WO2007124171A2 - Compositions and methods for treating trinucleotide repeat disorders - Google Patents

Abstract

This invention relates to compositions and methods for treating disorders resulting from protein-misfolding. We describe exemplary compounds, which may be contained in pharmaceutical compositions, the screening methods by which they were discovered, and their use as therapeutic or prophylactic agents.

Description

COMPOSITIONS AND METHODS FOR TREATING TRINUCLEOTIDE REPEAT DISORDERS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Application serial No. 60/793,474, filed on April 20, 2006. For the purpose of any U.S. patent that may issue based on the present application, U.S. Application Serial No. 60/793,474 is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

This invention relates to compositions and methods for treating cancer and neurodegenerative disorders and more particularly, methods of making and using compounds that modulate hsp90. We describe exemplary compounds, which may be contained in pharmaceutical compositions, the screening methods by which they were discovered, and their use as therapeutic or prophylactic agents.

BACKGROUND

Although the amino acid sequence of a protein ultimately dictates its native conformation, most polypeptides would fail to fold effectively in the highly concentrated, complex environment of the cell without the assistance of molecular chaperone proteins, a highly conserved group of proteins that serve to maintain the normal intracellular protein folding environment. Chaperone proteins generally function in multiprotein complexes. Such complexes play a central role in the folding of many proteins involved in a wide range of diseases including many cancers and neurodegenerative disorders.

SUMMARY

The present invention is based, in part, on our discovery of compositions and methods that can be used to treat or prevent diseases that are responsive to modulation of a heat shock protein (hsp), namely hsp90. The compositions and methods can, for example, be used in the treatment or prevention of cancers or neurological disorders. Cancers encompassed by the invention include breast cancer, ovarian cancer, endometrial cancer, prostate cancer and leukemia. Neurological disorders encompassed by the invention include disorders that are believed to be associated with the expression of a polypeptide containing an expanded polyglutamine (poly(Q)) repeat. These disorders include, for example, Huntington's disease, spinocerebellar ataxia, hemiplegϊc migraine, spinobulbar muscular atrophy (SBMA), dentatorubral- pallidoluysian atrophy, and amyloidosis

Although the compounds and compositions described herein are not limited to those that exert an effect by any particular cellular mechanism, they include those that modulate the activity of an hsp90. Hsp90 functions within the context of a multiprotein complex to assist in the folding of newly synthesized proteins and to stabilize and refold denatured proteins after cellular stress. The hsp90 chaperone complex targets a discrete subset of proteins, termed hsp90 client proteins, many of which have oncogenic potential. The hsp90 chaperone complex is referred to variously as the hsp90 chaperone complex, hsp90 molecular chaperone complex, the hsp90 chaperone machine, the hsp90 chaperone machinery, and the hsp90 molecular chaperone machinery. The multiprotein complex encompasses five particular proteins, hsp90, hsp40, hsp70, Hop and p23, as well as other co-chaperones (e.g., CDC37, Ahal and CHIP) and immunophilins (e.g., FKBP51, FKBP52, cyclopbilin- 40, UNC-45).

Hsp90 is highly conserved evolutionarily and eukaryotes express several isoforms. Hsp90 proteins affected, directly or indirectly, by a composition of the invention can include any of the various isoforms of hsp90. For example, the hsp90 can be an hsp90α, the major, inducible, form located in the cytosol; hsp90 β, the minor, constitutive, form located in the cytosol; or hsp90N, a membrane associated variant implicated in cellular transformation. The compound may also affect an hsp90 analogue, such as Grp94, a form localized in the endoplasmic reticulum or Hsp/754 TRAPl, a form localized in the mitochondrial matrix.

We may refer to a compound that modulates the activity of hsp90 as an hsp90 inhibitor. There are many ways in which the activity of hsp90 can be inhibited. We may refer to the protein that is affected by the compound as a target protein. The target protein can be an hsp90 protein. For example, an hsp90 inhibitor may inhibit hsp90 directly, for example, by inhibiting the ATP binding or the ATPase activity of hsp90, or by inhibiting hsp90 dimerization or the binding of hsp90 to a particular client protein. Alternatively, the target protein can be a protein that is active upstream or downstream in a biochemical pathway in which the hsp90 protein is active. The target protein can be another member of the hsp90 complex, for example, hsp40, hsp70, Hop and p23 or a co-chaperones (e.g. CDC37, Ahal and CHIP) or immunophilins {e.g., FKBP51, FKBP52, cyclophilin-40, UNC-45). Alternatively, the target protein can be an hsp90 client proteins, for example, a steroid hormone receptor (e.g., the estrogen receptor, the androgen receptor, or the progesterone receptor) or a protein having an expanded poly(Q) repeat. Alternatively, the target protein could be an enzyme that modulates the target protein (by inhibiting or promoting its activity) through a post-translational modification (e.g., phosphorylation, glycosylation, acetylation, alkylation, isoprenylation, or lipoylation).

A compound that inhibits hsp90 can result in the increased degradation of hsp90 client polypeptides associated with specific disorders, thereby relieving a sign, symptom or phenotype of the associated disorder. For example, increased degradation of a steroid hormone receptor may be useful in the treatment or prophylaxis of particular cancers associated with elevated levels of such receptors (e.g., breast cancer, prostate cancer or ovarian cancer). Alternatively, increased degradation of a polypeptide containing an expanded poly(Q) repeat may be useful in the treatment or prophylaxis of neurological disorders associated with the overexpression of a polypeptide containing an expanded poly(Q) repeat (e.g., Huntingdon's disease, spinocerebellar ataxia, hemiplegic migraine, spinobulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy, or amyloidosis).

These scenarios are meant to describe the manner in which the compounds of the invention may exert their effect on intracellular protein levels within a cell, but the invention is not so limited. The invention encompasses compounds according to the formulas described herein, compositions containing them (e.g., pharmaceutical formulations and kits), and methods of using them regardless of the mechanism by which they work.

The compounds useful for the pharmaceutical compositions described herein include compounds of Formula VI:

(VI) or pharmaceutically acceptable salts or prodrugs thereof, wherein the constituent members are defined herein. In some embodiments, the compositions can include compounds of Formula Via:

Via or pharmaceutically acceptable salts or prodrugs thereof, wherein the constituent members are defined herein. An exemplary compound conforming to Formula Via can have the structure 2-((E)-((E)-3-(4-chlorophenyl)allylidene)amino)phenol, listed as Compound A9 in Table 1.

In other embodiments, the compositions can include compounds of Formula VIb:

VIb or pharmaceutically acceptable salts or prodrugs thereof, wherein the constituent members are defined herein. An exemplary compound conforming to Formula VIb can have the structure 3-[2-(2,4-dimethyl-l,3-thiazol-5-yl)-2- oxoethylidene]-3,4-dihydro-2H-l,4-benzoxazin-2-one, listed as Compound A24 in Table 1. In other embodiments, the compositions can include biologically active portions of compounds of Formula VI. A compound that comprises a portion of a compound of Formula VI that retains the ability to function {e.g., retains sufficient activity to be used for one or more of the purposes described herein) is a biologically active portion. An exemplary biologically active fragment is 2-aminophenol. The invention also encompasses pharmaceutically acceptable salts or solvates of the compounds described above and further herein and prodrugs, metabolites, structural analogs, and other pharmaceutically useful variants thereof. These other variants may be, for example, a complex containing the compound and a targeting moiety, as described further below, or a detectable marker (e.g., the compound may be joined to a fluorescent compound or may incorporate a radioactive isotope). When in the form of a prodrug, a compound may be modified in vivo (e.g., intracellularly) after being administered to a patient or to a cell in culture. The modified compound (i.e., the processed prodrug) may be identical to a compound described herein and will be biologically active or have enough activity to be clinically beneficial. The same is true of a metabolite; a given compound may be modified within a cell and yet retain sufficient biological activity to be clinically useful.

The invention includes, for example, a pharmaceutical composition comprising a compound of Formula A9 (2-((E)-((E)-3-(4- chlorophenyl)allylidene)amino)phenol) (or a salt of the compound of Formula A9) or Formula A24 ((3-[2-(2,4-dimethyl-l,3-thiazol-5-yl)-2-oxoethylidene]-3,4-dihydro- 2H-l,4-benzoxazin-2-one) (or a salt of the compound of Formula A24). In one embodiment, the invention includes a pharmaceutical composition comprising a compound of Formula Al 8 (4-(4-(3,4-dichlorophenyl)thiazol-2-ylamino)phenol).

A pharmaceutical composition as described herein can be formulated for any acceptable route of administration, including oral and parenteral routes of administration.

Packaged products (e.g., sterile containers containing one or more of the compounds described herein and packaged for storage, shipment, or sale) and kits, including at least one compound of the invention and instructions for use, are also within the scope of the invention.

In one aspect, the invention features substantially pure preparations of the compounds described herein or combinations thereof. A naturally occurring compound is substantially pure when it is separated to some degree from the compound(s) or other entities (e.g., proteins, fats, or minerals) it is associated with in nature. For example, a naturally occurring compound described herein is substantially pure when it has been separated from 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the compound(s) or other moieties it is associated with in nature. While the compounds of the invention may be naturally occurring and may be purified using conventional techniques, they may also be non-naturally occurring and may be synthesized (naturally occurring compounds can be synthesized as well). Compounds prepared by chemical synthesis are substantially pure, as are compounds that have been separated from a library of chemical compounds. A substantially pure compound may be one that is separated from all the other members of the compound library or it may be one that has been separated to a limited extent (e.g., it may remain associated with a limited number (e.g., 1, 2, 3, 4, or 5-10) of other members of the library). A compound library is not a pharmaceutical or therapeutic composition.

Regardless of their original source or the manner in which they are obtained, the compounds of the invention can be formulated in accordance with their use. For example, the compounds can be formulated within compositions for application to cells in tissue culture or for administration to a patient. For example, the compounds can be mixed with a sterile, pharmaceutically acceptable diluent (such as normal saline). As noted below, and as known in the art, the type of diluent can vary depending upon the intended route of administration. The resulting compositions can include additional agents, such as preservatives. The compounds may also be applied to a surface of a device (e.g., a catheter) or contained within a pump, patch, or other drug delivery device.

The methods of the invention include methods for treating a subject (e.g., a human patient) with cancer or a neurological disorder. These methods can include the steps of a) identifying a subject who is experiencing or is likely to experience cancer or a neurological disorder; and b) providing to the subject a therapeutic amount of a pharmaceutically acceptable composition comprising compound of Formula VI. The compositions can be administered to a subject in a variety of ways. For example, the compositions can be administered transdermally or injected (infused) intravenously, subcutaneously, intracranially, intramuscularly, intraperitoneally, or intrapulmonarily. Oral formulations are also within the scope of the present invention. The dosage required will depend upon various factors typically considered by one of ordinary skill in the art. These factors include the route of administration, the nature of the formulation, the nature of the patient's illness, the subject's size, weight, surface area, age, gender, other drugs being administered to the patient, and the judgement of the attending physician. The compositions can be administered along with or in addition to standard treatments for particular cancers or neurological disorders, e.g., drug therapy, immunotherapy, radiation therapy, or surgery.

Cancers amenable to the diagnostic, therapeutic, and/or prognostic methods of the invention can be cancers that are responsive to the modulation of hsp90. While we believe we understand certain events that occur in the course of treatment, the compositions of the present invention are not limited to those that work by affecting any particular cellular mechanism. Any form of cancer which is associated with misregulation of hsp90 {e.g., overexpression or altered client protein binding or activity) is within the scope of the invention. Such cancers can include, but are not limited to, breast cancer, ovarian cancer, endometrial cancer, prostate cancer and leukemia. Cancers encompassed by the methods of the invention can also include, but are not limited to, cancers characterized by the misexpression of a polypeptide comprising an expanded poly(Q) repeat. Examples of such cancers include breast, ovarian and endometrial cancers, which may have altered expression of the estrogen or progesterone receptor or prostate cancer, which may have altered expression of the androgen receptor, or the TeI-AML- 1 gene fusion in leukemia.

The methods includes methods for identifying an agent that selectively decreases intracellular levels of a polypeptide comprising an expanded poly(Q) repeat. Agents or compounds identified by these methods may be useful hsp90 inhibitors. The method includes providing a first cell that expresses a first subunit of beta-galactosidase and includes a polynucleotide sequence including an inducible promoter operably linked to a polynucleotide encoding a first fusion polypeptide having an expanded poly(Q) repeat and a second, complementary subunit of beta-galactosidase. For example, the first cell can express the delta (Δ) subunit of beta-galactosidase, and the fusion polypeptide can include the alpha (α) subunit of beta-galactosidase. In the alternative, the first cell can express the α-subunit of beta-galactosidase, and the fusion polypeptide can include the Δ-subunit of beta-galactosidase. The method also includes providing a second cell that expresses a first subunit of beta-galactosidase and includes a polynucleotide sequence including an inducible promoter operably linked to a polynucleotide encoding a second fusion polypeptide having a wildtype poly(Q) repeat and a second, complementary subunit of beta-galactosidase, as described for the first cell. In each of the first and second cells, when the first subunits of beta-galactosidase and the second, complementary sub units of beta-galactosidase are expressed, functional beta-galactosidase is produced in the cells. The method further includes inducing expression of the fusion polypeptides in the first and second cells, contacting the first and second cells with a candidate agent, and determining the levels of functional beta-galactosidase in the cells. A decrease in the level of functional beta-galactosidase in the first cell relative to the level of functional beta- galactosidase in the second cell identifies the agent as one that selectively decreases intracellular levels of a polypeptide containing an expanded poly(Q) repeat.

The polypeptide having an expanded poly(Q) repeat can be a fragment of any polypeptide known to exhibit such repeats naturally. For example, the polypeptide can be a huntingtin polypeptide, or a fragment of a huntingtin polypeptide containing the expanded repeat. Alternatively, the polypeptide can be atrophin-1, ataxin-1, ataxin 2, ataxin 3, the αla-voltage dependent calcium channel, ataxin 7, AJBl, or the androgen receptor, or a fragment of any of these polypeptides containing the expanded repeat. The expanded poly(Q) repeat useful in the assay can include at least 40 consecutive glutamine residues (e.g., 40, 45, 50, 60, 70, 90, 100, 105, or 110 glutamine residues) and the wildtype poly(Q) repeat can include 35 or fewer consecutive glutamine residues (e.g., 30, 25, 23, 20, 15, or 10 residues). The inducible promoter can be any inducible promoter, such as an ecdysone-responsive promoter.

The cells useful in the assays can be any cell type, such as any mammalian cell type. The cells can be neuronal cells, such as PC 12 cells. The screening method can also be automated. Candidate agents useful for the described methods can be small organic or inorganic molecules, a protein, a peptide, or a nucleic acid.

The methods can also include further assays to determine the mechanism of action of an agent that decreases intracellular levels of a polypeptide containing an expanded poly-(Q) repeat, such as determining whether the agent inhibits a chaperone or co- chaperone protein, such as heat shock protein of the hsp90 family.

The agents identified in our screening assays were compounds that decreased intracellular levels of a polypeptide containing an expanded poly(Q) repeat. Many of the compounds also failed to cause a decrease in the intracellular levels of a polypeptide containing a "wildtype" poly(Q) repeat sequence. While these compounds may decrease intracellular levels of a polypeptide containing an expanded poly(Q) repeat by increasing the rate of degradation of these polypeptides, or by decreasing the rate of synthesis, the invention is not limited to compounds that exert their effect on the disease process by any particular mechanism. The compounds identified in our assays, and useful for the pharmaceutical compositions described herein, are shown in Table 1.

Table 1. Compounds

A compound that causes a decrease in intracellular levels of a polypeptide containing an expanded poly(Q) repeat may cause decreased transcription or translation as compared to a counterpart polypeptide that does not contain an expanded poly(Q) repeat, or that contains a wildtype poly(Q) repeat. Alternatively, the compound may increase the rate of degradation of a polypeptide containing an expanded poly(Q) repeat. An increased rate of degradation can be caused, for example, by an effect on the proteosome of the cell, or an effect on the way the polypeptide interacts with the proteosome, or an affect on a chaperone or co-chaperone. For example, a compound that targets a chaperone that participates in the folding of the polypeptide containing the expanded poly(Q) repeat may result in a higher percentage of misfolded polypeptide which then increases the amount of the polypeptide targeted for degradation. The mechanism of action of compounds that decrease intracellular levels of a polypeptide containing an expanded poly(Q) repeat include but are not limited to those described above.

"PolyQ-containing" polypeptides include a number of consecutive glutamine residues, which may be described in the art as homopolymeric polyQ regions. While the number of consecutive glutamine residues may be quite low (e.g., as few as 3-10 (e.g., five)), polyQ-containing polypeptides typically have about 26 or more consecutive amino acid residues (e.g., 28, 30, 33, 34, 35, 36, 37, 40, 42, 47, 50, 52, 60, 65, 70, 72, 75, 80, 85, 95, 100, 103, 104, 110, 119, 120, 130, 140, 144, 151, 160, 170, 180, 190, 191, 195, 200, 210, 230, 250, 270 or 300 consecutive glutamine residues). For example, a glutamine-rich polypeptide can have at least 35 consecutive glutamine residues. Polypeptides having such a region of consecutive glutamine residues may also be referred to as having an "expanded" polyglutamine region. PoIyQ containing polypeptides can be naturally occurring polypeptides such as the huntingtin protein, atrophin-1, ataxin-1, ataxin 2, ataxin 3, the αla-voltage dependent calcium channel, ataxin 7, AEBl, and the androgen receptor. "Expanded," as used herein, means that the number of glutamines in the poly(Q) repeat region is greater than normal. The expanded poly(Q) repeat may also result in a disease phenotype. "Wildtype" htt protein contains 6-35Q, while Huntington's Disease (HD) patients an have expanded poly(Q) region containing 40-10OQ. Thus, an expanded poly(Q) repeat in htt means an htt polypeptide having a poly(Q) region of greater than 35 glutamines (e.g., 36, 37, 38, 39, 40, 45, 50, 75, 100, 150, or more glutamines). Accordingly, the invention features compounds that decrease levels of polypeptides containing expanded poly(Q) repeats, and methods of treating a subject in which any one of those polypeptides disrupts cellular function and results in a disease state (e.g., a subject having HD, spinocerebellar ataxia, hemiplegic migraine, spinobulbar muscular atrophy (SBMA), cancer, amyloidosis, or dentatorubral-pallidoluysian atrophy). For example, one or more of the compounds of the invention may inhibit gene expression, such as by inhibiting translation or transcription, or one of more the compounds of the invention may increase degradation of the polypeptide containing the expanded poly(Q) repeat. The increased sensitivity to degradation can result, for example, from increased targeting to the proteasome or by increased or altered interaction with chaperone proteins that leads to polypeptide misfolding. Accordingly, the invention features compounds that decrease levels of any one of the aforementioned polypeptides and methods of treating a subject in which any one of those polypeptides is expressed to an extent that cellular function is disrupted and a disease state results.

Certain compounds of the invention can decrease intracellular levels of polypeptides containing expanded poly(Q) repeat regions by inhibiting chaperones or co-chaperones that interact with the polypeptides. Thus the compounds of the invention can be used for treating a subject having a disorder, or diagnosed as having a disorder or at risk for developing a disorder caused by or associated with a misfolded polypeptide. Protein-mi sfolding diseases include, for example, Alzheimer's Disease, Parkinson's Disease, Amyolotrophic Lateral Sclerosis, prion disease, and the poly(Q) repeat diseases described above. In addition to determining the effect of a compound on polypeptide levels (and, in animal models or clinical trials, the effect of a compound on the signs and symptoms of a disease), the assays or screens can include a step in which one determines cellular toxicity. One can also generate a dose response profile of putative assay hits and record the results in a screening database (which is also within the scope of the present invention).

In specific embodiments, the compositions of the present invention can be administered to a subject having Huntington's disease, any of the several types of spinocerebellar ataxias {e.g., SCAl, SCA2, SCA6, SCA7 and Machado- Joseph disease (MJD/SCA3)), hemiplegic migraine, spinobulbar muscular atrophy (SBMA; also known as Kennedy's disease), and dentatorubral-pallidoluysian atrophy, breast cancer, ovarian cancer, endometrial cancer, prostate cancer and leukemia, or amyloidosis, or a disorder with a similar underlying cellular basis (i.e., an expanded poly(Q) repeat). The compositions of the present invention can be administered to a subject having other diseases caused by, or associated with, protein misfolding, such as Alzheimer's Disease, Parkinson's Disease, amylotrophic lateral sclerosis, or a prion disease, such as Creutzfeldt- Jakob disease.

