WO2009140621A2 - Compositions and methods relating to heat shock transcription factor activating compounds and targets thereof - Google Patents

Abstract

The present invention relates to HSF activating compounds, methods for their discovery, and their research and therapeutic uses. In particular, the present invention provides compounds capable of facilitating HSFl activation, and methods of using such compounds as therapeutic agents to treat a number of conditions associated with protein misfolding.

Description

COMPOSITIONS AND METHODS RELATING TO HEAT SHOCK TRANSCRIPTION FACTOR ACTIVATING COMPOUNDS AND TARGETS

THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to pending U.S. Provisional Patent Application Serial No. 61/053,513, filed 05/15/2008, which is herein incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under GM059911-08 and

GM076954 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to Heat Shock Transcription Factor (HSF) activating compounds, methods for their discovery, and their research and therapeutic uses. In particular, the present invention provides compounds capable of facilitating HSFl activation, and methods of using such compounds as therapeutic agents to treat a number of conditions associated with diseases and other pathophysiological states caused by or associated with defective protein folding.

BACKGROUND OF THE INVENTION For a long time, protein folding was regarded as simply a theoretical problem.

Researchers investigated the mechanisms of protein folding to close the huge gap in our knowledge between the genetic blueprint of a protein and its biological function. Only in the 1990s did it become clear that wrongly folded proteins are involved in the development of many diseases. Protein folding has become a focus of attention in pharmaceutical research: it is probable that new approaches to the treatment of diseases such as Parkinson's disease and Alzheimer's disease are to be found within its complex pathways. Docket No. DUKE-30386/WO-l/ORD ^66

Protein folding diseases can be divided into two groups: in the first, excessive quantities of incorrectly folded proteins collect in the form of uncontrolled piles of molecular rubbish. This is a group of diseases known as amyloidoses, of which Alzheimer's disease is a well-known example. In the other, a small error in the genetic blueprint leads to incomplete folding of a protein, which affects its function.

A common characteristic of all amyloidoses is the collection of aggregates or plaques of insoluble protein in the extracellular tissue, which cannot be broken down by enzymes. Their ordered structure gives them crystal-like properties: they are made up of long filaments (fibrils) that are formed from densely packed β-pleated sheets of identical proteins. There are at least 20 different proteins that can act as the building blocks of these fibrils, each of which is associated with a different disease. In so-called systemic amyloidoses, the precursors of these plaques are transported through the bloodstream from their point of origin to their point of deposition. Localized amyloidoses are of greater clinical significance, as they affect the central nervous system, which is particularly susceptible to damage, as well as the heart and other organs and tissues.

What are needed are improved compositions and methods for treating diseases associated with improper protein folding, aggregation and/or the clearance of damaged proteins.

SUMMARY

The present invention relates to HSFl activating compounds, methods for their discovery, and their research and therapeutic uses. In particular, the present invention provides compounds capable of facilitating HSFl activation (e.g., homotrimerization), and methods of using such compounds as therapeutic agents to treat a number of conditions associated with protein misfolding.

Experiments conducted during the course of development of embodiments for the present invention developed a specialized high throughput screen for identifying small molecules capable of activating the human Heat Shock Transcription Factor 1 (HSFl) protein from complex chemical libraries. In addition, experiments conducted during the course of developing embodiments for the present invention identified molecules capable of activating the human Heat Shock Transcription Factor 1. It was shown that these molecules activate Docket No. DUKE-30386/WO-l/ORD 3/66

human HSFl when added to cells, and activate expression of heat shock proteins (e.g., HSP70, HSP25). In addition, it was shown that these molecules reduce aggregation of poly- glutamine proteins in neuronal cells.

As such, in certain embodiments, the present invention provides compositions capable of HSF activation. The compositions are not limited to a particular type of HSF. In some embodiments, the HSF is HSFl, HSF2 or HSF4. The compositions are not limited by the manner in which they result in HSF activation. In some embodiments, HSF activation includes, but is not limited to, activation of HSFl homo-trimerization, activation of HSF target protein expression (e.g., Heat Shock Proteins including but not limited to HSP70 and HSP25), activation of protein chaperone activity (e.g., increased protein folding, increased protein solubilization, protein degradation), and/or reducing protein aggregation (e.g., aggregation of poly-glutamine proteins in neuronal cells).

