cells, tamoxifen exhibits its antiproliferative, tumoristatic activities by binding to the receptor and competitively inhibiting estradiol binding (Jordan V C, Collin M M, Rowsby L and Prestwich G. J. Endocrinol 75: 305-316, 1977; Connor C E, Norris J D, Broadwater G, Willson T M, Gottardis M M, Dewhirst M W and McDonnell D P. Cancer Res 61: 2917-2922, 2001). However, a large percentage of patients with metastatic breast cancer are resistant to tamoxifen at diagnosis or become unresponsive to the antiestrogenic actions of tamoxifen. Consequently, metastatic breast cancer carries a poor prognosis and there is no effective treatment for late-stage disease.

[0074] The following example is based on the novel conceptual approach to treating breast cancer that centers on the ER polypeptide. We propose to target the ER for degradation via the ubiquitin-dependent proteolysis pathway (Deshaies R J. Annu Rev Cell Dev Biol 15: 435-467, 1999). Specifically, heterobifunctional small molecules (“Protacs”, for Proteolysis-Targeting Chimeric molecules) will by synthesized. One moiety of Protac binds to the ubiquitin ligase SCFbeta-TRCP, whereas the second moiety (Estradiol or E2) binds to ER. Via its two moieties, the Protac will tether ER to SCFbeta-TRCP, which in turn is will activate the ubiquitination and subsequent proteolysis of ER.

[0075] As proof of principle for Protac technology, we targeted methionine aminopeptidase-2 (MetAP-2) for ubiquitination and degradation using a Protac. MetAP-2 has not been reported to serve as a substrate for the SCF ubiquitin ligases. MetAP-2 is a naturally occurring ligand for the anti-angiogenic drug fumagillin, and forms a covalent linkage with both fumagillin and the related compound ovalicin. A chimeric compound (Protac) was synthesized that contained the IkBalpha (IkBa) phosphopeptide linked to ovalicin. MetAP-2 is ubiquitinated in the presence of Protac-1 and is degraded by proteasomes in Xenopus extracts (Sakamoto K M, Kim K B, Kumagai A, Mercurio F, Crews C M and Deshaies R J. Proc Natl Acad Sci U S A 98: 8554-8559, 2001). Since the last submission of this proposal, we have also demonstrated that the Protac technology can be applied to other ubiquitin ligases. Cbl is a single polypeptide ubiquitin ligase that regulates critical signaling molecules through ubiquitination and proteolysis. A zap70 phosphopeptide-ovalicin Protac can recruit MetAP-2 for ubiquitination (data not shown). Importantly, we have now demonstrated that a Protac containing the IkBa phosphopeptide linked to estradiol can promote ubiquitination and degradation of the estrogen receptor in vitro through noncovalent interactions.

[0076] Synthesis of IB-DHT

[0077] Dimethylformamide (DMF, 28 μl) was added to dichloromethane solution (20 ml) containing Fmoc-Gly-OH (1.06 g, 3.56 mmol) and oxalyl chloride (0.62 mL, 7.1 mmol) at 0° C. After 3 hr of stirring at room temperature under N2 atmosphere, dichloromethane was evaporated using a stream of nitrogen gas and further dried under high vacuum for 2 hours. The resulting solid residue was redissolved in dichloromethane (10 mL) and was combined with 5 α-dihydrotestosterone (DHT, from Sigma) (0.18 g, 0.61 mmol) and dimethylaminopyridine (0.58 g, 4.75 mmol) in dichloromethane (10 ml) at 0° C. The reaction mixture was stirred overnight at room temperature. After water (15 mL) was added to the reaction mixture, crude product was extracted with dichloromethane (15 mL×3), dried over MgSO4 and finally concentrated under reduced pressure. The resulting residue was flash-chromatographed to provide DHT-Gly-Fmoc (oil, 0.21 g, 0.37 mmol, 61%). Next, DHT-Gly-Fmoc (0.12 g, 0.21 mmol) in 2 mL of tetrahydrofuran (THF) was treated with 1 M solution of tetrabutylammonium fluoride (TBAF) in THF (0.3 mL) at room temperature for 20 min, and THF was removed under reduced pressure. The resulting crude product was flash-chromatographed to yield DHT-Gly-NH2 (a white solid, 49 mg, 67%). Next, DHT-Gly-NH2 (49 mg, 0.14 mmol) was dissolved in 1 ml of dimethylformamide (DMF), and disuccinimidyl suberate (0.265 g, 0.72 mmol) was added at room temperature. After 6 hour stirring at room temperature, DMF was removed under high vacuum, and the resulting crude product was flash-chromatographed to give DHT-Gly-monosuccinimidyl suberate (a white solid, 70 mg, 0.12 mmol, 86%).

[0078] DHT-Gly-monosuccinimidyl suberate (5.5 mg, 9.1 μmol) in DMSO (0.6 mL) was added to DMSO solution (1 mL) containing IB peptide (4.5 mg, 2.75 μmol) and dimethylaminopyridine (2 mg, 16 μmol). After 20 min stirring at room temperature, the coupling reaction was completed, which was confirmed by a Kaiser test. DMSO was removed under high vacuum, and the resulting crude product was repeatedly washed with dichloromethane and methanol to remove excess DHT-Gly-monosuccinimidyl suberate to give the final product, IB peptide-suberate-Gly-DHT (3 mg, 1.4 μmol, 51%). The final product was characterized by electrospray (ES) mass spectrometry. ES-MS (M+H)+ for ovalicinol-Gly-suberate-IB peptide/dimethylaminopyridine was 2,118.73 Da. All other intermediates were characterized by 400-MHz 1H NMR spectroscopy.

[0079] Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

	Number	Date	Country
Parent	09953473	Sep 2001	US
Child	10299203	Nov 2002	US

Proteolysis targeting chimeric pharmaceutical

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