The invention also includes methods of treating a subject who has been diagnosed as having, or who is at risk of developing, a disorder characterized by overexpression of a protein containing an expanded poly(Q) repeat. The methods generally include identifying the subject and administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein. For example, the treatment methods include administering a pharmaceutical composition to a subject diagnosed as having, or at risk of developing, Huntington's disease, spinocerebellar ataxia, hemiplegic migraine, spinobulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy, breast cancer, ovarian cancer, endometrial cancer, prostate cancer and leukemia, or amyloidosis.

In one embodiment, the invention includes a method of treating a subject who has a cancer characterized by misexpression of a polypeptide comprising an expanded poly(Q) repeat. The method includes administering to the patient a therapeutically effective amount of a pharmaceutically acceptable composition comprising a compound of Table 1, such as Formula Al 8 (4-(4-(3,4-dichlorophenyl)thiazol-2- ylamino)phenol) . Therapeutic methods featured in the invention can include the step of identifying a subject in need of treatment. The subject can be identified by, for example, a health care professional (e.g., a physician) on the basis of subjective or objective information (e.g., based on comments from the subject, a physical examination, and/or on measurable parameters (i.e., diagnostic tests)). Subjects who are treated with the compounds featured in the invention may have been diagnosed with any disease characterized by a polypeptide containing an expanded poly(Q) repeat or a misfolded polypeptide. Alternatively, the subject may be at risk for developing these disorders. For example, a subject may have a family history or a genetic mutation or element (e.g., an expanded trinucleotide repeat) that contributes to the development of disease. Human subjects, in consult with their physicians and/or other health care professionals, can decide whether their risk is great enough to undergo preventative care (as is the case for any prophylactic treatment or procedure). While the subjects of the preventative and/or therapeutic regimes described herein may be human, the compounds and compositions of the invention can also be administered to non-human subjects (e.g., domesticated animals (such as a dog or cat), livestock (e.g., a cow, pig, sheep, goat, or horse), or animals kept in captivity (e.g., in zoos or parks) or on preserves).

The prophylactic and therapeutic methods can be carried out by administering to the subject a pharmaceutical composition containing a therapeutically effective amount of one or more of the compounds described herein. While a single compound may be effective, the invention is not so limited. A subject can be treated with multiple compounds, administered simultaneously or sequentially (i.e., before or after a compound of the present invention). For example, a subject can be treated with one or more of the compounds described herein and, optionally, a chemotherapeutic agent, an analgesic, a bronchodilator, levodopa or a similar medication, haloperidol, or risperdone. In other embodiments, the "second" agent can be a vitamin, mineral, nucleic acid (e.g., an antisense oligonucleotide, siRNA, or microRNA), a therapeutic protein (e.g., a peptide), including therapeutic antibodies or antigen-binding portions thereof, or an anti- inflammatory agent. Compositions containing a compound of the invention and a second agent, as described herein, are also within the scope of the present invention.

The combination therapy will, of course, depend on the disorder being treated. Where a compound of the invention is administered to treat a patient with Huntington's disease, it may be combined with a medication to suppress chorea; where a compound of the invention is administered to treat a patient with a cancer, it may be combined with a known chemo therapeutic agent used to treat that type of cancer; and so forth.

Compounds that decrease intracellular levels of expanded poly(Q) polypeptides or modulate hsp90 can also be used to diagnose diseases characterized by expanded poly(Q) polypeptides and/or hsp90 misregulation. These methods can be carried out by providing a biological sample from a patient suspected of having a disease associated with an expanded poly(Q) polypeptide or hsp90 misregulation; exposing the sample to a compound of the invention; and determining whether the compound modulates the intracellular level of the expanded poly(Q) polypeptide or hsp90, or those compounds' activity, within the sample. The compound can be one that is known to interact directly with a primary target or one that interacts with a protein acting upstream or downstream from the primary target. For example, the compound can be one that is known to interact with a chaperone (e.g., hsp90) or a co- chaperone. The compound can also be one that is known to interact with proteins in the context of the suspected disease. For example, a compound that is known to decrease intracellular levels of huntingtin can be used to diagnose a patient suspected of having HD. The sample will be exposed to the compound for a time and under conditions (e.g., physiological conditions of temperature and pH) sufficient to permit the compound to affect proteins within the sample. The diagnostic methods can be carried out before, after, or in conjunction with other diagnostic tests, and their results can inform the subject's treatment regime. For example, where a compound is found to decrease intracellular levels of huntingtin proteins in a sample obtained from a patient suspected of having HD, that compound may then be used to treat the patient.

Further within the invention is the use of a compound that conforms to one of the exemplary compounds in Table 1 , or, as described herein, a salt, solvate, or other analog or variant thereof, for treating a subject who has been diagnosed as having, or who is at risk for developing, a disorder characterized by misexpression of a polypeptide containing an expanded poly(Q) repeat. For example, the compound can be Formula A9 (2-((E)-((E)-3-(4-chlorophenyl)allylidene)amino)phenol) (or a salt thereof), or Formula A24 ((3-[2-(2,4-dimethyt-l,3-thiazol-5-yl)-2-oxoethylidene]-3,4- dihydro-2H-l,4-benzoxazin-2-one) (or a salt thereof). Further within the invention is the use of a compound that conforms to one of the exemplary compounds in Table 1 , or, as described herein, a salt, solvate, or other analog or variant thereof, in the preparation of a medicament for treating a subject who has been diagnosed as having, or who is at risk for developing, a disorder characterized by misexpression of a polypeptide containing an expanded poly(Q) repeat. For example, the compound can be Formula A9 (2-((E)-((E)-3-(4- chlorophenyl)allylidene)amino)phenol) (or a salt thereof), or Formula A24 ((3-[2- (2,4-dimethyl-l,3-thiazol-5-yl)-2-oxoethylidene]-3,4-dihydro-2H-l,4-benzoxazin-2- one) (or a salt thereof).

Similarly, the invention features the use of a compound that conforms to one of the exemplary compounds in Table 1, or, as described herein, a salt, solvate, or other analog or variant thereof, for the preparation of a medicament for treating a subject who has been diagnosed as having, or who is at risk for developing, a disorder characterized by misexpression of a polypeptide containing an expanded poly(Q) repeat. For example, the compound can be Formula A9 (2-((E)-((E)-3-(4- chlorophenyl)allylidene)amino)phenol) (or a salt thereof), or Formula A24 ((3-[2- (2,4-dimethyl-l,3-thiazol-5-yl)-2-oxoethylidene]-3,4-dihydro-2H-l,4-benzoxazin-2- one) (or a salt thereof). The disease or disorder can be any of those specified herein, including Huntington's disease, any of the several types of spinocerebellar ataxias (e.g., SCAl, SCA2, SCA6, SCA7 and Machado- Joseph disease (MJD/SCA3)), hemiplegic migraine, spinobulbar muscular atrophy (SBMA; also known as Kennedy's disease), and dentatorubral-pallidoluysian atrophy, breast cancer, ovarian cancer, endometrial cancer, prostate cancer and leukemia, or amyloidosis, or a disorder with a similar underlying cellular basis (i.e., an expanded poly(Q) repeat).

Further within the invention is the use of a compound that conforms to one of the exemplary compounds in Table 1, or, as described herein, a salt, solvate, or other analog or variant thereof, for treating a subject who has been diagnosed as having, or who is at risk for developing, a disorder characterized by a polypeptide misfolding, including disorders characterized by the expression of a polypeptide containing an expanded poly(Q) repeat. Similarly, the invention features the use of a compound that conforms to one of the exemplary compounds in Table 1, or, as described herein, a salt, solvate, or other analog or variant thereof, for the preparation of a medicament for treating a subject who has been diagnosed as having, or who is at risk for developing, a disorder characterized by misfolded polypeptide, including disorders characterized by the expression of a polypeptide containing an expanded poly(Q) repeat. The disease or disorder can be any of those specified herein, including those mentioned above as being characterized by expression of a polypeptide containing an expanded poly(Q) repeat, as well as Alzheimer's Disease, Parkinson's Disease, amylotrophic lateral sclerosis, or a prion disease, such as Creutzfeldt- Jakob disease.

Other features and advantages of the invention will be apparent from the accompanying drawings and description, and from the claims.

DESCRIPTION OF DRAWINGS

Figure 1 is a graph showing that β-galactosidase induction in d-a23Q and d- a97Q was PonA dependent.

Figure 2 is a graph showing that removal of inducer resulted in reduction in β - galactosidase activity in d-a97Q (Figure 2A) and d-a23Q (Figure 2B) respectively.

Figure 3 depicts the result of an experiment analyzing the effect of Class I compounds on b-galactosiase activity in d-a97Q and d-a23Q cells.

Figure 4 depicts the result of an experiment analyzing the ability of Class I and Class II compounds to inhibit polyglutamine induced toxicity in Htt^Q103 PC 12 cells.

Figure 5 depicts the result of an experiment analyzing the effect of Maybridge compounds M4, M5 and M6 on b-galactosiase activity in d-a97Q and d-a23Q cells.

Figure 6 is a graph showing the results of a rabbit reticulocyte luciferase refolding assay used to monitor refolding of luciferase protein in vitro. "GA" is the known hsp90 inhibitor Geldanamycin.

Figure 7 is a graph showing the effect of the A9 analogs, A8 and A7, in the rabbit reticulocyte luciferase refolding assay.

Figure 8 is a graph showing a synergistic effect of A9 and geldanamycin in the rabbit reticulocyte luciferase refolding assay.

Figure 9 is a graph showing the activity of compounds in an hsp70 reporter assay in 3T3-Y9-B12 cells.

Figure 10 is a graph showing the activity of compounds in an hsp70 reporter assay in 3T3-Y9-B12-SD cells. Figure 11 is a graph showing the activity of compounds in an hsp90 ATPase assay.

Figure 12 is a graph showing the effect of compounds on Her2 levels in MCF7 cells.

Figure 13 is a graph showing the activity of compounds in an hsp90 chaperone pathway assay.

Figure 14A is a graph showing the activity of compounds in the d-a97Q β- galactosidase assay.

Figure 14B is a graph showing the activity of compound in an hap70 reporter assay.

DETAILED DESCRIPTION

The hsp90 chaperone complex mediates the folding of many proteins involved in the regulation of cell cycle progression, growth control, tissue invasion and metastasis, angiogenesis and programmed cell death. The hsp90 chaperone complex targets a discrete subset of proteins, termed hsp90 client proteins, many of which have oncogenic potential. Examples of hsp90 client proteins include steroid hormone receptors (the estrogen receptor, the progesterone receptor, the androgen receptor, the glucocorticoid receptor and the mineralocorticoid receptor), receptor tyrosine kinases (Her2, EGFR, IGFlR and FLT3), the SCR family kinases (SRC, LCK and FYN), serine/threonine kinases (Raf-1, AKT, and CDK4), cell cycle G2 checkpoint kinases (WEEl, MYT 1, and POLO-I), mutant fusion kinases (BCR-ABL and NPM-ALK), transcription factors (p53, HSF-I, and HIF-I) and telomerase (hTERT).

Hsp90 proteins of the invention can include any of the various isoforms of hsp90. Representative sequences of hsp90α (also referred to in the art as HSP 86, Heat shock protein HSP 90-alpha, NY-REN-38 antigen, heat shock protein 9OkDa alpha (cytosolic), class A member 1) include NP_001017963.1 GL63029937;

NP_005339.2 GL40254816. A representative sequences of hsp90 β (also referred to in the art as HSP 90, Heat shock protein HSP 90-beta, heat shock protein 9OkDa alpha (cytosolic), class B member 1) includes NP 031381.2 GI:20149594. A representative sequence of hsp90N includes NM_001017963.1 GI:63029936. A representative sequence of Grp94 (also referred to in the art as 94 kDa glucose- regulated protein, endoplasmin precursor, heat shock protein 90 kDa beta member, tumor rejection antigen, gp96 homolog) includes NM_003299.1 GI:4507676. A representative sequence of Hsp/754 TRAPl includes NM_016292.1 GI:7706484.

Hsp90 proteins typically include an ATP binding region, a client protein binding region and a dimerizing region. Three structural domains have been described: a highly conserved N-terminal domain of about 25 kDa, a middle domain of about 40 kDa, and a C-terminal domain of about 12 kDa; the N-terminus and the middle domains are connected by a "charged linker" region. Hsp90 constitutively forms homodimers with the contact sites are localized within the C-terminus. The ATP binding pocket is situated in the N-terminal domain. The hsp90 inhibitor geldanamycin also binds hsp90 at this site and acts as a competitive inhibitor of hsp90 ATPase activity. The middle domain is divided into three regions: a 3-layer α-β-α sandwich, a 3 -turn α-helix and irregular loops, and a 6-tum α-helix. The middle domain has been shown to be involved in client binding. The C-terminal domain comprises an alternative ATP-binding site, which becomes accessible when the N- terminal ATP-binding site is occupied. At the very C-terminal end of the protein is the tetratricopeptide repeat (TPR) motif recognition site, a conserved MEEVD pentapeptide, that is responsible for the interaction with co-factors including, for example, the immunophilins FKBP51 and FKBP52, the stress induced phosphoprotein 1 (Stil/Hop), cyclophilin-40, PP5, and Tom70.

The hsp90 chaperone complex includes at least five specific proteins, hsp90, hsp40, hsp70, Hop and p23, as well as other co-chaperones (e.g., CDC37, Ahal and CHIP) and immunophilins (e.g., FKBP51, FKBP52, cyclophilin-40, UNC-45). The co-chaperones hsp40, p23, FKBP51, FKBP52 facilitate interactions between hsp90 and the client protein; other co-chaperones (Hop and CHIP) enable molecular chaperones to interact with other proteins.

Compositions The compositions provided herein include the following:

A compound of Formula VI:

(VI) or a pharmaceutically acceptable salt thereof, wherein: a dashed line indicates an optional bond;

A is NR⁴ when the bond between A and R² is a single bond;

A is N when the bond between A and R² is a double bond;

R¹ is H, C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, C(O)-(C_1-6 alkyl), C(O)- (C_2-6 alkenyl), or C(O)-(C_2-6 alkynyl);

R² is =CR⁵R⁶ when the bond between A and R² is a double bond;

R² is -CR⁷=CR⁸R⁹ when the bond between A and R² is a single bond;

R³ is halo, C_]-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, C_1-6 haloalkyl, CN, NO₂, N₃, 0R^a, SR^a, C(O)R^b, C(O)NR^cR^d, C(O)OR^a, OC(O)R^b, OC(O)NR^cR^d, NR^cR^d, NR^cC(O)R^b, NR^cC(0)NR^cR^d, NR^cC(O)OR^a, S(O)R^b, S(O)NR^cR^d, S(O)₂R⁶, NR^cS(O)₂R^b, or S(O)₂NR^cR^d;

R⁴ is H or Ci_-6 alkyl;

R⁵ is -(L)_m-Cy;

R⁶ is H or Ci_-6 alkyl;

R⁷ is H, Ci_-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, or C(O)O-(Ci_-6 alkyl); or R¹ and R⁷ together form -(CR¹⁰R¹ ') -;

R⁸ is -(L)_m-Cy;

R⁹, R¹⁰, and R¹¹ are independently selected from H and Ci_-6 alkyl; or R¹⁰ and R¹ ' together form O or S;

L is Ci-6 alkylene, C_2-6 alkenylene, -C(O)-, -C(O)-(Ci_-6 alkylene)-, or - C(O)-(Ci_-6 alkenylene)-;

Cy is aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, each optionally substituted by 1, 2, or 3 substituents independently selected from halo, Ci_-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, C,.₆ haloalkyl, CN, NO₂, N₃, OR^aI, SR^al, C(O)R^bl, C(O)NR^clR^dl, C(O)OR^aI, OC(O)R^bl, OC(O)NR^CIR^dI, NR^clR^dl, NR^clC(O)R^bI, NR^CIC(O)NR^clR^dl, NR^clC(O)OR^al, S(O)R^bI, S(O)NR^clR^dl, S(O)₂R^bl, NR^clS(O)₂R^bl, and S(O)₂NR^clR^dI;

R »a^a, r R>a^al^l, R", R ,b^Dl¹, τ R> c^c, R ,cl, R^α, and R _>d^αlⁱ are independently selected from H, Ci-₆ alkyl, C₁^ haloalkyl, C_2-β alkenyl, C_2-O alkynyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl; n is 0, 1, 2, 3 or 4; and m is 0 or 1.

In some embodiments, the compound has Formula Via:

Via or a pharmaceutically acceptable salt thereof, wherein the variables are defined above.

In some embodiments, the compound has Formula VIb:

VIb or a pharmaceutically acceptable salt thereof, wherein the variables are defined above.

In some embodiments, the bond between A and R² is a single bond.

In some embodiments, the bond between A and R² is a double bond.

In some embodiments, A is NR⁴.

In some embodiments, A is N.

In some embodiments, R¹ is H or Ci-β alkyl.

In some embodiments, R is H. In some embodiments, R² is =CR⁵R⁶.

In some embodiments, R² is -CR⁷=CR⁸R⁹.

In some embodiments, R³ is halo or Ci -e alkyl.

In some embodiments, R³ is methyl.

In some embodiments, R⁴ is H.

In some embodiments, R⁶ is H.

In some embodiments, R⁷ is H;

In some embodiments, R¹ and R⁷ together form -(CR¹⁰R¹ ') -.

In some embodiments, R⁹, R¹⁰, and R¹ ' are each H.

In some embodiments, R¹⁰ and R¹¹ together form O.

In some embodiments, L is C2-6 alkenylene, -C(O)-, or -C(O)-(Ci -β alkenylene)-.

In some embodiments, L is C_2-6 alkenylene.

In some embodiments, L is -CH=CH-.

In some embodiments, L is -C(O)-.

In some embodiments, L is -C(O)-(Ci_-6 alkenylene)-.

In some embodiments, Cy is aryl or heteroaryl, each optionally substituted by 1, 2, or 3 substituents independently selected from halo, Q_-6 alkyl, C_2-6 alkenyl, C_2-O alkynyl, C_-6 haloalkyl, CN, NO₂, N₃, OR^al, SR^al, C(O)R^bI, C(O)NR^clR^dl, C(O)OR^al, OC(O)R^bl, OC(O)NR^clR^dl, NR^clR^dl, NR^clC(O)R^bl, NR^clC(O)NR^clR^dl, NR^clC(O)OR^al, S(O)R^bl, S(O)NR^clR^dl, S(O)₂R^bI, NR^clS(O)₂R^bl, and S(O)₂NR^clR^dl.

In some embodiments, Cy is phenyl optionally substituted by 1, 2, or 3 substituents independently selected from halo, Ci -β alkyl, C_2-6 alkenyl, C₂-₆ alkynyl, C_1-6 haloalkyl, CN, NO₂, N₃, OR^al, SR^al, C(0)R^bl, C(O)NR^clR^dl, C(O)OR^al, OC(O)R^bl, OC(O)NR^clR^dl, NR^clR^dl, NR^clC(O)R^bl, NR^clC(O)NR^clR^dl, NR^clC(O)OR^al, S(O)R^bl, S(O)NR^clR^dl, S(O)₂R^bl, NR^clS(O)₂R^b\ and S(O)₂NR^clR^dl.

In some embodiments, Cy is phenyl optionally substituted by 1, 2, or 3 substituents independently selected from halo, Ci _β alkyl, and OR^al.

In some embodiments, Cy is phenyl optionally substituted by 1 or 2 substituents independently selected from Cl, Br, and OH.

In some embodiments, Cy is heteroaryl optionally substituted by 1, 2, or 3 substituents independently selected from halo, Ci -₆ alkyl, C_2-6 alkenyl, C_2-O alkynyl, Ci_-6 haloalkyl, CN, NO₂, N₃, OR^al, SR^al, C(O)R^bl, C(O)NR^CIR^dl, C(O)OR^al, OC(O)R^bl, OC(O)NR^clR^dl, NR^clR^d1, NR^clC(O)R^bl, NR^clC(O)NR^CIR^dI, NR^clC(O)OR^al, S(O)R^bl, S(O)NR^clR^dl, S(O)₂R^bl, NR^clS(O)₂R^bl, and S(O)₂NR^clR^dl.