In certain embodiments, the composition comprises a compound described by the

following formula:

including salts, esters, and prodrugs thereof. The compound is not limited to particular definitions of Ri through R₉ groups. In some embodiments, the Ri through R₉ groups define a compound capable of HSF activation, identifiable using screening techniques

described herein. In some embodiments, Ri is

O^" , -OH, H, or halogen (e.g., chlorine). In some embodiments, Xi is Docket No. DUKE-30386/WO-l/ORD 4/66

, or Xi is absent. In some embodiments, X₂ is S or C. In some embodiments, X₃ is S or C. In some embodiments, R9 is -OCH₃. In some embodiments, R₂

. In some embodiments, R₅ is S or C. In some embodiments, R₆ is H or O . In some embodiments, R₇ is H or O . In Docket No. DUKE-30386/WO-l/ORD 5/66

some embodiments, Rg is or

In some embodiments, Rio is chlorine or CH₃. In some

embodiments, Rn is substituted or unsubstituted alkyl such as, for example,

or

Docket No. DUKE-30386/WO-l/ORD 6/66

In some embodiments, the compound is

Docket No. DUKE-30386/WO-l/ORD 7/66

Docket No. DUKE-30386/WO-l/ORD 8/66

In certain embodiments, the composition comprises a compound described by the compounds shown in Figure 7. Docket No. DUKE-30386/WO-l/ORD 9/66

In certain embodiments, the composition comprises a functional derivative of a

compound capable of HSF activation (e.g.,

a functional derivative of HSFlA).

In certain embodiments, the composition comprises a compound described by the

following formula:

; including salts, esters, and prodrugs thereof. The compound is not limited to a particular definitions of Rl through R8 groups. In some embodiments, the Rl through R8 groups define a compound capable of HSF activation, identifiable using screening techniques described herein. In some embodiments, Rl is

, H, or halogen (e.g., chlorine). In some embodiments, R2 is

or

In some embodiments, ^R3 R₄ I-S or Docket No. DUKE-30386/WO-l/ORD 10/66

. In some embodiments, R5 is S or C. In some embodiments, R6 is H or O . In some embodiments, R7 is H or O . In some embodiments, R8 is

or In some embodiments, R9 is chlorine or CH3. In some embodiments, RlO is substituted or unsubstituted alkyl such as, for example,