In some embodiments, Cy is thiazolyl optionally substituted by 1 , 2, or 3 substituents independently selected from halo, Ci-₆ alkyl, C_2-6 alkenyl, C_2-6 alkynyl, C_1-6 haloalkyl, CN, NO₂, N₃, OR^al, SR^al, C(O)R^bl, C(O)NR^clR^dl, C(O)OR^al, OC(O)R^bI, OC(O)NR^clR^dl, NR^CIR^dl, NR^clC(O)R^bl, NR^clC(O)NR^clR^dl, NR^clC(O)OR^aI, S(O)R^bl, S(O)NR^clR^dl, S(O)₂R^bl, NR^CIS(O)₂R^bI, and S(O)₂NR^CIR^dl.

In some embodiments, Cy is thiazolyl optionally substituted by 1 or 2 Q-s alkyl.

In some embodiments, Cy is thiazolyl optionally substituted by 1 or 2 methyl groups.

At various places in the present specification, substituents of compounds of the invention are disclosed in groups or in ranges. It is specifically intended that the invention include each and every individual subcombination of the members of such groups and ranges. For example, the term "Ci_-6 alkyl" is specifically intended to individually disclose methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl, and C₆ alkyl. The term "substituents" refers to a group "substituted" on, for example, an alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, heteroaralkyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group at any atom of that group. In one aspect, the substituents on a group are independently any one single, or any subset of, the aforementioned substituents. In another aspect, a substituent may itself be substituted with any one of the above substituents.

It is further appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.

The following definitions apply to Formula VI and Formulas Via and VIb.

As used herein, the term "alkyl" is meant to refer to a saturated hydrocarbon group which is straight-chained or branched. Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t- butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. An alkyl group can contain from 1 to about 20, from 2 to about 20, from 1 to about 10, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms.

As used herein, the term "alkylyene" refers to a linking alkyl group.

As used herein, "alkenyl" refers to an alkyl group having one or more double carbon-carbon bonds. Example alkenyl groups include ethenyl, propenyl, and the like.

As used herein, "alkenylene" refers to a linking alkenyl group.

As used herein, "alkynyl" refers to an alkyl group having one or more triple carbon-carbon bonds. Example alkynyl groups include ethynyl, propynyl, and the like.

As used herein, "haloalkyl" refers to an alkyl group having one or more halogen substituents. Example haloalkyl groups include CF₃, C₂F₅, CHF₂, CCI₃, CHCl₂, C₂Cl₅, and the like.

As used herein, "aryl" refers to monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, aryl groups have from 6 to about 20 carbon atoms.

As used herein, "arylene" refers to a linking aryl group.

As used herein, "cycloalkyl" refers to non-aromatic carbocycles including cyclized alkyl, alkenyl, and alkynyl groups. Cycloalkyl groups can include mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) ring systems, including spirocycles. In some embodiments, cycloalkyl groups can have from 3 to about 20 carbon atoms, 3 to about 14 carbon atoms, 3 to about 10 carbon atoms, or 3 to 7 carbon atoms. Cycloalkyl groups can further have 0, 1, 2, or 3 double bonds and/or 0, 1, or 2 triple bonds. Also included in the definition of cycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the cycloalkyl ring, for example, benzo derivatives of pentane, pentene, hexane, and the like. A cycloalkyl group having one or more fused aromatic rings can be attached though either the aromatic or non-aromatic portion. One or more ring-forming carbon atoms of a cycloalkyl group can be oxidized, for example, having an oxo or sulfido substituent. Example cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, adamantyl, and the like. As used herein, a "heteroaryl" group refers to an aromatic heterocycle having at least one heteroatom ring member such as sulfur, oxygen, or nitrogen. Heteroaryl groups include monocyclic and polycyclic (e.g., having 2, 3 or 4 fused rings) systems. Any ring-forming N atom in a heteroaryl group can also be oxidized to form an N-oxo moiety. Examples of heteroaryl groups include without limitation, pyridyl, N- oxopyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl, indolinyl, and the like. In some embodiments, the heteroaryl group has from 1 to about 20 carbon atoms, and in further embodiments from about 3 to about 20 carbon atoms. In some embodiments, the heteroaryl group contains 3 to about 14, 3 to about 7, or 5 to 6 ring- forming atoms. In some embodiments, the heteroaryl group has 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms.

As used herein, "heterocycloalkyl" refers to a non-aromatic heterocycle where one or more of the ring- forming atoms is a heteroatom such as an O, N, or S atom. Heterocycloalkyl groups can include mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) ring systems as well as spirocycles. Example "heterocycloalkyl" groups include morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, 2,3- dihydrobenzofuryl, 1,3-benzodioxole, benzo-l,4-dioxane, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, and the like. Also included in the definition of heterocycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the nonaromatic heterocyclic ring, for example phthalimidyl, naphthalimidyl, and benzo derivatives of heterocycles. A heterocycloalkyl group having one or more fused aromatic rings can be attached though either the aromatic or non-aromatic portion. Also included in the definition of heterocycloalkyl are moieties where one or more ring- forming atoms is substituted by 1 or 2 oxo or sulfido groups. In some embodiments, the heterocycloalkyl group has from 1 to about 20 carbon atoms, and in further embodiments from about 3 to about 20 carbon atoms. In some embodiments, the heterocycloalkyl group contains 3 to about 20, 3 to about 14, 3 to about 7, or 5 to 6 ring-forming atoms. In some embodiments, the heterocycloalkyl group has 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms. In some embodiments, the heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, the heterocycloalkyl group contains 0 to 2 triple bonds.

As used herein, "halo" includes fluoro, chloro, bromo, and iodo.

The compounds described herein, including those conforming to any formula, can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. The present compounds that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated for the present compounds. Cis and trans geometric isomers of the present compounds are described and may be isolated as a mixture of isomers or as separated isomeric forms.

The compounds described herein, including any of those conforming to Formulas I- VI also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Exemplary prototropic tautomers include ketone — enol pairs, amide - imidic acid pairs, lactam — lactim pairs, amide - imidic acid pairs, enamine — imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, for example, IH- and 3H-imidazole, IH-, 2H- and 4H- 1 ,2,4-triazole, IH- and 2H- isoindole, and IH- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

Compounds of the invention also include all isotopes of atoms occurring in the intermediate or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.

The term, "compound," as used herein with respect to any compound conforming to one of Formulas I- VI, is meant to include all stereoisomers, geometric iosomers, tautomers, and isotopes of the structures depicted. All compounds, and pharmaceuticaly acceptable salts thereof, are also meant to include solvated or hydrated forms.

The compounds of the present invention can be prepared in a variety of ways known to one of ordinary skill in the art of organic synthesis. The compounds of the present invention can be synthesized using the methods as hereinafter described below, together with synthetic methods known in the art of synthetic organic chemistry or variations thereon as appreciated by one of ordinary skill in the art.

The present compounds can be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one of ordinary skill in the art by routine optimization procedures.

Regardless of their original source or the manner in which they are obtained, the compounds of the invention can be formulated in accordance with their use. For example, the compounds can be formulated within compositions for application to cells in tissue culture or for administration to a patient. When employed as pharmaceuticals, any of the present compounds can be administered in the form of pharmaceutical compositions. These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular, oral or parenteral. Methods for ocular delivery can include topical administration (eye drops), subconjunctival, periocular or intravitreal injection or introduction by balloon catheter or ophthalmic inserts surgically placed in the conjunctival sac. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular administration. Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, and the like. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

This invention also includes pharmaceutical compositions which contain, as the active ingredient, one or more of the compounds described herein in combination with one or more pharmaceutically acceptable carriers. In making the compositions of the invention, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material (e.g., normal saline), which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10 % by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. As is known in the art, the type of diluent can vary depending upon the intended route of administration. The resulting compositions can include additional agents, such as preservatives. The compounds may also be applied to a surface of a device (e.g., a catheter) or contained within a pump, patch, or other drug delivery device. The therapeutic agents of the invention can be administered alone, or in a mixture, in the presence of a pharmaceutically acceptable excipient or carrier (e.g., physiological saline). The excipient or carrier is selected on the basis of the mode and route of administration. Suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences (E. W. Martin), a well-known reference text in this field, and in the USP/NF (United States Pharmacopeia and the National Formularly).

In preparing a formulation, the active compound can be milled to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it can be milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size can be adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh. Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. The pharmaceutical compositions can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.

The compositions can be formulated in a unit dosage form, each dosage containing from about 5 to about 100 mg, more usually about 10 to about 30 mg, of the active ingredient. The term "unit dosage forms" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

The active compound can be effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. It will be understood, however, that the amount of the compound actually administered will usually be determined by a physician, according to the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, the active ingredient is typically dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the active ingredient of the present invention. The tablets or pills of the present invention can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

The liquid forms in which the compounds and compositions of the present invention can be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described herein and/or known in the art. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in can be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device can be attached to a face masks tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered orally or nasally from devices which deliver the formulation in an appropriate manner.

The amount of compound or composition administered to a patient will vary depending upon what is being administered, the purpose of the administration, such as prophylaxis or therapy, the state of the patient, the manner of administration, and the like. In therapeutic applications, compositions can be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. As noted, effective doses can vary and will depend on the disease condition being treated as well as by the judgment of the attending clinician depending upon factors such as the severity of the disease, the age, weight and general condition of the patient, and the like. An effective dose is a dose that produces a desirable clinical outcome by, for example, improving a sign or symptom of the disease being treated or slowing its progression.

The compositions administered to a patient can be in the form of one or more of the pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization techniques or may be sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the compound preparations typically will be between about 3 and 11, more preferably between 5 to 9 and most preferably between 7 and 8. It will be understood that use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of pharmaceutical salts.

The proportion or concentration of a compound of the invention in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration. For example, the compounds of the invention can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w/v of the compound for parenteral adminstration. Some typical dose ranges are from about 1 μg/kg to about 1 g/kg of body weight per day. In some embodiments, the dose range is from about 0.01 mg/kg to about 100 mg/kg of body weight per day. The dosage is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, formulation of the excipient, and its route of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

The present compounds can also be formulated in combination with one or more additional active ingredients, which can include any pharmaceutical agent such as anti- viral agents, antibodies, immune suppressants, anti-inflammatory agents, chemotherapeutics, and the like.

Using assays such as those described herein, we have identified certain compounds, which are categorized according to one of Formulas I- VI, including Formulas Via and VIb. The invention encompasses these compounds in, for example, a substantially pure form, as well as various compositions containing one or more of them (e.g., pharmaceutical formulations, packaged products, and kits) and methods of using them. Formulas VI , Via, and VIb appear above. Formulas I-V follow.

Formula I

Compounds that can be used in practicing the invention can have the general formula of Formula I, where Z¹ is N, or NH; Y¹ is a bond, or C1-C₃ alkenyl; X¹ is a bond, N, or carboxy or carbonyl; W¹ is aryl, heteroaryl, or heterocyclyl including 5-6 atoms, each of which is optionally substituted with 1-5 R³; W² is OH, or O; R¹ is H, or carboxy, which when taken together with W², Yi, Zi, and the phenyl to which W² is attached forms a heterocyclyl including 6 atoms; R² is H or C1-C6 alkyl; each of 1-5 R³ is independently H, C1-C6 alkyl, halo, aryl, oxo, or -C(=S)NR⁴R⁵; each of R⁴ and R⁵ is independently H, or C1-C6 alkyl; and p is an integer from 0-4. The W¹ of Formula I can be a phenyl group, and the phenyl group can be substituted with 1 to 4 chloro and/or bromo. The W¹ of Formula I can be a 5-thiazole, and the 5-thiazoIe can be substituted with Ci-C₆ alkyl. The W¹ of Formula I can be a 4-pyrazole, and 4-pyrazole can be substituted with 1-3 R³. The R¹ of Formula I can be carboxy, and can form a ring when taken together with OH, Yi, Zj, and the phenyl to which it is attached. A compound of Formula I can be a salt.

Formula II

Compounds that can be used in practicing the invention can have the general formula of Formula II, where Z² is aryl, or heteroaryl including 5-6 atoms; optionally substituted with 1-3 R⁸; each of R⁶ and R⁷ is independently H, hydroxyl, or when taken together with the phenyl to which they are attached form a bicyclic aryl including 6-10 atoms; each of 1-3 R⁸ is independently nitro, R⁹, or NR¹⁰R¹ ' ; R⁹ is phenyl, optionally substituted with 1-5 halo; R¹⁰ is H; R^u is H, or -C(=O)CH₂R¹²; and R¹² is aryloxy including 7-11 atoms, optionally substituted with C1-C6 alkyl. The Z² of Formula II can be pyrimidine optionally substituted with 1-3 R⁸, and each of 1-3 R⁸ can be independently nitro or amino. The Z² of Formula II can be thiazole optionally substituted with R⁹, and R⁹ can be phenyl optionally substituted with 1-5 chloro. The compound of Formula II can be a salt.

Formula EQ

Compounds that can be used in practicing the invention can have the general formula of Formula III, where R¹³ is H, or halo; R¹⁴ is H, or hydroxy; and each of R¹⁵, R¹⁶, R¹⁷, or R¹⁸ is independently H, or C1-C6 alkyl. The R¹³ of Formula III can be iodo. The compound of Formula III can be a salt.

Formula IV

Compounds that can be used in practicing the invention can have the general formula of Formula IV, where each of R¹⁹ and R²⁰ is independently H, or C1-C6 alkoxy; and each of R²¹ and R²² is independently H, or C1-C6 alkyl. The compound of Formula TV can be a salt.

Formula V

Compounds that can be used in practicing the invention can have the general formula of Formula V, where each of R²³, R²⁵, R²⁶ is independently H, or C1-C6 alkyl; R²⁴ is H, or C1-C6 alkyl; R²⁷ is H, or C1-C6 alkoxy; and R²⁸ is H, or halo. The R²⁸ of Formula V can be iodo. The compound of Formula V can be a salt.

The compounds and compositions of the invention can be, or can include: 2-((E)-((E)-3-(3-bromophenyl)allylidene)amino)-4-methylphenol; (E)-2-(4- chlorostyryl)phenol; 2-((E)-((E)-3-(4-chlorophenyl)allylidene)amino)phenol; 3-[2- (2,4-dimethyl- 1 ,3-thiazol-5-yl)-2-oxoethylidene]-3,4-dihydro-2H- 1 ,4-benzoxazin-2- one; 4-(4-(3,4-dichlorophenyl)thiazol-2-ylamino)phenol; (E)-8,9-dimethoxy-5,5- dimemyl-5,6-dmydrobenzo[d]pyrazolo[4,3-b]azepin-3(2H)-one; N-(4- (phenylamino)phenyl)-2-(o-tolyloxy)acetainide; 4-[(2-hydroxyphenyl)hydrazono]-5- oxo-3-phenyl-4,5-dihydro-lH-pyrazole-l-carbothioamide; N-2-naphthyl-5-nitro-4,6- pyrimidinediamine; l-ethyl-4-iodo-2-methyl-lH-benzimidazol-5-ol; or 8-iodo-7- methoxy-4-methyl-2H-chromen-2-one. For example, the compounds and compositions of the invention can be, or can include a compound shown in Table 1.

The following definitions apply to Formulas I- V.

The term "halo" or "halogen" refers to any radical of fluorine, chlorine, bromine or iodine. The terms "cyclylalkyl" and "cycloalkyl" refer to saturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon groups. Any atom can be substituted by, for example, one or more substituents. Cycloalkyl groups can contain fused rings, which share a common carbon atom. Cycloalkyl moieties can include, for example, cyclopropyl, cyclohexyl, methylcyclohexyl (the point of attachment to another moiety can be either the methyl group or a cyclohexyl ring carbon), adamantyl, and norbornyl.

The term "alkenyl" refers to a straight or branched hydrocarbon chain containing 2-20 carbon atoms and having one or more double bonds. Any atom can be substituted by one or more substituents. Alkenyl groups can include, for example, allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. One of the double bond carbons can optionally be the point of attachment of the alkenyl substituent. The term "alkynyl" refers to a straight or branched hydrocarbon chain containing 2-20 carbon atoms and having one or more triple bonds. Any atom can be substituted by one or more substituents. Alkynyl groups can include, for example, ethynyl, propargyl, and 3-hexynyl. One of the triple bond carbons can optionally be the point of attachment of the alkynyl substituent.

The term "alkoxy" refers to an -O-alkyl radical. The term "heterocyclyl" refers to a monocyclic, bicyclic, tricyclic or other polycyclic ring system having: 1-4 heteroatoms if monocyclic; 1-8 heteroatoms if bicyclic; or 1-10 heteroatoms if tricyclic. The heteroatoms can be O, N, or S (e.g., carbon atoms and 1-4, 1-8, or 1-10 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively). The heteroatom can optionally be the point of attachment of the heterocyclyl substituent. Any atom can be substituted, by, for example, one or more substituents. The heterocyclyl groups can contain fused rings, which share a common carbon atom. Heterocyclyl groups can include, for example, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino, pyrrolinyl, and pyrrolidinyl.

The term "heteroaryl" refers to an aromatic monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon groups having: 1-4 heteroatoms if monocyclic; 1-8 heteroatoms if bicyclic; or 1-10 heteroatoms if tricyclic. The heteroatoms can be O, N, or S {e.g., carbon atoms and 1-4, 1-8, or 1-10 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively). Any atom can be substituted by, for example, one or more substituents. Heteroaryl groups can contain fused rings, which share a common carbon atom. Heteroaryl groups include pyridyl, thienyl, furanyl, imidazolyl, and pyrrolyl.

The term "oxo" refers to an oxygen atom, which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.

As noted, the present compounds, including those just described, can be variously formulated for oral or parenteral administration to a patient.

Each of the variables designated by, for example, R, X, Y, m, and n in any of the formulas disclosed herein can be selected independently. While we tend to use the term "compound(s)", we may also use terms like "agent(s)" to refer to the molecules described herein.

The invention also encompasses pharmaceutically acceptable salts or solvates of a compound of any of Formulas I- VI, and prodrugs, metabolites, structural analogs, and other pharmaceutically useful variants thereof. These other variants may be, for example, a complex containing the compound and a targeting moiety, as described further below, a second therapeutic agent or a detectable marker (e.g. , the compound may incorporate a radioactive isotope or be joined to a fluorescent compound). When in the form of a prodrug, a compound may be modified in vivo (e.g., intracellularly) after being administered to a patient or to a cell in culture. The modified compound (i.e., the processed prodrug) may be identical to a compound described herein and will be biologically active or have enough activity to be clinically beneficial. The same is true of a metabolite; a given compound may be modified within a cell and yet retain sufficient biological activity to be clinically useful.

A salt, for example, can be formed between an anion and a positively charged substituent (e.g., amino) on a compound described herein. Suitable anions include chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, and acetate. Likewise, a salt can also be formed between a cation and a negatively charged substituent {e.g. , carboxylate) on a compound described herein. Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as tetramethylammonium ion.

Examples of prodrugs include esters and other pharmaceutically acceptable derivatives, which, upon administration to a subject, are capable of providing active compounds. A "prodrug" may be any pharmaceutically acceptable salt, ester, salt of an ester, or other derivative of a compound of this invention (for example an imidate ester of an amide), which, upon administration to a recipient, is capable of providing (directly or indirectly) a compound of this invention. Particularly favored derivatives and prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal {e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment {e.g., the brain or lymphatic system) relative to the parent species. Preferred prodrugs include derivatives where a group which enhances aqueous solubility or active transport through the gut membrane is appended to the structure of formulae described herein.

The present compounds may be modified by appending appropriate functionalities to enhance selected biological properties {e.g., targeting to a particular tissue). Such modifications are known in the art and include those which increase biological penetration into a given biological compartment {e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.

The present compounds may contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are expressly included in the present invention. The present compounds may also contain linkages {e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage (e.g., restriction resulting from the presence of a ring or double bond). Accordingly, all cis/trans and E/Z isomers are expressly included in the present invention. The present compounds may also be represented in multiple tautomeric forms, in such instances, the invention expressly includes all tautomeric forms of the compounds described herein, even though only a single tautomeric form may be represented (e.g. , alkylation of a ring system may result in alkylation at multiple sites, the invention expressly includes all such reaction products). All such isomeric forms of such compounds are expressly included in the present invention. All crystal forms of the compounds described herein are expressly included in the present invention.

As noted, the compounds of the invention may be mixed with or joined to a detectable marker or tag, to another therapeutic agent, or to a moiety that facilitates passage across the blood-brain barrier.

The compounds described herein can be packaged in suitable containers labeled, for example, for use as a therapy to treat a disease or disorder characterized by a polypeptide containing an expanded poly(Q) repeat. The containers can include the compound (i.e., the diagnostic/prophylactic/therapeutic agent) and one or more of a suitable stabilizer, carrier molecule, flavoring, and/or the like, as appropriate for the intended use. Accordingly, packaged products (e.g., sterile containers containing one or more of the compounds described herein and packaged for storage, shipment, or sale at concentrated or ready-to-use concentrations) and kits, including at least one compound of the invention and instructions for use, are also within the scope of the invention. A product can include a container (e.g., a vial, jar, bottle, bag, or the like) containing one or more compounds of the invention and a legend (e.g., a printed label or insert or other medium describing the product's use (e.g., an audio- or videotape)). The legend can be associated with the container (e.g., affixed to the container) and can describe the manner in which the compound therein should be administered (e.g., the frequency and route of administration), indications therefore, and other uses. The compounds can be ready for administration (e.g., present in dose-appropriate units), and may include a pharmaceutically acceptable adjuvant, carrier or other diluent and/or an additional therapeutic agent. Alternatively, the compounds can be provided in a concentrated form with a diluent and instructions for dilution.

It is further intended that the compounds of the invention are stable. As used herein "stable" refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and preferably capable of formulation into an efficacious therapeutic agent. Combinations of substituents and variables envisioned by this invention are only those that result in the formation of stable compounds. Stable compounds are those that are stable enough to allow manufacture and that maintain their integrity for a sufficient period of time to be useful for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject).

In one aspect, the invention features substantially pure preparations of the compounds described herein or combinations thereof. A naturally occurring compound is substantially pure when it is separated to some degree from the compound(s) or other entities (e.g., proteins, fats, or minerals) it is associated with in nature. For example, a naturally occurring compound described herein is substantially pure when it has been separated from 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the compound(s) or other moieties it is associated with in nature. These degrees of purity are not limiting, however, the compounds of the invention need be only as pure as necessary to cause a beneficial clinical result and to conform with good manufacturing practices. While the compounds of the invention may be naturally occurring and may be purified using conventional techniques, they may also be non-naturally occurring and may be synthesized (naturally occurring compounds can be synthesized as well; see below). Compounds prepared by chemical synthesis are substantially pure, as are compounds that have been separated from a library of chemical compounds. A substantially pure compound may be one that is separated from all the other members of the compound library or it may be one that has been separated to a limited extent (e.g., it may remain associated with a limited number (e.g., 1, 2, 3, 4, or 5-10) of other members of the library. As noted, while more than one of the agents described herein can be formulated within the same composition, and while the compositions can also include a second therapeutic agent (as described herein), the pharmaceutical compositions of the invention expressly exclude extremely heterogeneous mixtures, such as libraries (e.g., combinatorial or compound libraries, including those that contain synthetic and/or natural products, and custom analog libraries, which may contain compounds based on a common scaffold). Such libraries can include hundreds or thousands of distinct compounds or random pools thereof. Whether or not commercially available, such libraries are excluded from the meaning of a pharmaceutical composition. Screening assays

Also featured are in vitro model systems that recapitulate key features of disease pathology and that are adaptable to high throughput screening against a large collection of chemical compounds. Using our assays, we have identified compounds we believe are capable of modulating intracellular levels of polypeptides containing an expanded poly(Q) repeat (either directly or indirectly) including those that cause pathological disorders such as Huntington's disease or cancer and the other diseases referred to herein (we tend to use the term "disease" to refer to any disorder, unwanted condition, or syndrome). The compounds described herein can be used to modulate (e.g., decrease) intracellular levels of polypeptides containing an expanded poly(Q) repeat, such as polyQ-containing polypeptides that are associated with pathological disorders, as well as non-naturally occurring polypeptides (e.g., polyQ-containing polypeptides that are used in disease models, such as models of HD).

The invention includes an assay for identifying, testing and/or monitoring the effect of a compound or other moiety on levels of an expanded poly(Q) polypeptide. The invention includes a method of identifying an agent that selectively decreases intracellular levels of a polypeptide comprising an expanded poly(Q) repeat. The method includes providing a first cell that expresses a first subunit of beta-galactosidase and includes a polynucleotide sequence including an inducible promoter operably linked to a polynucleotide encoding a first fusion polypeptide having an expanded poly(Q) repeat and a second, complementary subunit of beta-galactosidase. For example, the first cell can express the delta (Δ) subunit of beta-galactosidase, and the fusion polypeptide can include the alpha (α) subunit of beta-galactosidase. In the alternative, the first cell can express the α-subunit of beta-galactosidase, and the fusion polypeptide can include the Δ- subunit of beta-galactosidase. The method also includes providing a second cell that expresses a first subunit of beta-galactosidase and includes a polynucleotide sequence including an inducible promoter operably linked to a polynucleotide encoding a second fusion polypeptide having a wildtype poly(Q) repeat and a second, complementary subunit of beta-galactosidase, as described for the first cell. In each of the first and second cells, when the first subunits of beta-galactosidase and the second, complementary subunits of beta-galactosidase are expressed, functional beta-galactosidase is produced in the cells. The method further includes inducing expression of the fusion polypeptides in the first and second cells, contacting the first and second cells with a candidate agent, and determining the levels of functional beta-galactosidase in the cells. A decrease in the level of functional beta-galactosidase in the first cell relative to the level of functional beta-galactosidase in the second cell identifies the agent as one that selectively decreases intracellular levels of a polypeptide containing an expanded poly(Q) repeat.

The polypeptide having an expanded poly(Q) repeat can be a fragment of any polypeptide known to exhibit such repeats. For example, the polypeptide can be a . huntingtin polypeptide, or a fragment of a huntingtin polypeptide containing the expanded repeat. The expanded poly(Q) repeat useful in the assay can include at least 40 consecutive glutamine residues (e.g., 40, 45, 50, 60, 70, 80, 90, 97, 100, 105, 103, 107, or 110 glutamine residues) and the wildtype poly(Q) repeat can include 35 or fewer consecutive glutamine residues (e.g., 30, 25, 23, 20, 15, or 10 residues). The inducible promoter can be any inducible promoter, such as an ecdysone-responsive promoter.

The cells useful in the assays can be any cell type, such as any mammalian cell type. The cells can be neuronal cells, such as PC 12 cells. The screening method can also be automated.

Candidate agents useful for the described methods can be small organic or inorganic molecules, a protein, a peptide, or a nucleic acid.

The methods can also include further assays to determine the mechanism of action of an agent that decreases intracellular levels of a polypeptide containing an expanded poly-(Q) repeat, such as determining whether the agent inhibits a chaperone or co- chaperone protein, such as heat shock protein (e.g., a heat shock protein of the hsp90 family).

To most accurately assess a compound, beta-galactosidase expression can be assayed before the cell is exposed to the compound, just after the cell has been exposed to the compound (i.e., before any significant incubation has occurred), and again after a period of incubation. When the method is carried out in this way, the first reading will provide an assessment of steady state fusion polypeptide levels in the cell. Signal generated by beta-galactosidase just after the cell has been exposed to the compound more accurately reflects any signal intensity caused by the compound per se. After a period of incubation with a test compound, cells are lysed and the beta-galactosidase assay is performed to assess the effect of the compound on intracellular fusion polypeptide levels. In this method, as well as the others described herein, the compound can be virtually any substance (e.g., the compound can be a biological molecule, such as a polypeptide expressed in the cell, a chemical compound, or a nucleic acid). Libraries that encode or contain candidate compounds are available to those of ordinary skill in the art through charitable sources (e.g., ChemBridge Corporation (San Diego, CA) (which provides useful information about chemical libraries on the worldwide web)) and commercial suppliers.

The cells that can be used in the methods and assays described herein can be mammalian cells (e.g., the cell of a rodent, non-human primate, or human) or yeast cells (of any strain). Regardless of the cell type used, the recombinant proteins (e.g., fusion proteins) they express can be placed under the control of an inducible promoter. Many useful inducible promoters are known in the art. For example, in the event mammalian cells are employed, the fusion protein can be placed under the control of an ecdysone inducible promoter (see, e.g., Rossi et al., Trends in Cell Biol. 10:119-122, 2000).

The assays can be used as a high throughput screen using methods know to those in the art. Typical high throughput screening assays include robotic instrumentation for plating cells into multiwell plates. The multiwell plates can be 96-, 384- or 1536- well plates. Concentrated stocks of compounds can be added from stock plates. In general, each assay plate includes both positive and negative control compounds. High throughput screening systems general can analyze between 10,000 and 100,000 compounds per day.

Compounds identified as capable of decreasing intracellular levels of a polypeptide containing an expanded poly(Q) repeat can be tested to determine the mechanism of action of the compound. For example, the compounds can be assayed for an effect on decreased protein synthesis (such as through pulse-labeling experiments) or increased protein degradation (such as by rate of decreased β- galactosidase signal in the presence of a test compound). The compounds can also be tested for a decrease in the rate of synthesis of a polypeptide containing an expanded poly(Q) repeat. Characterization of nascent polypeptide chain interactions inside the ribosome during synthesis provide strong evidence for distinctive chaperone activity within the ribosome tunnel as a nascent chain is undergoing elongation. The events which occur during the folding and processing of nascent chains could be the sites of specificity for the action of compounds which impact on the instantaneous rate of synthesis of the polypeptide. The compounds can also be tested for an increase in the rate of degradation of a polypeptide containing an expanded poly(Q) repeat.

Experiments can be performed to elucidate what aspects of degradation are affected by a compound that causes an increased degradation rate of an expanded po Iy(Q) repeat. For example, assays performed in the presence of a proteasome inhibitor will indicate if a degradation pathway is proteosome dependent. If β- galactosidase activity in cells contacted with a proteasome inhibitor and a test compound is decreased as compared to cells contacted with a proteasome inhibitor in the absence of the compound, then the pathway of degradation is proteasome- independent.

A test compound can also be tested for an effect on chaperone activity. For example, rabbit reticulocyte assays are useful for assaying the ability of a compound to inhibit protein refolding. Rabbit reticulocytes contain chaperone and co-chaperone proteins that facilitate protein refolding. Therefore, a compound that inhibits refolding of a polypeptide containing an expanded poly(Q) repeat in rabbit reticulocytes may target a chaperone or co-chaperone. Further experiments, such as coimmunoprecipitation experiments, can verify whether a particular chaperone, such as hsp90, interacts with the polypeptide containing an expanded poly(Q) repeat. In vitro assays of chaperone activity can also be useful in determining the impact of compounds on these pathways.

Other assays for evaluating the effect of a compound on chaperoning activity include measuring the levels of specific hsp90 client proteins in cells that have been treated with a test compound. For example, one population of cells can be teated with one or more concentrations of the test compound, while a second, control population, is treated with vehicle alone, and a third population of cells is treated with a known hsp90 inhibitor, for example, geldanamycin. The resulting levels of a specific client protein or proteins can be assayed by any method known in the art, for example, and without limitation, immunoprecipitaion, immunoblotting, ELISA , enzymatic assays for the activity of the specific protein or protein chip assays. Alternatively, or in addition , the compounds be assayed for the ability to inhibit the ATPase activity of hsp90, using any ATPase assays known to those of skill in the art, for example, the yeast hsp90 malachite green assay. Test compounds can also be assayed for the ability to induce the synthesis of hsp70, using for example, immunoprecipitation or hsp70 reporter assays.

In vitro assays using reconstituted chaperoning systems are also useful for more clearly defining the precise molecular target of a particular compound. One such assay includes a reconstituted progesterone receptor chaperoning assay which measures the capacity of purified hsp90, hsp70, hsp40, Hop and p23 to efficiently assemble the progesterone receptor.

Further validation of compounds in additional cell-based and animal assay systems are within the scope of the invention.

For use in the screening methods, both naturally occurring and non-naturally occurring polypeptides containing expanded poly(Q) repeats can be produced recombinantly. Such recombinant proteins, nucleic acid sequences encoding them, and expression vectors useful in mediating expression are also within the scope of the present invention. The cell in which the recombinant polypeptide is produced can be used directly in the methods of the invention, or the recombinant polypeptide can be purified from the culture medium or from a lysate of the cells. Cells that include an expanded poly(Q) repeat polypeptide and, optionally, a compound disclosed herein (e.g. , a compound of Tables 1 or a salt, solvate, biologically active variant or other analog thereof) are within the scope of the present invention, as are arrays of such cells.

Variants of the expanded poly(Q) repeat polypeptides can also be targets of the compounds of the invention, or used in the assays described herein. Variants can be prepared by substituting selected amino acid residues in the polypeptides. A variant of an expanded poly(Q) repeat polypeptide includes a polypeptide that has high sequence identity (e.g., 60, 70, 80, 90, 95, 96, 97, 98, or 99%) to an expanded poly(Q) repeat polypeptide.

Isolated nucleic acid molecules that encode naturally occurring, expanded poly(Q) repeat polypeptides, variants thereof, or non-naturally occurring expanded poly(Q) repeat polypeptides are useful in the methods of the invention and in the assays described herein. Naturally occurring nucleic acid sequences that encode expanded poly(Q) repeat polypeptides are well known in the art and can be obtained, for example, from GENBANK™. The nucleic acid triplet that encodes the amino acid glutamine can be either CAA or CAG. Typically, expressing an expanded poly(Q) repeat polypeptide in a cell involves inserting a sequence encoding a poly(Q) repeat polypeptide into a vector, where it is operably linked to one or more expression control sequences. The need for, and identity of, expression control sequences will vary according to the type of cell in which the poly(Q) repeat polypeptide sequence is to be expressed. Examples of expression control sequences include transcriptional promoters, enhancers, suitable mRNA ribosomal binding sites, and sequences that terminate transcription and translation.

Suitable expression control sequences can be selected by one of ordinary skill in the art. Standard methods can be used by the skilled person to construct expression vectors. See, generally, Sambrook et al., 1989, Cloning — A Laboratory Manual (2^nd Ed), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Useful vectors include plasmid vectors and viral vectors. Viral vectors can be, for example, those derived from retroviruses, adenoviruses, adeno-associated virus, SV40 virus, pox viruses, or herpes viruses. Once introduced into a host cell {e.g., a bacterial cell, a yeast cell, an insect cell, an avian cell, or a mammalian cell), the vector can remain episomal, or be incorporated into the genome of the host cell.

While pharmaceutical formulations are described further below, we note here, that the compounds of the invention, including those just described, can be formulated for oral or parenteral administration to a patient. Likewise, while methods are described further elsewhere herein, we note that the invention encompasses methods of treating a subject who has, who has been diagnosed as having, or who is at risk of developing, a disorder characterized by a polypeptide containing an expanded poly(Q) repeat. The methods can include the step of identifying the subject (or patient) and administering to the subject a therapeutically effective amount of a pharmaceutical composition that includes any of the compounds described herein {e.g., a compound conforming to Formulas I- V). The subject may have been diagnosed as having, or at risk of developing, Huntington's disease, spinocerebellar ataxia, hemiplegic migraine, spinobulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy, breast cancer, ovarian cancer endometrial cancer, prostate cancer, leukemia, or amyloidosis. The invention also encompasses methods of treating a subject who has, who has been diagnosed as having, or who is at risk of developing, a disorder characterized by protein misfolding. The methods can include the step of identifying the subject (or patient) and administering to the subject a therapeutically effective amount of a pharmaceutical composition that includes any of the compounds described herein (e.g., a compound conforming to Formulas I- V). The subject may have been diagnosed as having, or at risk of developing, a disease characterized by a polypeptide containing an expanded poly(Q) repeat, such as those listed above, or, for example, Alzheimer's Disease, Parkinson's Disease, amyotrophic lateral sclerosis, or prion disease.

Each of the variables designated by, for example, R, X, Y, m, and n in any of the formulas disclosed herein can be selected independently. While we tend to use the term "comρound(s)", we may also use terms like "agent(s)" to refer to the molecules described herein.

The compounds identified by the methods described herein (which may also be referred to herein as "therapeutic agents") may be used to treat a variety of disorders, including Huntington's disease. For example, the compounds, described herein can be included as therapeutic agents in pharmaceutical compositions to treat HD and other conditions described herein that are characterized by expression of a polypeptide containing an expanded poly(Q) repeat.

Treating a subject can encompass administration of a therapeutic agent as a prophylactic measure to prevent the occurrence of disease or to lessen the severity or duration of the symptoms associated with the disease. Physicians and others of ordinary skill in the art routinely make determinations as to the success or failure of a treatment. Treatment can be deemed successful despite the fact that not every symptom of the disease is totally eradicated. Treatment can also be deemed successful despite side- effects.

It is usual in the course of developing a therapeutic agent that tests of that agent in vitro or in cell culture are followed by tests in animal models of human disease, and further, by clinical trials for safety and efficacy in humans. Accepted animal models for many diseases are now known to those of ordinary skill in the art. For example, therapeutic agents of the present invention can be screened in a Drosophila model of neurodegeneration as well as in more evolutionarily advanced animals.

Mammalian models for Huntington's disease are available. To generate similar animal models, a homolog of the expanded poly(Q) repeat polypeptide is first cloned from the genome of the selected mammal using standard techniques. For example, the sequence can be amplified by PCR or obtained by screening an appropriate library under conditions of low stringency (as described, e.g., in Sambrook et al. supra.). Subsequently, trinucleotide repeats can be introduced into the gene by molecular cloning and mutagenesis techniques. For example, in a HD model, CAG repeats can be introduced in the HD gene. The site for insertion of the repeat sequence can be located by alignment of the cDNA from the desired mammal with the human cDNA for the expanded poly(Q) polypeptide. The modified gene with artificially expanded repeats can be reintroduced into the mammal using standard methods for transgenesis.

Methods for generating transgenic mice are routine in the art {See, e.g. , Hogan et al., Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1994)). As an example, a mouse bearing a transgene comprising the HD gene and expanded CAG repeats has symptoms similar to the human disease. Murine symptoms can include hyperactivity, circling, abnormal gait, tremors, learning deficits, hypoactivity, and hypokinesis. Neuropathological symptoms include general brain atrophy, progressive striatal atrophy, neuropil aggregates, inclusions in the striatum, reduced dendritic spines, cell loss in the cortex, and striatum.

Any of these behavioral or physiological deficits can be assessed in order to determine the efficacy of a given therapeutic agent of the invention. For example, the agent can be administered to a transgenic mouse model, generated as described above. The symptoms of a treated mouse can be compared to untreated mice at various times during and after treatment. In addition, treated and untreated mice can be sacrificed at various intervals after treatment, and the neuropathology of the brain can be analyzed. Thus, the efficacy of the treatment can be evaluated readily by comparing the behavioral symptoms, neuropathological symptoms, and clinical symptoms of treated and untreated mice.

Subjects who are treated with the compounds of the invention may have been diagnosed with any disease characterized by expression of a polypeptide containing an expanded poly(Q) repeat. Alternatively, the subject may be at risk for developing these disorders. For example, a subject may have a family history or a genetic mutation or element (e.g., an expanded trinucleotide repeat) that contributes to the development of disease. Human subjects, in consult with their physicians and/or other health care professionals, can decide whether their risk is great enough to undergo preventative care (as is the case for any prophylactic treatment or procedure). While the subjects of the preventative and/or therapeutic regimes described herein may be human, the compounds and compositions of the invention can also be administered to non-human subjects. The prophylactic and therapeutic methods can be carried out by administering to the subject a pharmaceutical composition containing a therapeutically effective amount of one or more of the compounds described herein. While a single compound may be effective, the invention is not so limited. A subject can be treated with multiple compounds, administered simultaneously or sequentially. For example, a subject can be treated with one or more of the compounds described herein and, optionally, a chemotherapeutic agent, an analgesic, a bronchodilator, levodopa or a similar medication. The combination therapy will, of course, depend on the disorder being treated. Where a compound of the invention is administered to treat a patient with a cancer, it may be combined with a known chemotherapeutic agents used to treat that type of cancer; where a compound of the invention is administered to treat a patient with Huntington's disease, it may be combined with a medication to decrease chorea; and so forth.