In certain embodiments, the compound is

Docket No. DUKE-30386/WO-l/ORD 11/66

In certain embodiments, the present invention provides methods for treating a condition associated with protein misfolding. The present invention is not limited to a particular method for treating a condition associated with abnormal protein folding. In some embodiments, the methods comprise administering to a subject (e.g., human being, cat, dog, mouse, rat, ape, monkey) having misfolded proteins a composition capable of facilitating HSF (e.g., HSFl) activation. The methods are not limited to treating a particular condition associated with protein misfolding. In some embodiments, conditions associated with irregular HSF (e.g., HSFl) activity include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington disease, Amyotrophic Lateral Sclerosis, a prion-based disease, cataract, age-related cataract, glaucoma, macular degeneration, age-related macular degeneration, retinitis pigmentosa, cardiovascular disease and stroke, heat stroke, spinocerebellar ataxia, Machado Joseph disease, stress-related neuronal degeneration, aging, cancer, and type 2 diabetes mellitus. In some embodiments, the methods are directed towards crystallins in age-related cataracts. In some embodiments, the methods are directed towards myocillin in glaucoma. In some embodiments, the composition is co-administered with one or more therapeutic agents (e.g., anticonvulsant agents, antipsychotic agents, rauwolfia alkaloids, antidepressants, dopamine prodrugs, dopamine agonists, catechol-O- methyltransferase (COMT) inhibitors, anticholinergics, MAO-B inhibitors, N-methyl-D- aspartic acid inhibitors, AChE inhibitors, NMDA antagonists, free-radical scavengers, glutamate pathway antagonists, antispastic agents, Congo red and its analogs, anthracyclines, amphotericin B and its analogs, sulfated polyanions, tetrapyrroles, sulfonylurea agents, meglitinides, biguanides, thiazolidinediones, dipeptidyl peptidase IV (DPP-4) inhibitors, incretin mimetics, amylin analogs, and alpha-glucosidase inhibitors, Levobunolol (Betagan), Timolol maleate/hemihydrate (Timoptic Timoptic XE, Betimol, Istalol), Carteolol (Cartrol, Ocupress), Betaxolol (Betoptic-S), Metipranolol hydrochloride (OptiPranolol), Levobetaxolol (Betaxon), Brimonidine (Alphagan-P), Apraclonidine (Iopidine), Dipivefrin (AKPro, Propine), Epinephrine (Epifrin), Memantine (Namenda, Axura), Dorzolamide HCl (Trusopt), Brinzolamide (Azopt), Acetazolamide (Diamox), Methazolamide (Neptazane), Dorzolamide HCl/timolol maleate (Cosopt), Latanoprost (Xalatan), Bimatoprost (Lumigan), Travoprost ophthamic solution (Travatan), Unoprostone (Rescula), Pilocarpine (Pilocar, Pilagan, Pilogel, Ocusert), Isosorbide (Ismotic), Mannitol (Osmitrol, Resectisol), Glycerin (Ophthalgan, Docket No. DUKE-30386/WO-l/ORD 1^66

Osmoglyn), Brimonidine/timolol (Combigan), Phenylephrine HCl (Neo-Synephrine), Prednisolone acetate (AK-Pred, Pred Forte), Dexamethasone (Ocu-Dex), Ciprofloxacin (Ciloxan), Erythromycin (E-Mycin), Nepafenac ophthalmic (Nevanac), Verteporfm (Visudyne), Pegaptanib (Macugen), Ranibizumab (Lucentis), Vitamin A (Aquasol A, DeI-Vi-A), Vitamin E (Aquasol E, Vitec), Ascorbic acid (Cebid, Ascorbicap, Cevalin, Cecon), Lutein or Zeaxanthin, Bilberry, Beta-carotene, Diltiazem (Cardizem, Dilacor, Tiamate), Acetazolamide (Diamox, Diamox Sequels), and Methazolamide (Neptazane)).

In certain embodiments, the present invention provides methods for identifying HSFl activating agents. The present invention is not limited to a particular method for identifying HSFl activating agents. In some embodiments, the method comprises a) providing a yeast yhsfΔ strain expressing human HSFl, wherein the yhsf. Δ strain comprises a yeast HSF gene coupled with an inducible promoter (e.g., GAL promoter); b) growing the yhsfΔ strain on a medium having the inducer (e.g., galactose); c) exposing the yhsfΔ strain to a candidate compound; d) switching the yhsfΔ strain to a repressive growth medium; e) assessing the growth of the yhsfΔ strain; and f) characterizing the candidate compound as a HSFl activating agent if the yhsfΔ strain grows on the non-inducer medium. In some embodiments, the human HSF is expressed via a pRS424-GPD-hHSFl plasmid. In some embodiments, the repressive medium is a glucose medium.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a model for the activation of human HSFl. HSFl is shown as an inactive monomer in the cytoplasm in the absence of stress. HSFl is localized to the cytoplasm as an inactive monomer. Upon stress-dependent stimulation, HSFl homotrimerizes, localizes to the nucleus, binds to DNA Heat Shock Elements (HSEs) becomes hyperphosphorylated, and activates gene transcription. Black oval: the HSFl DNA binding domain; hatched rectangles: LZ 1-3 (large), LZ4 (small), which form intermolecular coiled-coils in the homo-trimer; P: phosphorylation. HSFllz4m harbors a point mutation in LZ4 that renders HSFl constitutive Iy homo-trimerized, perhaps by breaking intramolecular coiled-coil interactions. Docket No. DUKE-30386/WO-l/ORD 13/66

Figure 2 shows yeast-based screen for small molecule activators of human HSFl (hHSFl). Yeast cells expressing the essential yeast Heat Shock Transcription Factor (yHSF) under control of the repressible GALl promoter are dependent on galactose for growth. Upon shifting the cells to glucose containing growth media, where the GALl promoter is shut off, the cells become dependent on activation of hHSFl for growth. Small molecules able to activate hHSFl allow growth on glucose.