Compounds that decrease intracellular levels of polypeptide containing an expanded poly(Q) repeat can also be used to diagnose diseases characterized by expression of an expanded poly(Q) repeat polypeptide. These methods can be carried out by providing a biological sample from a patient suspected of having a disease associated with an expanded poly(Q) repeat polypeptide; exposing the sample to a compound of the invention; and determining whether the compound modulates intracellular levels the expanded poly(Q) repeat polypeptide within the sample. The compound can be one that is known to interact directly with a primary target or one that modulates protein levels by acting upstream from the primary target. The compound can also be one that is known to interact with proteins in the context of the suspected disease. For example, a compound that is known to decrease intracellular levels of huntingtin can be used to diagnose a patient suspected of having HD. The sample will be exposed to the compound for a time and under conditions (e.g., physiological conditions of temperature and pH) sufficient to permit the compound to affect proteins within the sample (e.g., huntingtin proteins within cells within the sample). The diagnostic methods can be carried out before, after, or in conjunction with other diagnostic tests, and their results can inform the subject's treatment regime. For example, where a compound is found to modulate intracellular levels of huntingtin proteins in a sample obtained from a patient suspected of having HD, that compound may then be used to treat the patient.

The blood-brain barrier is an obstacle for the delivery of drugs from circulation in the bloodstream to the brain. The endothelial cells of brain capillaries are connected by tight intercellular junctions, which inhibit the passive movement of compounds out of the blood plasma into the brain. These cells also have reduced pinocytic vesicles in order to restrict the indiscriminate transport of materials intracellularly. These features of the brain regulate the exchange of materials between plasma and the central nervous system. Both active and passive transport mechanisms operate to exclude certain molecules from traversing the barrier. For example, lipophilic compounds are more permeable to the barrier than hydrophilic compounds (Goldstein et al., Scientific American 255:74-83, 1996; Pardridge et al., Endocrin. Rev. 7:314-330, 1996).

However, the blood-brain barrier must also allow for the selective transport of desired materials into the brain in order to nourish the central nervous system and to remove waste products. The mechanisms by which this is accomplished can provide the means for supplying the therapeutic agents described herein.

The compositions of the invention can be delivered to the CNS following conjugation with other compounds as follows (and as described further in, for example, U.S. Patent No. 5,994,392). In one instance, polar groups on a compound are masked to generate a derivative with enhanced lipophilic qualities. For example, norepinephrine and dopamine have been modified with diacetyl and triacetyl esters to mask hydroxyl groups. An implementation of this strategy has been previously used to create a pro-drug derivative of dopamine (see U.S. Patent No. 5,994,392). The modified drugs are generally referred to as pro-drugs, and the compounds of the invention encompass those described herein in which polar groups are masked. This method may have the additional advantage of providing an inactive species of the compound in the general circulation. After crossing the blood-brain barrier, enzymes present in the central nervous system are able to hydrolyze the linkages (e.g., ester linkages), thereby unmasking the compound and liberating the active drug. Thus, compounds of the invention can be chemically modified to create pro-drugs by, e.g., conjugation to a lipophilic moiety or carrier. A compound or a variant thereof having at least one free hydroxyl or amino group can be coupled to a desired carrier (e.g., a fatty acid, a steroid, or another lipophilic moiety).

More specifically, and for example, the hydroxyl groups can first be protected with acetonide. The protected agent is then reacted with the desired carrier in the presence of a water-extracting compound (e.g., dicyclohexyl carbodiiamide), in a solvent (e.g., dioxane, tetrahydrofurane), or N,N dimethylformamide at room temperature. The solvent is then removed, and the product is extracted using methods routinely used by those of ordinary skill in the art. Amine groups can be coupled to a carboxyl group in the desired carrier. An amide bond is formed with an acid chloride or low carbon ester derivative of the carrier. Bond formation is accompanied by HCl and alcohol liberation. Alcohol groups on the compound can be coupled to a desired carrier using ester bonds by forming an anhydride derivative, i.e. the acid chloride derivative, of the carrier. One of ordinary skill in the art of chemistry will recognize that phosphoramide, sulfate, sulfonate, phosphate, and urethane couplings are also useful for coupling a therapeutic agent (e.g., a compound described herein) to a desired carrier. A useful and adaptable method for lipidation of antibodies is described by Cruikshank et al. (J. Acquired Immune Deficiency Syndromes and Human Retrovirology .14:193, 1997).

Procedures for delivering therapeutic agents (or "compounds") of the invention to the CNS can also be carried out using the transferrin receptor as described, for example, in U.S. Patent No. 6,015,555. To implement this procedure, the agents are conjugated to a molecule that specifically binds to the transferrin receptor (e.g., an antibody or antigen- binding fragment thereof, or transferrin). Methods for obtaining antibodies against the transferrin receptor and for coupling the antibodies to a desired compound are also described in U.S. Patent No. 6,015,555.

Monoclonal antibodies that specifically bind to the transferrin receptor include OX-26, T58/30, and B3/25 (Omary et al, Nature 286:888-891, 1980), T56/14 (Gatter et al., J. Clin. Path. 36:539-545, 1983), OKT-9 (Sutherland et al., Proc. Natl. Acad. ScL USA 78:4515-4519, 1981), L5.1 (Rovera, Blood 59:671-678, 1982) and 5E-9 (Haynes et al., J. Immunol. 127:347-351, 1981). In one embodiment, the monoclonal antibody OX- 26 is used. The antibody of choice can be an Fab fragment, a F(ab')2 fragment, a humanized antibody, a chimeric antibody, or a single chain antibody.

The antibody to the transferrin receptor is conjugated to a desired compound with either a cleavable or non-cleavable linker. The preferred type of linker can be determined without undue experimentation by making cleavable and non-cleavable conjugates and assaying their activity in, for example, an in vitro or cell culture assay described herein. The conjugates can be further tested in vivo (e.g., in a animal model of a disease of interest). Examples of chemical systems for generating non-cleavable linkers include the carbodiimmide, periodate, sulfhydryl-maleimide, and N-succinimidyl-3-(2-puridyldithio) propionate (SPDP) systems. Carbodiimide activates carboxylic acid groups, which then react with an amino group to generate a noncleavable amide bond. This reaction may be especially useful for coupling two proteins. Periodate is used to activate an aldehyde on an oligosaccharide group such that it can react with an amino group to generate a stable conjugate. Alternatively, a hydrazide derivative of the desired compound can be reacted with the antibody oxidized with periodate. Sulfhydryl-maleimide and SDPD use sulfhydryl chemistry to generate non-cleavable bonds. SDPD is a heterobifunctional crosslinker that introduces thiol-reactive groups. In the sulfhydryl-maleimide system, an NHS ester (e.g., gamma- maleimidobutyric acid NHS ester) is used to generate maleimide derivative, for example, of a protein drug or antibody. The maleimide derivative can react with a free sulfhydryl group on the other molecule.

Cleavable linkers are also useful. Cleavable linkers include acid labile linkers such as cis-aconitic acid, cis-carboxylic alkadienes, cis-carboxylic alkatrienes, and polypeptide-maleic anhydrides (see U.S. Patent No. 5,144,011).

In one embodiment, the compound is a compound having one of the structures shown in Table 1. Such a compound can be covalently attached to an antibody specific for the transferrin receptor. In one embodiment, use of a single chain antibody is preferred in order to facilitate covalent fusion with the therapeutic agent.

The targeting antibody can be linked covalently to the therapeutic agent (or "compound") of the invention. A protease recognition site can be included in the linker if cleavage of the antibody is required after delivery.

The efficacy of strategies to deliver a desired compound across the blood-brain barrier can, of course, be monitored. The desired compound, conjugated for delivery across the blood-brain barrier, is administered to a test mammal (e.g. , a rat, a mouse, a non-human primate, a cow, a dog, a rabbit, a cat, or a sheep). One of ordinary skill in the art will, however, recognize that the permeability of the blood-brain barrier varies from species to species, with the human blood-brain barrier being the least permeable. The mode of administration can be the same as the desired mode of treatment (e.g., intravenous). For a comprehensive analysis, a set of test mammals is used. The test mammals are sacrificed at various times after the agent is administered and are then perfused through the heart with, e.g., Dulbecco's phosphate-buffered saline (DPBS) to clear the blood from all organs. The brain is removed, frozen in liquid nitrogen, and subsequently sectioned in a cryostat. The sections are placed on glass microscope slides. The presence of the desired agent is then detected in the section, for example with an antibody, or by having administered a radiolabeled or otherwise tagged compound (such labeled therapeutic compounds as described above). Detection is indicative of the compound having successfully traversed the blood-brain barrier. If a method of enhancing the compounds permeability to the blood-brain barrier is being assessed, then the amount of the agent detected in a brain section can be compared to the amount detected in a brain section from an animal treated with the same compound without the enhancing method.

The terms "blood-brain barrier permeant" or "blood-brain barrier permeable" are qualities of a compound for which the ratio of a compound's distribution at equilibrium in the cerebrospinal fluid (CSF) relative to its distribution in the plasma (CSF/plasma ratio) is greater than at least (or about) 0.01, 0.02, 0.05, or 0.1. While lower ratios are generally preferred, any ratio that allows a compound to be used clinically is acceptable.

To facilitate targeting to a polypeptide of interest {e.g., to a huntingtin protein), the compound (e.g., a compound conforming to any of Formulas I- V) can include a moiety that specifically binds to the target protein. For example, a compound conforming to Formula I can be joined to an antibody or an antigen-binding portion thereof (e.g., a single chain antibody) that specifically binds the target protein (e.g., huntingtin). Targeting moieties are described further below.

A therapeutic vector can be administered to a subject, for example, by intravenous injection, by local administration (see U.S. Patent No. 5,328,470) or by stereotactic injection (see e.g., Chen et αl., Proc. Nαtl. Acαd. Sci. USA 91:3054-3057, 1994). The compound can be further formulated, for example, to delay or prolong the release of the active agent by means of a slow release matrix.

Regardless of whether or not the compound is to cross the blood-brain barrier, it can be conjugated to a targeting agent that facilitates interaction with a target protein (e.g., huntingtin). As noted, the compound can be directly or indirectly joined to an antibody (e.g., a single chain antibody) or an antigen-binding fragment thereof that specifically binds the target protein.

An appropriate dosage of the therapeutic agents of the invention must be determined. An effective amount of a therapeutic compound is the amount or dose required to ameliorate a symptom of a disorder associated with expression of a polypeptide containing an expanded poly(Q) repeat. Determining the amount required to treat a subject is routine to one of ordinary skill in the art (e.g., a physician, pharmacist, or researcher). First, the toxicity and therapeutic efficacy of an agent (i.e. a tri-domain molecule) is determined. Routine protocols are available for determining the LD_5O (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population) in non-human animals. The therapeutic index is measured as the ratio of the LD₅₀/ED50. Compounds, formulations, and methods of administration with high therapeutic indices are preferable as such treatments have little toxicity at dosages that provide high efficacy. Compounds with toxic or undesirable side effects can be used, if means are available to deliver the compound to the affected tissue, while minimizing damage to unaffected tissue.

In formulating a dosage range for use in humans, the effective dose of a therapeutic agent can be estimated from in vitro cell studies and in vivo studies with animal models. If an effective dose is determined for ameliorating a symptom in cell culture, a dose can be formulated in an animal in order to achieve a circulating plasma concentration of sodium butyrate that falls in this range. An exemplary dose produces a plasma concentration that exceeds the IC₅₀ {i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture assays. The circulating plasma concentration can be determined, for example, by administering a labeled therapeutic composition to the test animal, obtaining a blood sample, and quantitating the amount of labeled compound present at various times after administration.

An appropriate daily dose of a therapeutic agent can be between about 0.1 mg/kg of body weight to about 500 mg/kg, or between about 1 mg/kg to about 100 mg/kg. The dose can be adjusted in accordance with the blood-brain barrier permeability of the compound. For example, a therapeutic compound can be administered at a dosage of 50 mg/kg to 100 mg/kg in order to treat the brain. The dose for a patient can be optimized while the patient is under care of a physician, pharmacist, or researcher. For example, a relatively low dose of a tri-domain therapeutic can be administered initially. The patient can be monitored for symptoms of the disorder being treated (e.g., HD). The dose can be increased until an appropriate response is obtained. In addition, the specific dose level for any particular subject can vary depending on the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and other drugs provided in combination.

As occurs in the course of all drug development, optimal treatment regimes will emerge through further modeling and clinical trials. It may be, for example, that a patient will receive a combination of compounds that act synergistically to decrease intracellular levels of an expanded poly(Q) repeat polypeptide by the same or different mechanisms of action. Combination therapies may also rely on administration of a compound that interferes with gene transcription (e.g., a small molecule or a nucleic acid that mediates RNAi) and a compound that facilitates degradation of any remaining unwanted polypeptide-containing complexes.

The efficacy of a dose of any therapeutic agent can be determined in a subject, For example, the subject can be monitored for clinical symptoms, for example, a symptom of a trinucleotide repeat disease, such as a symptom of HD. Behavioral symptoms of HD include irritability, apathy, lethargy, depression, hostile outbursts, loss of memory and/or judgment, loss of ability to concentrate, anxiety, slurred speech, difficulty swallowing and/or eating, and inability to recognize persons. Clinical symptoms of HD include loss of coordination, loss of balance, inability to walk, uncontrolled movements of the fingers, feet, face, and/or trunk, rapid twitching, tremors, chorea, rigidity, and akinesia (severe rigidity).

The compounds of the invention or biologically active variants thereof (e.g., salts) may be synthesized in vitro, produced in vivo (e.g., produced within the body (e.g., intracellularly) following administration to a patient), or produced following application to a cell in culture. Accordingly, the present invention features methods of making the compounds and compositions of the present invention. The compounds can be synthesized using routine techniques known to one of ordinary skill in the art. For example, the compounds can be made by providing a starting compound or intermediate and reacting the compound or intermediate with one or more chemical reagents in one or more steps to produce a compound described herein (e.g., a compound of any of Formulas I-V).

Some of the compounds described herein can be obtained from commercial sources. As noted, others can be synthesized by conventional methods using commercially available starting materials and reagents. The compounds described herein can be separated from a reaction mixture and further purified by a method such as column chromatography, high-pressure liquid chromatography, or recrystallization. As can be appreciated by one of ordinary skill in the art, further methods of synthesizing the compounds of the formulae herein are available. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof. Techniques useful for the separation of isomers, for example, stereoisomers are within skill of the art and are described in Eliel, E.L.; Wilen, S.H.; Mander, L.N. Stereochemistry of Organic Compounds, Wiley Interscience, NY, 1994. For example compounds can be resolved via formation of diasteromeric salts, for example, with a chiral base, for example, (+) or (-) a- methylbenzylamine, or via high performance liquid chromatography using a chiral column.

In an alternate embodiment, the compounds described herein may be used as platforms or scaffolds that may be utilized in combinatorial chemistry techniques for preparation of derivatives and/or chemical libraries of compounds. Such derivatives and libraries of compounds have biological activity and are useful for identifying and designing compounds possessing a particular activity. Combinatorial techniques suitable for utilizing the compounds described herein are known in the art as exemplified by Obrecht, D. and Villalgrodo, J.M., "Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular- Weight Compound Libraries", Pergamon-Elsevier Science Limited (1998), and include those such as the "split and pool" or "parallel" synthesis techniques, solid-phase and solution-phase techniques, and encoding techniques (see, for example, Czarnik, Curr. Opin. Chem. Bio. 1:60, 1997). Thus, one embodiment relates to methods of using the compounds described herein for generating derivatives or chemical libraries. The methods can be carried out by performing these, and optionally additional, steps: (1) providing a body comprising a plurality of wells; (2) providing one or more compounds identified by methods described herein in each well {e.g., any of the compounds of Formulas I-V; (3) providing an additional one or more chemicals in each well, where the compound, upon exposure to the chemical(s) may produce one or more products; and (4) isolating the resulting one or more products from each well. We may refer to the original compound as the "first" compound and to the chemical as the "second" compound. The order in which the first and second compounds are added to the wells can vary, and the methods can be carried out in vitro or in cell culture. Lead derivatives can be further tested in animal models.

In alternate embodiments, the methods of using the compounds described herein for generating derivatives or chemical libraries can be carried out using a solid support. These methods can be carried out by, for example: (1) providing one or more of the compounds described herein attached to a solid support; (2) treating the one or more compounds identified by methods described herein attached to a solid support with one or more additional compounds or chemicals; (3) isolating the resulting one or more products from the solid support. In these methods, "tags" or identifier or labeling moieties may be attached to and/or detached from the compounds described herein or their derivatives, to facilitate tracking, identification or isolation of the desired products or their intermediates. Such moieties are known in the art and exemplary tags are noted above. The chemicals (or "second" compound(s)) used in the aforementioned methods may include, for example, solvents, reagents, catalysts, protecting group and deprotecting group reagents, and the like. Examples of such chemicals are those that appear in the various synthetic and protecting group chemistry texts and treatises which are known in the art and may be referenced herein.

In one aspect, the invention includes cell-based and in vitro assays {e.g., high throughput screens) that can be used with essentially any compound collection. Following an assay, the result can be recorded in a database, and such databases are also within the scope of the present invention. For example, the invention features a computer-readable database that includes a plurality of records. Each record includes (a) a first field that includes information reflecting the identity of an agent {e.g., an agent within one of the types of libraries described herein) and (b) a second field that includes information concerning the impact of the agent on intracellular polypeptide levels. Additional fields may include the results of toxicity tests, dose-response tests, and the like. The information contained with the fields can be obtained in any order {e.g., the information reflecting intracellular protein levels can be obtained first). However, to help ensure the integrity of the database, the information should be obtained independently (or "blindly"). The database can also include a field comparing the agent to a clinical outcome {e.g., an improvement in a sign or symptom associated with Huntington's disease, cancer, or any of the other disorders described herein). The number of records can be, but is not necessarily, great. For example, a useful database can include at least 10, 25, 50, 100, 250, 500, 1000, 1500, 1800, 2000, or 2500 records.

Methods of Treating/Preventing Cancer

Provided herein are methods for treating and/or preventing cancer or symptoms of cancer in a subject comprising administering to the subject a therapeutically effective amount of a composition comprising Formula VI. The methods disclosed herein are generally useful for inhibiting cancer cell growth and as prophylactic therapeutics. We may refer to prophylaxis as the complete prevention of the symptoms of a disease, a delay in onset of the symptoms of a disease, or a lessening in the severity of subsequently developed disease symptoms. As used herein, "therapy" can mean a complete abolishment of the symptoms of a disease or a decrease in the severity of the symptoms of the disease.

In some embodiments, the cancer is a cancer associated with misregulation of hsp90 or an hsp 90 client protein. There are many ways in which such misregulation can occur. In some cancers, the intracellular levels of hsp90 may be increased or decreased resulting in alterations in the levels of certain oncogenic hsp90 client proteins. In other cancers, the hsp90 client proteins are either mutated or overexpressed or both. In the normal healthy cell, the interactions between the hsp90 complex and the client proteins are typically low-affinity. Many oncogenic forms of hsp90 client proteins display unusually stable interactions with the hsp90 chaperone complexes. These associations impair normal proteolytic turnover of these cancer related molecules and seems to be important for their transforming activity. Oncogenic mutations generally occur in proteins that regulate cell growth and differentiation. A proto-oncogene is a normal gene that can become an oncogene, either after mutation or increased expression. Proto-oncogenes are often involved in signal transduction and execution of mitogenic signals, usually through their protein products. Upon activation, a proto-oncogene (or its product) becomes a tumor inducing agent, an oncogene. Specific mutations kinds of mutations can include for example, without limitation, point mutations, translocations, or expansion of polyglutamine repeat sequences. In some embodiments, the cancer is breast, endometrial, prostate cancer or leukemia. In other embodiments, the cancer is neuroblastoma or colon cancer. In some embodiments the subject has been diagnosed as having a cancer or as being predisposed to cancer.

Symptoms of cancer are well-known to those of skill in the art and include, without limitation, unusual mole features, a change in the appearance of a mole, including asymmetry, border, color and/or diameter, a newly pigmented skin area, an abnormal mole, darkened area under nail, breast lumps, nipple changes, breast cysts, breast pain, death, weight loss, weakness, excessive fatigue, difficulty eating, loss of appetite, chronic cough, worsening breathlessness, coughing up blood, blood in the urine, blood in stool, nausea, vomiting, liver metastases, lung metastases, bone metastases, abdominal fullness, bloating, fluid in peritoneal cavity, vaginal bleeding, constipation, abdominal distension, perforation of colon, acute peritonitis (infection, fever, pain), pain, vomiting blood, heavy sweating, fever, high blood pressure, anemia, diarrhea, jaundice, dizziness, chills, muscle spasms, colon metastases, lung metastases, bladder metastases, liver metastases, bone metastases, kidney metastases, and pancreatic metastases, difficulty swallowing, and the like.