Figure 3 shows microtiter plate analysis of human HSFl activation. Yeast hsfΔ cells harboring the G^ZJ -yeast HSF (yHSF) plasmid. On galactose (gal) all cells were viable due to galactose-inducible expression of yHSF. On glucose (glu) expression of yHSF was extinguished and cells were inviable when they express wild type human HSFl or the empty vector. However, yhsfΔ cells expressing a constitutively trimerized human HSFl protein (HSFllz4m) were viable in the absence of yeast HSF. All wells within a given row of the microtiter plate section contained the same yeast strain to show consistency. Plasmids transformed into the strain and carbon sources were indicated. Figures 4 show growth of the yhsfΔ: human HSFl yeast strain in the presence of library compounds (Figure 4A) and in the presence of HSFlA derivatives (Figure 4B). Yeast cells expressing hHSFl were seeded into 96-well plates at a concentration of -1,000 cells/well in glucose and treated with 10 micromolar of various compounds from a chemical library or the DMSO solvent control. Growth was monitored by determining the optical density (O.D.600) for 96 hours.Note that compounds in the 1391 and 1393 series are structurally related but distinct from the 1261 series.

Figure 5 shows structures of three independent compounds positive in the yeast screen for human HSFl activator molecules, designated HSFl-A, HSFl-B and HSFl-C. Yeast cell growth in the presence of these molecules at 10 uM is shown in Figure 4. Figure 6 shows synthetic routes for HSFlA, HSFlB and HSFlC. The structural relatedness of the three lead compounds simplified synthesis as a two-step reaction.Figure 7 shows HSFl activating compounds.

Figure 7 shows compounds identified as HSFl activating compounds.

Figure 8 shows HSFl activating compounds identified thorugh screens conducted with compounds from the LOPAC and Prestwick chemical libraries Docket No. DUKE-30386/WO-l/ORD 14/66

Figure 9 shows that HSFlA activates the expression of the HSP70 and HSP25 heat shock protein (chaperone) genes in Mouse Embryonic Fibroblast (MEF) cells.

Figure 10 shows HSFlA dependent activation of HSP70 is dependent on the presence of the gene encoding HSFl. Figure 11 shows HSFlA acts synergistically with heat shock to activate expression of the HSP70 protein chaperone. Figure 12A and 12B show that HSFlA promotes expression of HSP70 and reduces the aggregation of poly-glutamine (polyQ) proteins in rat neuronal precursor (PC- 12) cells.

Figure 12 shows that HSFlA promotes expression of HSP70 and reduces aggregation of poly-glutamine (polyQ) proteins in rat neuronal precursor (PC- 12) cells.

Figure 13 shows that HSFlA functions to ameliorate eye degeneration in a fruit fly model of poly glutamine disease (Example VIII).

Figure 14 shows that compositions of some embodiments of the present invention activate HSFl independently of HSP90 binding (Example IX).

DETAILED DESCRIPTION OF THE INVENTION

The proper synthesis, folding, trafficking, modifications, interactions, biochemical activities and eventual clearance of cellular proteins is essential for normal growth, development and maintenance during the life cycle of all organisms. Inappropriate folding, aggregation and accumulation of abnormal proteins is proteo-toxic to cells due to their dominant affects of insolubility, inappropriate interactions and long half-lives (see, e.g., Johnson, J.L., and Craig, E.A. (1997) Cell 90(2): 201-204; Bukau, B., et al, (2000) Cell 101(2): 119-122; Hartl, F.U. (1996) Nature 381 : 571-580; Deuerling, E., and Bukau, B. (2004) Crit. Rev. Biochem. MoI. Bio. 39: 261-277; Bukau, B., et al., (2006) Cell 125(3): 443- 451; Dickey, C.A., et al., (2007) Trends in MoI. Med. 13(1): 32-38; each of which are herein incorporated by reference in their entireties). Neuronal tissues and cells are exquisitely sensitive to defects in protein folding, aggregation and clearance and these defects are causally or correlatively associated with diseases that include Huntington's disease, Parkinson's disease, Alzheimer's disease, Amyotropic Lateral Sclerosis, prion diseases and other neurodegenerative disorders (see, e.g., Bonini, N. M. (2002) Proc. Natl. Acad. ScL, USA 99:16407-16411; Muchowski, P.J. (2002) Neuron 35: 9-12; Morimoto, R. I. (2006) Docket No. DUKE-30386/WO-l/ORD 15/66