In some embodiments compositions comprising two or more anticancer agents are contemplated. One or more hsp90 inhibitors may also be administered with another therapeutic agent, such as a cytotoxic agent, an antibody or cancer chemotherapeutic. Concurrent administration of two or more therapeutic agents does not require that the agents be administered at the same time or by the same route, as long as there is an overlap in the time period during which the agents are exerting their therapeutic effect. Simultaneous or sequential administration is contemplated, as is administration on different days or weeks.

In some embodiments the methods provided contemplate the administration of combinations, or "cocktails", of different antibodies. Such antibody cocktails may have certain advantages inasmuch as they contain antibodies which exploit different effector mechanisms or combine directly cytotoxic antibodies with antibodies that rely on immune effector functionality. Such antibodies in combination may exhibit synergistic therapeutic effects. Useful antibodies can include antibodies that target the EGF receptor, e.g. , Cetuximab (Erbitux™), antibodies that target VEGF, e.g. , Bevacizumab (Avastin™) and antibodies that target Her-2, e.g., trastuzimab (Herceptin™)

A cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes {e.g., ¹³¹1, ¹²⁵I, ⁹⁰Y and ¹⁸⁶Re), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin or synthetic toxins, or fragments thereof. A non-cytotoxic agent refers to a substance that does not inhibit or prevent the function of cells and/or does not cause destruction of cells. A non-cytotoxic agent may include an agent that can be activated to be cytotoxic. A non-cytotoxic agent may include a bead, liposome, matrix or particle.

In some embodiments, conventional cancer medicaments are administered with the compositions disclosed herein. Highly suitable agents include anti- angiogenic agents. Anti-angiogenic agents block the ability of tumors to stimulate new blood vessel growth necessary for their survival. Any anti-angiogenic agent known to those in the art can be used, including agents such as Bevacizumab (Avastin®, Genentech, Inc.) that block the function of vascular endothelial growth factor (VEGF). Other examples include, without limitation, Dalteparin (Fragmin®), Suramin ABT-510, Combretastatin A4 Phosphate, Lenalidomide, LY317615(Enzastaurin), Soy Isoflavone (Geni stein; Soy Protein Isolate) AMG-706, Anti-VEGF antibody, AZD2171, Bay 43-9006 (Sorafenib tosylate), PI-88, PTK787/ZK 222584 (Vatalanib), SUl 1248 (Sunitinib malate), VEGF-Trap, XLl 84, ZD6474, Thalidomide, ATN-161, EMD 121974 (Cilenigtide) and Celecoxib (Celebrex®).

Other useful agents include those agents that promote DNA-damage, e.g., double stranded breaks in cellular DNA, in cancer cells. Any form of DNA-damaging agent know to those of skill in the art can be used. DNA damage can typically be produced by radiation therapy and/or chemotherapy. Examples of radiation therapy include, without limitation, external radiation therapy and internal radiation therapy (also called brachytherapy). Energy sources for external radiation therapy include x- rays, gamma rays and particle beams; energy sources used in internal radiation include radioactive iodine (iodine¹²⁵ or iodine¹³¹), and from strontium⁸⁹, or radioisotopes of phosphorous, palladium, cesium, indium, phosphate, or cobalt. Methods of administering radiation therapy are well know to those of skill in the art. Examples of DNA-damaging chemotherapeutic agents include, without limitation, Busulfan (Myleran), Carboplatin (Paraplatin), Carmustine (BCNU), Chlorambucil (Leukeran), Cisplatin (Platinol), Cyclophosphamide (Cytoxan, Neosar), Dacarbazine (DTIC -Dome), Ifosfamide (Ifex), Lomustine (CCNU), Mechlorethamine (nitrogen mustard, Mustargen), Melphalan (Alkeran), and Procarbazine (Matulane)

Other cancer chemotherapeutic agents include, without limitation, alkylating agents, such as carboplatin and cisplatin; nitrogen mustard alkylating agents; nitrosourea alkylating agents, such as carmustine (BCNU); antimetabolites, such as methotrexate; folinic acid; purine analog antimetabolites, mercaptopurine; pyrimidine analog antimetabolites, such as fluorouracil (5-FU) and gemcitabine (Gemzar®); hormonal antineoplastics, such as goserelin, leuprolide, and tamoxifen; natural antineoplastics, such as aldesleukin, interleukin-2, docetaxel, etoposide (VP- 16), interferon alfa, paclitaxel (Taxol®), and tretinoin (ATRA); antibiotic natural antineoplastics, such as bleomycin, dactinomycin, daunorubicin, doxorubicin, daunomycin and mitomycins including mitomycin C; and vinca alkaloid natural antineoplastics, such as vinblastine, vincristine, vindesine; hydroxyurea; aceglatone, adriamycin, ifosfamide, enocitabine, epitiostanol, aclarubicin, ancitabine, nimustine, procarbazine hydrochloride, carboquone, carboplatin, carmofur, chromomycin A3, antitumor polysaccharides, antitumor platelet factors, cyclophosphamide (Cytoxin®), Schizophyllan, cytarabine (cytosine arabinoside), dacarbazine, thioinosine, thiotepa, tegafur, dolastatins, dolastatin analogs such as auristatin, CPT-11 (irinotecan), mitozantrone, vinorelbine, teniposide, aminopterin, carminomycin, esperamicins (See, e.g., U.S. Patent No. 4,675,187), neocarzinostatin, OK-432, bleomycin, furtulon, broxuridine, busulfan, honvan, peplomycin, bestatin (Ubenimex®), interferon-β, mepitiostane, mitobronitol, melphalan, laminin peptides, lentinan, Coriolus versicolor extract, tegafur/uracil, estramustine (estrogen/mechlorethamine).

Additional agents which may be used as therapy for cancer patients include EPO, G-CSF, ganciclovir; antibiotics, leuprolide; meperidine; zidovudine (AZT); interleukins 1 through 18, including mutants and analogues; interferons or cytokines, such as interferons α, β, and γ hormones, such as luteinizing hormone releasing hormone (LHRH) and analogues and, gonadotropin releasing hormone (GnRH); growth factors, such as transforming growth factor-β (TGF-β), fibroblast growth factor (FGF), nerve growth factor (NGF), growth hormone releasing factor (GHRF), epidermal growth factor (EGF), fibroblast growth factor homologous factor (FGFHF), hepatocyte growth factor (HGF), and insulin growth factor (IGF); tumor necrosis factor-α & β (TNF-α & β); invasion inhibiting factor-2 (IIF-2); bone morphogenetic proteins 1-7 (BMP 1-7); somatostatin; thymosin-α-1; γ-globulin; superoxide dismutase (SOD); complement factors; and anti-angiogenesis factors.

Prodrugs include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, b-lactam- containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized into a prodrug form for use herein include, but are not limited to, those chemotherapeutic agents described above.

The methods disclosed herein can be applied to a wide range of species, e.g., humans, non-human primates (e.g- , monkeys), horses, cattle, pigs, sheep, deer, elk, goats, dogs, cats, mustelids, rabbits, guinea pigs, hamsters, rats, and mice.

The duration of treatment with any composition provided herein can be any length of time from as short as one day to as long as the life span of the host (e.g., many years). For example, hsp90 chaperone-modulating compositions can be administered once a month for three months or once a year for a period often years. It is also noted that the frequency of treatment can be variable. For example, hsp90 chaperone -modulating compositions can be administered once (or twice, three times, etc.) daily, weekly, monthly, or yearly. hsp90 chaperone modulating compositions can be administered together, i.e., at the same point in time or sequentially.

An effective amount of any composition provided herein can be administered to a host. The term "effective" as used herein refers to any amount that induces a desired therapeutic response while not inducing significant toxicity in the host. Such an amount can be determined by assessing a host's response after administration of a known amount of a particular composition. In addition, the level of toxicity, if any, can be determined by assessing a host's clinical symptoms before and after administering a known amount of a particular composition. It is noted that the effective amount of a particular composition administered to a host can be adjusted according to a desired outcome as well as the host's response and level of toxicity. Significant toxicity can vary for each particular host and depends on multiple factors including, without limitation, the host's disease state, age, and tolerance to pain.

The invention is further illustrated by the following examples, which should not be construed as further limiting.

EXAMPLES Example 1 : Materials and Methods

Cloning. The β-galactosidase Δ-subunit, lacking the first 500 bp of β-galactosidase, was cloned into a pIND vector (Invitrogen), which contains five E/GREs and a promoter for induction with Ponasterone A. The β-galactosidase α- subunit, the first 500 bp of β-galactosidase, was cloned upstream of the first 600 amino acids of Htt containing either 97Q or 23Q, with a FLAG-tag in between. These constructs, α 97Q and α 23Q, were then cloned into a pIND/Hygro vector (Invitrogen), containing five E/GREs and a promoter for induction with Ponasterone A.

Generation of stable PCl 2 cell lines. PC 12 cells stably transfected with pVgRXR (Ecdysone Receptor expression vector), was used as the parental cell line and is referred to as EcR PC 12. Lipofectamine 2000 (Invitrogen) was used to stably transfect β-galactosidase Δ-subunit in pIND and α 97Q or α 23 Q in pIND/Hygro into the EcR PC 12 cells.

In order to make stable cell lines that express both the β-galactosidase Δ- subunit and the α Htt fusion constructs, two rounds of stable transfections were performed. In the first step, the β-galactosidase Δ-subunit was stably transfected into PC 12 cells containing the expression vector for the Ecdysone Receptor, EcR PC 12 cells. Small colonies of these EcR-Δ cells were individually picked and expanded. After 24 hours of induction with PonA, 3 out of 31 clones were found to alpha- complement with transiently transfected α 23Q, i.e., to produce cell lines that had β- galactosidase activity. Clone 600-2 was chosen to continue with the next round of stable transfections because it most closely resembled the morphology of the parental cell line. Clone 600-2 was then stably transfected with either the α 97Q or α 23Q to make Δ-α 97Q or Δ-α 23 Q cells, respectively. Individual colonies were picked and expanded. Cell lines Δ-α 97Q #46 and Δ-α 23 Q #44 were selected for the assay based on their similarities in cell size, morphology, doubling time, amount of substrate hydrolyzed and β-galactosidase staining patterns.

Cell culture. Δ-α 97Q and Δ-α 23Q PC 12 cells were grown in DME medium with 15% fetal bovine serum, 2 mM penicillin-streptomycin, 2 mM L-glutamine at 37°C with 5% CO₂. The p VgRXR, pIND β-galactosidase Δ-subunit, and pIND/Hygro α 97Q or α 23Q constructs were maintained with 0.2 mg/ml Zeocin, 0.25 mg/ml Geneticin, and 0.1 mg/ml Hygromycin, respectively. β-galactosidase activity assay. Δ-α 97Q and Δ-α 23Q cells were seeded at 50 x 10⁴ cells/ml in 96-well format, induced with 5 μM Ponasterone A (AG Scientific) in DMSO, and then grown at 37°C for 24 hours. Each well was then rinsed with PBS, and 10 μl of Modified RIPA was added (150 mM NaCl, 50 mM Tris HCl pH 7.4, 1 mM EDTA, 1% NP-40, 1% w/v Na-deoxycholate, stored at 4°C). 67 μl of a master mix (5 μl 1Ox Cleavage Buffer, 0.135 μl 14.3 M β-mercaptoethanol, 44.865 μl dH₂O, 17 μl of 4 mg/ml ONPG) from Invitrogen's β-galactosidase Assay Kit was added to each well and incubated at 37°C for 30-60 minutes. The ONPG cleavage products were stabilized by the addition of 125 μl of STOP Buffer (I M Na₂CO₃) and quantified spectrophotometrically at 405 run on a plate reader. β-galactosidase activity is reported in nmoles of ONPG hydrolyzed. The absorbance at 405 run can be converted to nmoles of ONPG hydrolyzed by the following formula: nmoles of ONPG hydrolyzed = (OD (S). 405 nπO * (final vol =1.92x IQ³ nH = (OD_405H11O * (42.667 nmole)

(4500 nl/nmole-cm) * (1 cm)

The data from the experimental wells was normalized by subtracting the amount of β-galactosidase activity in un-induced wells.

The Z' factor, a useful indicator of the quality of a assay for high throughput screening ( Zhang et al., J. Biomol. Screen. 4(2):67-73, 1999), for Δ-α 97Q cell population was 0.73, which is well within the desirable range of 0.5 or higher. The Z' factor was determined by treating Δ-α 97Q cells with increasing concentrations of PonA and analyzing beta-galactosidase activity at several time points. Z' values were then calculated for each concentration of inducer at each time point using the following equation: Z' = 1 — (3σC+ + 3σC— )/(μC+ - μC— ), where μC+ and μC— are the mean maximum and minimum values of beta-galactosidase signal, respectively, and σC+ and σC— are the standard deviations of those means. For this assay, maximum signal was defined as beta-galactosidase assay readings from cells induced to express beta-galactosidase, and minimum signal was that obtained from uninduced cells.

Cell viability. Cell viability was evaluated using an MTS assay. Δ-α polyQ cells were seeded at 10 x 10⁴ cells/96-well or Htt^Ql03 PC12 cells at 2 x 10⁴ cells/96- well and then grown for either 24 hours or 72 hours, respectively. 40 μl of MTS/PMS (Promega) was added to each well and incubated for 2-4 hours. The co_.lorimetric change of MTS converted was read at 490 nm on a plate reader. The data from the experimental wells was normalized by subtracting the reading from medium-only wells.

Immunofluorescence. Δ-α polyQ cells were seeded at 50 x 10⁴ cells/ml in chamber slides and grown for 24 hours. The cells were then fixed with 4% paraformaldehyde and blocked with 10% goat serum in PBS for 1 hour. MAB2166 Htt (1:500 Chemicon) or β-galactosidase (1:2000 MP Bio) antibodies and secondary antibodies (Alexa-488 and -594 1:200) were diluted in 10% goat serum and 0.2% Tween in PBS and incubated for 1 hour.

Immunoblots. Either Δ-α 97Q or Δ-α 23Q cells were seeded in 12-well plates at 50 x 10⁴ cells/ml and grown at 37°C for 24 hours. Proteins were extracted from cells with Lysis Buffer (50 itiM Tris pH8, 100 mM NaCl, 5 mM MgCl₂, 0.5% NP-40) and Complete Protease Inhibitors (Roche) at 2x, on ice 30 min. Protein concentration was determined using Protein Assay and BSA standards (Bio Rad). 10-15 μg of protein in SDS loading buffer with B-mercaptoethanol was heated at 80⁰C for 5 minutes, then loaded on an 8.5% acrylamide gel in a PROTEAN II system (Bio Rad). Proteins were transferred to PVDF (Millipore) using 15% MeOH in transfer buffer (25 mM Tris, 190 mM glycine), at 80 V for 2 hours in 4°C.

PVDF blots were blocked in PBST with 0.5% milk for 1 hour. Actin (1:500, Sigma), β-galactosidase (1 :2000, MP Bio), or MAB2166 Htt (1 :2000, Chemicon) antibodies were diluted in PBST with 0.5% milk and incubated for 2 hours at room temperature or overnight at 4°C. HRP-conjugated secondary antibodies were diluted in PBST with 0.5% milk and incubated with blots for 30-45 min. Proteins were visualized with ECL Plus (Amersham Biosciences), and blots exposed to MR film (Kodak). Example 2: Characterization of Δ-α 97Q and Δ-α 23Q cell lines

The Δ-α 97Q and Δ-α 23 Q cell lines were further analyzed to confirm that the beta-galactosidase activity levels in the lines correlated with the production of PonA indicible β-galactosidase Δ and α Htt protein levels. Δ-α 97Q and Δ-α 23Q cell lines were treated with increasing concentrations of PonA for 24 hours, lysed and the lysates assayed for β-galactosidase activity. As shown in Figure 1, beta-galactosidase activity in both cell lines was PonA dose dependent. Furthermore, immunoblots showed that the protein levels of β-galactosidase Δ-subunit and α 97Q or α 23Q increased with the increase in PonA.

The capacity of Δ-α 97Q and Δ-α 23Q cells to degrade the induced α 97Q or α 23Q proteins was also analyzed. Cells were seeded and treated with 5 uM PonA for 24 hours. The medium was then removed from all wells and replaced with either medium without PonA (washed) or medium with PonA (un-washed). Cells were analyzed for beta-galactosidase activity or beta-galactiosidase and htt protein levels at 0, 24, 30, 36, 42 or 48 hours after removal of Pon A. As shown in Figure 2, by 12 hr after removal of the inducer {i.e., 36 hours after induction), the β-galactosidase activity was the same as in un-induced (0 hr) d-a97Q and d-a23Q cells (Figure 2A and Figure 2B, respectively). Corresponding immunoblots showed that the reduction in beta-galactosidase activity was correlated with a reduction in immunoreactive beta- galactosidase protein.

Example 3: High throughput screening of compounds: Chembridge library

Combinatorial compound libraries were purchased from Chembridge Corp. (San Diego,CA; 30,000 compounds comprising the Diverse™ and CNS™ Sets). Compounds were obtained from vendors in bar-coded 96-well microplates, each of which contained 80 unique compounds arrayed in 10 columns; the extreme left and right columns of each plate contained solvent only (DMSO), leaving the corresponding wells on assay plates available for assay controls.

For high throughput screening, Δ-α 97Q cells were seeded on 96-well plates at 10 x 10⁴ cells per well using an automated cell dispenser (Multi-Drop,TiterTek, Huntsville, AL) in 200 μl of media without selection drugs. The following day, β-galactosidase Δ-α 97Q expression was induced by the addition of PonA to a final concentration of 3 μM. Compounds were added to assay plates using a robotic liquid handler platform with a 96-well dispensing head (Evolution P3, PerkinElmer). On each plate, eight wells of PonA induced cells treated with vehicle, i.e., DMSO alone, in the extreme right-hand column served as positive controls and eight wells of uninduced, DMSO treated cells in the extreme left-hand column served as negative controls for β-galactosidase background activity level. The middle 10 columns received compounds and PonA simultaneously. Compounds were screened in triplicate at a single doses of 5-10 μM concentrations. Following addition of compounds, assay plates were returned to the incubator and cells were cultured at 37⁰C for 24 hours. The media was then removed from the wells using an automated plate washer (BioTek), each well was rinsed with PBS and cells were lysed in 10 μL Modified RJPA buffer and assayed for beta-galactosidase activity as described in Example 1. Assay results were collected using a Victor2-V multilabel plate reader (Perkin Elmer).

A collection of 1 14 compounds previously shown to decrease an N-terminal Htt fragment (Htt 1-17 aa) with 103Q fused to EGFP was tested (Coufal et al, J.Biomolecular Screening 1_2:351 -360, 2007). Thirty four of these compounds reduced beta-galactosidase activity to 75% or less of the beta-galactosidase activity in the untreated control cells and were selected for further analysis.

Dose-response curves for these 34 compounds were generated in Δ-α 97Q cells in a low-throughput screen. At 10 μM, 17 compounds were found to have more than 101% of the β-galactosidase activity in the untreated control cells, five compounds with 76-100% β-galactosidase activity in the untreated control cells, and one could not provide reproducible results. These 23 compounds were not further analyzed. The remaining 11 compounds showed a linear dose response, and at 10 μM reduced β-galactosidase activity to less than 75% of the value in the corresponding untreated control cells. Specifically, one compound reduced β-galactosidase activity to between 0-25% of the value in the corresponding untreated control cells, five reduced β-galactosidase activity to between 26-50% of the value in the corresponding untreated control cells, and five reduced β-galactosidase activity to between 51-75% of the value in the corresponding untreated control cells.

Example 4: Four classes of compounds identified by an Δ-α 23Q counter-screen The 11 compounds that reduced β-galactosidase activity in Δ-α 97Q cells to less than 75% of the value in the corresponding untreated control cells were then assayed in Δ-α 23Q cells to evaluate their selectivity for expanded Htt. The compounds were categorized into four classes based upon their relative effects on beta-galactosidase activity in the expanded poly(Q) cell line, Δ-α 97Q, and the unexpended poly(Q) cell line, Δ-α 23Q . (See Table 1 for compound structures.) Class I compounds (A7, A8, A9, and A24) decreased β-galactosidase activity in Δ-α 97Q cells, but increased it in Δ-α 23Q cells as compared to induced cells without compound. The Class II compound (Al 8) had complex effects on both cell lines. Class III compounds (A25 and A31) decreased β-galactosidase activity in Δ-α 97Q cells but had no effect on Δ-α 23Q cells. Finally, Class IV compounds (A14, Al 5, A20 and A29) decreased β-galactosidase activity in both cell lines.

Dose-response curves for the class I compounds on Δ-α 97Q and Δ-α 23Q cells are shown in Figure 3. The structures of the respective compounds, A7, A8, A9, and A24, are shown in the left-hand column. For each set of dose response curves in Figure 3, the x-axis indicates μM compound concentration; the y-axis indicates relative beta-galactosidase activity. Significant differences (p< 0.005) between the values for the expanded poly(Q) Δ-α 97Q cells and the unexpanded poly(q) Δ-α 23Q cells are marked in the graphs by an asterisk. For the right hand column of graphs, each data point represented as average of 5 replicate samples; for the left hand column each data point represented an average of 3 replicate samples.

The Class I, II, and III compounds were assayed for their effects on cell viability in both the Δ-α 97Q and Δ-α 23Q cell lines using an MTS assay and described in Example 1. Cells were induced with PonA and either 5 or 10 μM compound for 24 hours, at which time the MTS solutions were added. 100% MTS activity was defined as the activity of induced cells without compound. Of the seven compounds tested (A7, A8, A9, A24, A18, A25 and A31) only compound A18 had any significant effect on cell viability.

Example 5: Effect of class I, II, and III compounds on β-galactosidase and Htt protein levels

The effect of the seven compounds (A7, A8, A9, A24, Al 8, A25 and A31) on beta-galactosidase and Htt protein levels was assayed by immunofluorescence (according to the method in Example 1) on Δ-α 97Q and Δ-α 23 Q cell lines that had been induced and treated with 10 μM compounds for 24 hours. None of the compounds had an effect on the level of β-galactosidase proteins in either cell line. Compounds A9 and A24 decreased the Htt fluorescent signal in Δ-α 97Q but not the Δ-α 23Q cell line. None of the compounds had an effect on the cytoplasmic localization of Htt or β-galactosidase proteins in either cell line.

Example 6: Effect of Class I, H, and HI compounds on Htt^Q103 PC12 cell toxicity

The compounds were effective inhibiting polyglutamine induced cytotoxicity in a neuronal-like PC- 12 cell line. This cell line, Htt^Q103 ,has been described in Aiken et al. (Neurobiology of Disease, 16:546-555, 2004). Specifically, class I, II, and III compounds were assayed for the ability to inhibit cellular toxicity due to expressed expanded Htt in Htt^{QI 03} PC12 cells. Expression of expanded Htt was induced in the presence of either 1, 5, 10, or 25 μM compound for 72 hours. The ability of the compounds to rescue toxicity was assessed by MTS as described in Example 1.

As shown in Figure 4, the Class I compounds showed a dose dependent ability to rescue toxicity Htt^QI03 cells, a model cell line that expresses an inducible GFP tagged with an expanded poly(Q) repeat. The y-axis in Figure 3 indicated relative cell viability; the value for uninduced cells was set at 100%. Induction of HttQ 103 reduced viability by about 50 %. Significant differences (p< 0.005) between the values induced DMSO-treated control cells and the induced, compound treated cells are marked in the graphs by asterisks. Each data point represented the average of 6 replicates, except the 25 uM concentration , which was an average of 3 replicates.

The effect of compounds A9 and A24 on the cellular distribution of the GFP- tagged Htt^¹⁰³ was analyzed by fluorescence microscopy. DMSO-treated control cells had multiple aggregates per cell, infrequently had either one or no aggregates, and never showed diffuse GFP. Cells treated with A9 mainly had either one or no aggregates, some had diffuse GFP, and rarely were there multiple aggregates per cell. Cells treated with A24 mainly had a single aggregate, occasionally had multiple aggregates per cell, and sometimes had diffuse GFP. The screening methods described above can include a step in which the effect of a given compound or compounds is assessed with respect to its/their effect on cell viability, proliferation, or aggregate inclusion. Example 7: High throughput screening of compounds: Maybridge library

A combinatorial compound library of 5000 diverse heterocyclic chemicals was purchased from Maybridge Corp. (Comwell, UK). Compounds were obtained from vendors in bar-coded 96-well microplates, each of which contained 80 unique compounds arrayed in 10 columns; the extreme left and right columns of each plate contain solvent only (DMSO), leaving the corresponding wells on assay plates available for assay controls.

The 5,000 compound library was screened on Δ-α 97Q as described above in Example 3. Thirteen compounds were found to decrease β-galactosidase activity to below 75% of induced cells without compound.

Dose response curves on Δ-α 97Q cells were used to confirm the screening results. Twelve of the compounds showed linear dose responses. Of these twelve, three reduced β-galactosidase activity to between 0-25% of the value in the corresponding untreated control cells, three reduced β-galactosidase activity to between 26-50% of the value in the corresponding untreated control cells, and six reduced β-galactosidase activity to between 51-75% of the value in the corresponding untreated control cells.

The 12 compounds that reduced β-galactosidase activity in Δ-α 97Q cells to less than 75% of the value in the corresponding untreated control cells were then assayed in Δ-α 23Q cells to evaluate their selectivity for expanded Htt. Three compounds did not decrease the β-galactosidase activity of Δ-α 23Q cells below 75% and were considered compounds with specificity to expanded Htt. The three compounds (M4, M5, and M6) each caused no change in β-galactosidase activity in Δ-23Q cells, and thus were categorized into Class III of compounds described in Example 4 above. Structures for compounds M4, M5 and M6 and their corresponding dose response curves are shown in Figure 5. For each set of dose response curves in Figure 3, the x-axis indicates μM compound concentration; the y- axis indicates relative beta-galactosidase activity. Significant differences (p< 0.005) between the values for the expanded poly(q) Δ-α 97Q cells and the unexpended poly(q) Δ-α 23Q cells are marked in the graphs by an asterisk. Each data point represented as average of two replicate samples. The remaining nine active compounds from the Maybridge library screen decreased β-galactosidase activity in Δ-α 23 Q cells and were categorized into Class IV of compounds as described in Example 4.

Example 8: Effect of compounds on luciferase refolding

The ability of the compounds to modulate protein chaperone activity was assayed in a rabbit reticulocyte based luciferase refolding assay. The assay was performed essentially according to methods described in Bodner et al. (Proc. Natl. Acad. Sd 103: 4246-4251 (2006), and Thulasiraman and Marts, Methods MoI Biol. 102:129-141 (1998), as modified by Schumacher et al, {Biochemistry 35:14889- 14898 (1996). Briefly, Quantilum recombinant firefly luciferase (Promega) was diluted to 100 nM in stability buffer: 25 mM Tricine-KOH, pH 7.8, 8 mM MgSO₄, 0.1 It)M EDTA, 10% glycerol, 0.25% Triton X-100, and 10 mg/ml BSA. The luciferase was denatured at 40⁰C for 15 minutes, followed by incubation for 10 minutes on ice and five-fold dilution into Tris Buffer (TB): 10 mM Tris-HCl, pH 7.5, 3 mM MgCl₂, 50 mM KCl, 2 mM DTT, 2 mM ATP, 10 mM phosphocreatine, and 35 U/ml creatine kinase. The denatured luciferase was then added at a final concentration of 10 nM to reactions containing TB, 10% untreated rabbit reticulocyte lysate (Promega), and DMSO, compounds, or geldanamycin (Calbiochem), a known hsp90 inhibitor that served as a positive control, in a final volume of 100 μl. All conditions were done in duplicate. Reactions were incubated at 25°C. At time-points from 0-80 minutes, 5 μL of renaturation sample was added to 120 μL of Luciferase Assay Reagent (Promega). Light production was measured for 10 seconds in a luminometer (MGM Instruments, Optocomp I). Renaturation activity was expressed as a percentage of the total signal from native luciferase incubated in reticulocyte lysate, as above.

Nine of the 11 compounds identified in the β-galactosidase complementation assay (A7, A8, A9, A14, A15, A18, A20, A24, and A31) inhibited luciferase refolding in the rabbit reticulocyte system. Compounds A25 and A29 were inactive. Further analysis indicated that compound Al 8 inhibited luciferase activity of the native luciferase enzyme.

As shown in Figure 6, 10 μM A9 and A24 each inhibited luciferase refolding at every time point assayed; kinetics for A9 were similar to those of the geldanamycin positive control. Figure 7 shows that at 5 μM, A7, A8 and A9 also inhibited luciferase refolding. Further experiments confirmed the activity of A9 at 5 μM and indicated that A9 was also active at 1 μM, and 0.8 μM. Similar experiments showed that Al 8, A31 , A14, A15, and A20 also inhibited refolding.

Compound A9 was also assayed in the presence of geldanamycin. As shown in Figure 8, a combination of 5 μM A9 and 360 nM geldanamycin (GA) resulted in about 50% more inhibition than was observed for either compound alone. The synergistic effect of A9 and geldanamycin may indicate that the two compounds have different molecular targets.

Example 9: The effect of compounds on induction of hsp 70 expression

The ability of compounds to induce hsp 70 expression was assayed both in an hsp70 reporter system (Compounds A9, A24 and A25) and by immunoblotting (Compound A9).

We used the Hsp70 reporter cell line, 3T3-Y9-B12, an NTH 3T3 cell line expressing a reporter construct encoding EGFP (Clontech, Palo Alto, CA) under the transcriptional control of a 400-bρ promoter fragment derived from the HSP70B gene. Construction of the cell line is described in Bagatell et al. (Clinical Cancer Research 6: 3312-3318, 2000).

Hsp70 reporter assays were performed essentially as described in Turbyville et al. (J. Nat. Prod. 69(2): 178 -184 (2006)). 3T3-Y9/B12 reporter cells were seeded into flat-bottomed 96-well plates (Falcon; Becton Dickinson, Lincoln Park, NJ) at a density of 20,000 cells/well and allowed to attach overnight (one column of wells was left empty to serve as blank controls). On the following day, serial 2-fold dilutions of compounds were added in triplicate to cell-containing wells. Triplicate wells were also treated with DMSO vehicle alone (volume not to exceed 0.1%) to serve as a negative control. Cells were incubated overnight, the medium was removed, and wells were rinsed once with PBS, followed by addition of 150 μL of PBS to each well. Fluorescence was determined on a Fluoroskan (LabSystems) or Analyst AD (LJL Biosystems) plate reader equipped with filter sets for excitation at 485 nm and emission at 525 nm. Mean florescence and standard deviations of triplicate determinations were calculated and plotted.

As shown in Figure 9, A9 treatment upregulated hsp70 promoter mediated fluorescence, indicating that A9 unregulated HSFl -dependent expression of hsp70. Although the effect was below the sensitivity of the plate reader, visual inspection indicated that A24 also upregulated HSFl dependent expression of hsp70; compound A25 was inactive in the assay. As shown in Figure 10, when A24 was assayed on a more sensitive subclone of 3T3-Y9-B12, 3T3-Y9-B12-SD cells, a dose-dependent induction of hsp70 was observed.

The ability of A9 to induce hsp70 was also assayed by immunoblotting according to the method described in Example 1. PC 12 cells were incubated with either DMSO or 10 or 20 μM A9, lysed and immunoblotted. Immunoblots were probed with Hsp70 (Hsp72) mouse monoclonal antibody (Clone #C92F3A-5) (Assay Designs). No detectable hsp70 protein was observed in the DMSO-treated control samples; a dose-dependent increase in immunoreative hsp70 was observed in the A9 treated samples.

Example 10: The effect of compounds on hsp90 ATPase activity

The effect of the compounds A9, A 18, A31, A14 or A24 on hsp90 ATPase activity was assayed with Malachite green essentially according to the method described in Aheme et al. {Methods MoI. Med. 85:149-161, 2003). The materials included 1) 96-well plates with black or white sides; 2) Assay buffer (stored at 4°C): 100 mM Tris-HCl, pH 7.4, 20 mM KCl, 6 mM MgCl₂, prepared by mixing per 100 ml: 10 ml IM Tris, 2 ml 1 M KCl, 600 μl IM MgCl₂, 87.4 ml H₂O; 3) Malachite green (Aldrich 213020), 0.0812% w/v (stored at RT) prepared by dissolving 81.2 mg in 100 ml; 4) Polyvinyl alcohol, USP (Spectrum Pl 180-08), 2.32% w/v, stored at room temperature (RT), prepared in boiling water; stirred for several hours to dissolve, and cooled to RT; 5) ammonium molybdate (Sigma- Aldrich Al 343), 5.72% w/v in 6M HCl, stored at RT, prepared by diluting 37% (12M) HCl 1 :1 with water to make 6 M HCl and then adding 2.86 g ammonium molybdate per 50 ml solution; 6) sodium citrate, 34% w/v, stored at RT (34 g/100 ml); 7) ATP, disodium salt, special quality (Roche 10519979001), stored at 4°C; 7) Yeast Hsp90 protein, (Axxora ALX- 201-138-C025)(25 μg; will need 2.5 μg/well).

The assay was performed as follows:

1. on the day of use, malachite green reagent was prepared from stock solution:

2 parts malachite green 3.0 ml 1 part polyvinyl alcohol 1.5 ml

1 part ammonium molybdate 1.5 ml

2 parts H₂O 3.0 ml and incubated at room temperature for about 2 hours, until the color changes from dark brown to golden yellow.

2. Compounds were added to the plate, 5 μl/well (matched at 1% DMSO; 0.2% final DMSO)

3. The ATP was dissolved in assay buffer to 2.5 mM stock and stored at room temperature until use, then 10 μl ATP stock/well was added to the plate (final cone of 1 mM).

4. Just before use, the HSP90 protein was thawed on ice and suspended in chilled assay buffer to a stock of 0.25 mg/ml and kept on ice. Incubation was initiated by adding 10 μl of stock HSP90 per well, except background wells which received 10 μl assay buffer. Final volume was 25 μl/well.

5. The plates were agitated for 2 minutes, sealed with plastic film, and incubated for 3 hours at 37°C.

6. The reaction was stopped by the addition of 80 μl of the malachite green solution to each well and the plates were agitated again.

7. 10 μl of sodium citrate was added per well as quickly as possible after the malachite green to reduce the non-enzymatic hydrolysis of ATP and the plates were agitated. Controls became blue-green; backgrounds were yellowish. Once the sodium citrate was added, the color was stable for up to 4 hours at room temperature.

8. Absorbance was measured at 610-620 run. Control absorbance was about 0.7-1.0, while background was about 0.15-0.20.

A malachite green assay indicated that neither A9, Al 8, A31 , A14 nor A24 inhibited the ATPase activity of hsp90. Representative data for A9 are shown in Figure 11; no inhibition of yeast hsp 90 ATPase activity was observed at either 10 or 50 μM A9; in contrast the known hsp90 inhibitor, geldanamycin showed a dose- dependent inhibition of hsp90 ATPase activity. These data suggested that the mechanism of action of A9 may be distinct from that of geldanamycin.

Example 11: The effect of compounds on hsp90 client proteins The effect of compound A9 on hsp90 client proteins was evaluated in two classes of hsp90 client proteins: steroid hormone receptors and kinases. A9 reduced the levels of the steroid hormone receptors, but had no effect on representative hsp90 client kinases.

For the steroid hormone receptor analysis, steady-state levels of two steroid hormone receptors, the estrogen receptor and the ecdysone receptor, were assayed by immunoblotting extracts from A9 treated MCF7, a human breast cancer cell line, and 14A2.6 cells, respectively. Cells were plated and treated for 24 hours with either DMSO, geldanamycin (360 nM) or A9 (20 uM). Cell lysates were immunob lotted as described in Example 1. Immunoblots were probed for either the ecdysone receptor (using the antibody clone DDA2.7 from Iowa Developmental Studies Hybridoma Bank) or the estrogen receptor (using the antibody Ab 14, Cat#MS-750-S0, from Neomarkers/Labvision at a 1:500 dilution). The immunoblotting experiments indicated that A9 treatment reduced the levels of estrogen receptor to below the limits of detectability in MCF-7 cells; a similar reduction was observed on MCF-7 cells that were treated with the known hsp90 inhibitor, geldanamycin. A9 also reduced the level of the ecdysone receptor relative to the levels found in both the DMSO and the geldanamycin-treated control cells.

The effect of A9 on client kinases Her-2, IGFl -receptor (IGFIR) and AKT was assayed by flow cytometry in the human breast cancer cell lines MCF-7 (Her2) and BT474 (IGFlR) and a human cervical carcinoma cell line, HeIa (AKT). Cells were cultured on 10 cm plates for 24 hours in the presence of either DMSO, geldanamycin, or 20 μM A9 for 24 hours. Cells were removed from the plates with 1 ml trypsin; trypsinization was stopped by the addition of 10 volumes media. Cells were collected by centrifugation, resuspended in PBS + 2% serum; centrifuged again and the cell pellets were resuspended in primary antibody diluted in PBS + 2% serum. The antibodies were as follows: Her-2, was detected with Anti-ErbB2 from Neomarkers catalogue number 9G6.10 at a 1:40 dilution prepared by diluting 2.5 μl of antibody stock per 100 μl of media; IGFlR was detected with Anti-IGFIR (BD Pharmingen; Anti-human CD221 Cat#55600) at a 1:50 dilution prepared by diluting 2 μl of antibody stock per 100 μl of media. Cells were aliquotted at 105-106 cells/100 μl/well in a round bottom plate or tubes, incubated for 60 minutes on ice, collected by centrifugation and washed once in blocking buffer, then resuspended in PE- conjugated secondary antibody, R-phycoerythroetin goat anti-mouse antibody (Jackson Labs, catalogue number 115-116-068) diluted 1:100 in PBS + 2% serum. Cells were incubated for 30 minutes on ice, in the dark, washed once in PBS/2% serum, then resuspended in PBS and analyzed on Becton Dickinson FACScan.

A9 treatment had no effect on the FACS signal, relative to the DMSO control, the kinases Her2, IGFlR, and AKT. As shown in Figure 12, A9 treatment had no effect on the Her-2 signal in MCF7 cells. In contrast, the known hsp90 inhibitor, geldanamycin sharply reduced the Her2 signal.

The effect of A9 on the levels of another kinase, CDK4, was analyzed by immunoblotting according to the method described above. HeIa cells were treated with either DMSO, geldanamycin or 10 or 20 μM A9. No effect on the levels of the CDK4 signal were observed with either concentration of A9; geldanamycin reduced the signal to below the levels of detectability on the immunoblot.

Example 12: The effect of compounds progesterone receptor assembly

Compounds A9 and A24 were evaluated for their ability to inhibit the hsp90 chaperoning pathway according to the in vitro model system for progesterone receptor assembly described in NeIa et al. (J. Biol. Chem. 281_.(36): 26235-26244, 2006).

Antibody resin was prepared by incubating PR22 (a mouse monoclonal antibody that recognizes the chicken progesterone receptor (PR)) with a slurry of protein A-Sepharose CL-4B (Amersham Biosciences) in PBS for 30 minutes at room temperature prior to use. Proportions were 7 μl of PR22 ascites for every 20 μl of resin volume. The conjugated resin was washed three times in PBS and then resuspended as a 1 : 1 slurry with ice-cold stripping buffer (20 mM Tris, pH 7.5, 500 mM KCl, 5 mM MgCl₂, 0.1% Nonidet P40, 1 mM dithiothreitol). When PR was isolated from SF9 cells, it was accompanied by an assortment of chaperones, mainly Hsp90, Hsp70, and p23. These chaperones were removed by treatment with high salt, ATP, and detergent (stripping buffer) while the immunoisolation is taking place. For the purification of PR, 40 μl of PR22/protein A resin slurry was added to 0.07 ml of salt-treated lysate. This mixture was incubated on ice for 1.5 hours. Receptor/resin complexes were washed three times with 1 ml of cold stripping buffer and once with reaction buffer, with brief centrifugation to pellet the resin. Resin pellets were used in reconstitution or binding reactions. For the progesterone receptor reconstitution assay, PR resin (20 μl) was suspended with 200 μl of cold reaction buffer containing 20 μg of Hsp70, 5 μg of Ydjl, DjAl, or DjBl, 5 μg of Hop, 20 μg of Hsp90, 5 μg of p23, and 5 mM ATP unless otherwise noted. Incubation proceeded at 30⁰C for 20 minutes. The samples were chilled on ice for 2 minutes and supplemented with 100 nM [³H]progesterone (American Radiolabeled Chemicals, Inc, St. Louis, MO, 50 Ci/mmol) plus 100 nM of unlabeled progesterone. The samples were incubated for 3 hours on ice with gentle resin suspension and then washed four times with 1 ml of reaction buffer. During the fourth suspension, 100 μl were removed for the measurement of [³H]progesterone. The final samples were suspended in 20 μl of SDS sample buffer, heated for 5 minutes at 95°C, and analyzed by SDS-PAGE. Analysis of complex formation after reconstitution assays was performed in 10% acrylamide gels.

The results of an hsp90 chaperone pathway assay performed in the presence of increasing concentrations (1, 2, 10, 20, and 50 uM) of compounds A9 and A 24 are shown in Figure 13. Compound A9 inhibited assembly in a dose-dependent fashion; inhibition was not observed for A24.

Example 12: The effect of A9 fragments on chaperoning assays

The A9 fragments, 2-aminophenol (2-AP) and 4-chlorocinnamaldehyde (4-cc), were assayed for their activity in two chaperone pathway assays, the beta- galactosidase screening assay described in Example 1 and the hsp70 reporter assay described in Example 9. Dose-response data for (1, 5, 8, and 10 uM) either A9, 2-AP, 4-cc, or a mixture of 2-AP and 4-cc in are shown in Figure 13a for the beta- galactosidase assay and in Figure 13b for the hsp70 reporter assay. Labels on the x- axis for both graphs are as follows: A9 and A9N refer to A9 from different sources. 2-ap refers to 2-aminophenol; 4-cc refers to 4-chlorocinnamaldehyde; and 2-ap+4cc refers to a mixture of the two compounds.

As indicated in Figures 14A and 14B, respectively, 2-AP showed a dose- dependent inhibition of both beta-galactosidase activity and hsp70 reporter activity. No inhibition was observed for 4-cc in either assay. A combination of the two compounds did not generate a dose dependent inhibition in the beta-galatosidase assay, but was active in the hsp70 reporter assay. Example 13: Effect of compounds A9 and A24 on chaperon e function

The activity of compounds A9 and A24 on chaperone function assays is summarized and compared with that of geldanamycin, a known hsp90 inhibitor, in Table 2. Table 2: Chaperone Function Assay Summary for Compounds A9 and A24

Example 14: Formatting the high throughput screen in a human breast cancer cell lin<

In this prophetic example, MCF-7, a human breast cancer cell line is obtained from the American Type Culture Collection and maintained in minimum essential medium (Eagle) with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids and 1 mM sodium pyruvate and supplemented with 0.01 mg/ml bovine insulin, 90%; fetal bovine serum, 10% at 37°C in a 5% CO₂ atmosphere.

The MCF7 cell line is stably transfected with pVgRXR (Ecdysone Receptor expression vector) using Lipofectamine 2000 (Invitrogen) to generate EcR MCF-7 cells. Construction of stable MCF-7 cell lines expressing either Δ-α 97Q or Δ-α 23Q is performed according to the two-step method described in Example 1. In the first step, the β-galactosidase Δ-subunit is stably transfected into EcR MCF-7 cells. Individual colonies are selected, expanded and then transiently transfected with α 23Q. Following induction with PonA, clones are screened for beta-galactosidase activity according to the method described in Example 1. An active clone is then stably transfected with either the α 97Q or α 23Q to make Δ-α 97Q or Δ-α 23Q cells, respectively. Individual colonies are picked and expanded. The MCF-7 Δ-α 97Q or Δ-α 23 Q cell lines are characterized according to the methods described in Example 2. High throughput screening is performed according to the method described in Example 3.

Claims

WHAT IS CLAIMED IS:

1. A compound of Formula VI:

(VI) or pharmaceutically acceptable salt thereof, wherein: a dashed line indicates an optional bond;

A is NR⁴ when the bond between A and R² is a single bond;

A is N when the bond between A and R² is a double bond;

R¹ is H, Ci-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, C(O)-(Ci_-6 alkyl), C(O)- (C_2-6 alkenyl), or C(O)-(C_2-6 alkynyl);

R² is =CR⁵R⁶ when the bond between A and R² is a double bond;

R² is -CR⁷=CR⁸R⁹ when the bond between A and R² is a single bond;

R³ is halo, Ci_-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, Ci_₆ haloalkyl, CN, NO₂, N₃, OR\ SR^a, C(O)R^b, C(O)NR⁰R", C(O)OR⁸, OC(O)R^b, OC(O)NR^cR^d, NR^cR^d, NR^cC(0)R^b, NR^cC(0)NR^cR^d, NR^cC(O)OR^a, S(O)R^b, S(O)NR^cR^d, S(O)₂R^b, NR^cS(O)₂R^b, or S(O)₂NR^cR^d;

R⁴ is H or Ci_-6 alkyl;

R⁵ Is -(L)_1n-Cy;

R⁶ is H or Ci_-6 alkyl;

R⁷ is H, Ci_-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, or C(O)O-(Ci_-6 alkyl); or R¹ and R⁷ together form -(CR¹⁰R¹ ') -;

R⁸ is -(L)_1n-Cy;

R⁹, R¹⁰, and R¹¹ are independently selected from H and C_1-6 alkyl; or R¹⁰ and R¹ ' together form O or S;

L is Ci_-6 alkylene, C_2-6 alkenyl ene, -C(O)-, -C(O)-(Ci_-6 alkylene)-, or - C(O)-(Ci_-6 alkenylene)-;

Cy is aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, each optionally substituted by 1, 2, or 3 substituents independently selected from halo, Ci_-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, C₆ haloalkyl, CN, NO₂, N₃, OR^al, SR^al, C(O)R^bl, C(O)NR^clR^dl, C(O)OR³¹, OC(O)R"¹, OC(O)NR^clR^dI, NR^clR^dl, NR^clC(O)R^bl, NR^CIC(O)NR^clR^dl, NR^clC(O)OR^aI, S(O)R^bl, S(O)NR^clR^dl, S(O)₂R⁶¹, NR^clS(O)₂R^bl, and S(O)₂NR^clR^dl;

R^a, R^al, R^b, R^bl, R^c, R^cl, R^d, and R^dl are independently selected from H, Ci-₆ alkyl, Ci.₆ haloalkyl, C_2-6 alkenyl, C_2-6 alkynyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl; n is O, 1, 2, 3 or 4; and m is O or 1.

2. The compound of claim 1, wherein the compound has Formula Via:

Via or a pharmaceutically acceptable salt thereof.

3. The compound of claim 1, wherein the compound has Formula VIb:

VIb or a pharmaceutically acceptable salt thereof.

4. The compound of claim 1, wherein the bond between A and R2 is a single bond.

5. The compound of claim 1, wherein the bond between A and R2 is a double bond.

5. The compound of claim 1, wherein the bond between A and R2 is a double bond.

6. The compound of claim 1, wherein A is NR⁴.

7. The compound of claim 1, wherein A is N.

8. The compound of claim 1, wherein R¹ is H or Ci .₆ alkyl.

9. The compound of claim 1, wherein R¹ is H.

10. The compound of claim 1, wherein R² is =CR⁵R⁶.

11. The compound of claim 1 , wherein R² is -CR⁷=CR⁸R⁹.

12. The compound of claim 1, wherein R³ is halo or C_1-^ alkyl.

13. The compound of claim 1 , wherein R³ is methyl.

14. The compound of claim 1, wherein R is H.

15. The compound of claim 1, wherein R⁶ is H.

16. The compound of claim 1, wherein R⁷ is H.

17. The compound of claim 1, wherein R¹ and R⁷ together form -(CR¹⁰R¹¹) -.

18. The compound of claim 1, wherein R⁹, R¹⁰, and R¹¹ are each H.

19. The compound of claim 1, wherein R¹⁰ and Rⁿ together form O.

20. The compound of claim 1, wherein L is C₂-₆ alkenylene, -C(O)-, or -C(O)- (Ci .₆ alkenylene)-.

21. The compound of claim 1, wherein L is C₂-₆ alkenylene.

22. The compound of claim 1, wherein L is -CH=CH-.

23. The compound of claim 1, wherein L is -C(O)-.

24. The compound of claim 1, wherein L is -C(O)-(Ci -_<s alkenylene)-.

25. The compound of claim 1, wherein Cy is aryl or heteroaryl, each optionally substituted by 1, 2, or 3 substituents independently selected from halo, Ci-₆ alkyl, C_2-6 alkenyl, C_2-6 alkynyl, Ci_-6 haloalkyl, CN, NO₂, N₃, OR^al, SR^al, C(O)R^bl, C(O)NR^clR^dl, C(O)OR³¹, OC(O)R⁶¹, OC(O)NR^clR^dl, NR^clR^dl, NR^clC(O)R^bl, NR^clC(O)NR^clR^dl, NR^cIC(O)OR^al, S(O)R^bl, S(O)NR^clR^dl, S(O)₂R^bl, NR^clS(O)₂R^bl, and S(O)₂NR^clR^dl.

26. The compound of claim 1, wherein Cy is phenyl optionally substituted by 1, 2, or 3 substituents independently selected from halo, Ci-₆ alkyl, C_2-6 alkenyl, C_2-6 alkynyl, C_1-6 haloalkyl, CN, NO₂, N₃, OR^aI, SR^al, C(O)R^bl, C(O)NR^clR^dl, C(O)OR^aI, OC(O)R^bl, OC(O)NR^cIR^dl, NR^clR^dl, NR^clC(O)R^bl, NR^{C 1}C(O)NR⁰¹R"¹, NR^clC(O)OR^al, S(O)R^bl, S(O)NR^clR^dI, S(O)₂R^bl, NR^cl S(O)₂R¹", and S(O)₂NR^clR^dl.

27. The compound of claim 1, wherein Cy is phenyl optionally substituted by 1, 2, or 3 substituents independently selected from halo, Ci_-6 alkyl, and OR^al.

28. The compound of claim 1, wherein Cy is phenyl optionally substituted by 1 or 2 substituents independently selected from Cl, Br, and OH.

29. The compound of claim 1, wherein Cy is heteroaryl^'optionally substituted by 1, 2, or 3 substituents independently selected from halo, Ci_-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, Ci_-6 haloalkyl, CN, NO₂, N₃, OR^al, SR^al, C(O)R^bl, C(O)NR^clR^dl, C(O)OR³¹, OC(O)R^bl, OC(O)NR^clR^dl, NR^clR^dl, NR^eIC(O)R^bl, NR^{C 1}C(O)NR⁰¹R¹", NR^clC(O)OR^al, S(O)R^bl, S(O)NR^CIR^dl, S(O)₂R¹", NR^clS(O)₂R^bl, and S(O)₂NR⁰ ' R^d '.

30. The compound of claim 1, wherein Cy is thiazolyl optionally substituted by 1, 2, or 3 substituents independently selected from halo, Ci-^ alley], C_2-O alkenyl, C₂-6 alkynyl, C_1-6 haloalkyl, CN, NO₂, N₃, OR^al, SR^al, C(O)R^bl, C(O)NR^clR^dl, C(O)OR³¹, OC(O)R^bl, OC(O)NR^clR^dl, NR^clR^dl, NR^c1C(O)R^bl, NR^clC(O)NR^clR^dI, NR^clC(O)OR^al, S(O)R^bl, S(O)NR^clR^dl, S(O)₂R^bl, NR^clS(0)₂R^bl, and S(0)₂NR^clR^dl.

31. The compound of claim 1 , wherein Cy is thiazolyl optionally substituted by 1 or 2 Ci_-6 alkyl.

32. The compound of claim 1, wherein Cy is thiazolyl optionally substituted by 1 or 2 methyl groups.

33. A compound of formula I that is (2-((E)-((E)-3-(4- chlorophenyl)allylidene)amino)phenol) or a pharmaceutically acceptable salt thereof.

34. A compound of formula VI that is ((3-[2-(2,4-dimethyl-l,3-thiazol-5-yl)- 2-oxoethylidene]-3,4-dihydro-2H-l,4-benzoxazin-2-one) or a pharmaceutically acceptable salt thereof.

35. A pharmaceutical composition comprising a compound of any of claims 1-34 or a salt thereof.

36. The pharmaceutical composition of claim 35, wherein the compound comprises Formula A9 (2-((E)-((E)-3-(4-chlorophenyl)allylidene)amino)phenol).

37. The pharmaceutical composition of claim 35, wherein the compound comprises Formula A24 ((3-[2-(2,4-dimethyl-l,3-thiazol-5-yl)-2-oxoethylidene]-3,4- dihydro-2H- 1 ,4-benzoxazin-2-one).

38. The composition of claim 35, wherein the composition is formulated for oral administration to a patient.

39. The composition of claim 35, wherein the composition is formulated for parenteral administration to a patient.

40. A method of treating a subject who has been diagnosed as having, or who is at risk of developing cancer, the method comprising

(a) identifying the subject; and

(b) administering to the subject a therapeutically effective amount of the pharmaceutical composition of any of claims 35-39.

41. The method of claim 40, wherein the cancer is breast cancer, ovarian cancer, endometrial cancer, prostate cancer, or leukemia.

42. The method of claim 41, wherein the cancer is breast cancer.

43. A method of treating a subject who has been diagnosed as having or who is at risk of developing, a disorder characterized by overexpression of a protein comprising an expanded poly-glutamine (poly(Q)) repeat, the method comprising

(a) identifying the subject; and

(b) administering to the subject a therapeutically effective amount of the pharmaceutical composition of any of claims 35-39.

44. The method of claim 43, wherein the subject has been diagnosed as having or is at risk of developing, Huntington's disease.

45. The method of claim 43, wherein the subject has been diagnosed as having or is at risk of developing, spinocerebellar ataxia, hemiplegic migraine, spinobulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy, breast cancer, ovarian cancer, prostate cancer, endometrial cancer, leukemia or amyloidosis.

46. The method of claim 45, wherein the protein comprises huntingtin.

47. The method of claim 45, wherein the protein comprises a steroid hormone receptor.

48. The method of claim 47, wherein the steroid hormone receptor is selected from the group consisting of an estrogen receptor, a progesterone receptor, an androgen receptor, a progesterone receptor, a glucocorticoid receptor and a mineralocorticoid receptor.

49. A method of treating a subject who has been diagnosed as having, or who is at risk of developing, a disorder characterized by misregulation of an hsp90 polypeptide or an hsp90 client polypeptide, the method comprising

(a) identifying the subject; and

(b) administering to the subject a therapeutically effective amount of the pharmaceutical composition of any of claims 35-39.

50. The method of claim 49, wherein the hsp90 client polypeptide comprises a steroid hormone receptor.

51. The method of claim 50, wherein the steroid hormone receptor is selected from the group consisting of an estrogen receptor, a progesterone receptor, an androgen receptor, a progesterone receptor, a glucocorticoid receptor and a mineralocorticoid receptor.

52. A method of modulating the activity of an hsp90 polypeptide comprising contacting a composition comprising the hsp90 polypeptide with a compound of Formula VI.

53. Use of a compound of Formula VI or a salt thereof in the preparation of a medicament.

54. Use of a compound of Formula A9 (2-((E)-((E)-3-(4- chlorophenyl)allylidene)amino)phenol) or a salt thereof or Formula A24 ((3-[2-(2,4- dimethyl-1 ,3-thiazol-5-yl)-2-oxoethylidene]-3,4-dihydro-2H-l ,4-benzoxazin-2-one) or a salt thereof in the preparation of a medicament.

55. Use of a compound of Formula A9 (2-((E)-((E)-3-(4- chlorophenyl)allylidene)amino)phenol) or a salt thereof or Formula A24 ((3-[2-(2,4- dimethyl-l,3-thiazol-5-yl)-2-oxoethylidene]-3,4-dihydro-2H-l,4-benzoxazin-2-one) or a salt thereof in the preparation of a medicament for treating a cancer.

56. Use of a compound of claim 55, wherein the cancer is breast cancer, ovarian cancer, endometrial cancer, prostate cancer, or leukemia.

57. Use of a compound of Formula A9 (2-((E)-((E)-3-(4- chlorophenyl)allylidene)amino)phenol) or a salt thereof or Formula A24 ((3-[2-(2,4- dimethyl-l,3-thiazol-5-yl)-2-oxoethylidene]-3,4-dihydro-2H-l,4-benzoxazin-2-one) or a salt thereof in the preparation of a medicament for treating a disorder characterized by overexpression of a protein comprising an expanded poly-glutamine (poly(Q)) repeat.

58. Use of a compound of claim 57, wherein the disorder is Huntington's disease, spinocerebellar ataxia, hemiplegic migraine, spinobulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy, breast cancer, ovarian cancer, prostate cancer, endometrial cancer, leukemia or amyloidosis.

59. A method of identifying an agent that selectively decreases intracellular levels of a polypeptide comprising an expanded poly(Q) repeat, the method comprising:

(a) providing a first cell that (i) expresses a first subunit of beta-galactosidase and (ii) comprises a polynucleotide sequence comprising an inducible promoter operably linked to a polynucleotide encoding a first fusion polypeptide having an expanded poly(Q) repeat and a second, complementary subunit of beta-galactosidase;

(b) providing a second cell that (i) expresses a first subunit of beta-galactosidase and (ii) comprises a polynucleotide sequence comprising an inducible promoter operably linked to a polynucleotide encoding a second fusion polypeptide having a wildtype poly(Q) repeat and a second, complementary subunit of beta-galactosidase; wherein the first subunits of beta-galactosidase and the second, complementary subunits of beta-galactosidase produce functional beta-galactosidase;

(c) inducing expression of the first fusion polypeptide and the second fusion polypeptide; (d) contacting the first cell and the second cell with a candidate agent; and

(e) determining the levels of functional beta-galactosidase in the first and second cells, wherein a decrease in the level of functional beta-galactosidase in the first cell contacted with the candidate agent relative to the level of functional beta-galactosidase in the second cell contacted with the candidate agent indicates that the candidate agent following contacting the cell with the candidate agent identifies the agent as one that selectively decreases intracellular levels of a polypeptide comprising an expanded poly(Q) repeat.

60. The method of claim 59, wherein the polypeptide having an expanded poly(Q) repeat is a huntingtin polypeptide.

61. The method of claim 59 or 60, wherein

(a) when the first subunit of beta-galactosidase is the alpha-subunit (α), the second, complementary subunit of beta-galactosidase is the delta-subunit (Δ), and

(b) when the first subunit of beta-galactosidase is the delta-subunit (Δ), the second, complementary subunit of beta-galactosidase is the alpha-subunit (α).

62. The method of any of claims 59-61, wherein the expanded poly(Q) repeat comprises at least 40 consecutive glutamine residues and the wildtype poly(Q) repeat comprises 35 or fewer consecutive glutamine residues.

63. The method of any of claims 59-62, wherein the inducible promoter is an ecdysone-responsive promoter.

64. The method of any of claims 59-63, wherein the cell is a mammalian cell.

65. The method of any of claims 59-64, wherein the cell is a human cell.

66. The method of any of claims 59-64, wherein the cell is a PC12 cell.

67. The method of any of claims 59-66, wherein the method is automated.

68. The method of any of claims 59-67, wherein the candidate agent is a small organic or inorganic molecule, a protein, a peptide, or a nucleic acid.

69. The method of any of claims 59-68, further comprising a step of determining whether the agent inhibits an hsp90 protein.

70. An agent identified by the method of any of claims 59-69.

71. The agent of claim 70, wherein the agent is a compound of Formula VI.

72. The agent of claim 70, wherein the agent is a compound of Formula A9 (2-((E)-((E)-3-(4-chlorophenyl)allylidene)amino)phenol) or a salt thereof or Formula A24 ((3-[2-(2,4-dimethyl-l,3-thiazol-5-yl)-2-oxoethylidene]-3,4-dihydro-2H-l,4- benzoxazin-2-one) or a salt thereof.

73. A method of treating a subject who has a cancer characterized by misexpression of a polypeptide comprising an expanded poly(Q) repeat, the method comprising administering to the patient a therapeutically effective amount of a pharmaceutically acceptable composition comprising a compound of Formula Al 8 (4- (4-(3 ,4-dichlorophenyl)thiazol-2-ylamino)phenol).

74. A method of treating a subject who has been diagnosed as having, or who is at risk of developing cancer, the method comprising

(a) identifying the subject; and

(b) administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising 2-aminophenol (H₂NCeHUOH).

75. A method of treating a subject who has been diagnosed as having or who is at risk of developing, a disorder characterized by overexpression of a protein comprising an expanded poly-glutamine (poly(Q)) repeat,

(a) identifying the subject; and

(b) administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising 2-aminophenol.