New England J. Med. 355: 2254-2255; Finkbeiner, S., et al, (2006) J. Neurosci. 26(41): 10349-10357; Furukawa, Y., et al., (2006) PNAS 103(18): 7148-7153; Gidalevitz, T., et al., (2006) Science 311 : 1471-1474; each of which are herein incorporated by reference in their entireties). Many of these are diseases occur frequently in the elderly and result in a variety of symptoms due to loss of function of motor, dopaminergic and other neurons essential for a normal healthy life (see, e.g., Cummings, CJ. and Zoghbi, H.Y. (2000) Hum. MoI. Genet. 9: 909-916; each of which is herein incorporated by reference in its entirety). Defects in protein folding, aggregation and clearance have also been implicated in type 2 diabetes mellitus (see, e.g., Chung, J., et al. (2008) PNAS 105(5) 1739-1744; each of which is herein incorporated by reference in its entirety).

While many aspects of these complex processes are incompletely understood, a variety of individual protein chaperones and co-chaperone complexes function to fold, process, mature and degrade cellular proteins (see, e.g., Johnson, J.L., and Craig, E.A. (1997) Cell 90(2): 201-204; Bukau, B., et al., (2000) Cell 101(2): 119-122; Hartl, F.U. (1996) Nature 381 : 571-580; Deuerling, E., and Bukau, B. (2004) Crit. Rev. Biochem. MoI. Bio. 39: 261- 277; Bukau, B., et al., (2006) Cell 125(3): 443-451; Dickey, C.A., et al., (2007) Trends in MoI. Med. 13(1): 32-38; each of which are herein incorporated by reference in their entireties). Many neurodegenerative diseases are caused, for example, by genetically programmed changes in specific proteins, such as through the addition of poly glutamine (polyQ) coding sequences, by genetic defects in the protein folding and processing machinery, or by as yet poorly understood mechanisms by which abnormal protein conformations can be propagated in a protein-catalyzed fashion (see, e.g., Bonini, N. M. (2002) Proc. Natl. Acad. ScL, USA 99:16407-16411; Muchowski, P.J. (2002) Neuron 35: 9- 12; Morimoto, R. I. (2006) New England J. Med. 355: 2254-2255; Finkbeiner, S., et al., (2006) J. Neurosci. 26(41): 10349-10357; Furukawa, Y., et al., (2006) PNAS 103(18): 7148- 7153; Gidalevitz, T., et al., (2006) Science 311 :1471-1474; Cummings, CJ. and Zoghbi, H.Y. (2000) Hum. MoI. Genet. 9: 909-916; each of which are herein incorporated by reference in their entireties).

Protein chaperones facilitate the folding, stabilization, solubilization and degradation of cellular proteins and are often included in the group of Heat Shock Proteins (Hsps) because Docket No. DUKE-30386/WO-l/ORD 16/66

their synthesis is elevated in response to heat and other stresses known to induce protein unfolding, aggregation and degradation (see, e.g., Morimoto, R.I., Tissieres, A., and Georgopoulos, C. (1994) The Biology of Heat Shock Proteins and Molecular Chaperones, Cold Springs Harbor Laboratory Press, Cold Springs Harbor New York; Lindquist, S. (1992) Curr. Opinion in Genet, and Develop. 2: 748-755; Feige, U., et al, (eds.) Stress-inducible cellular responses. VoI 77, Birkhauser, Verlag, Boston; Lindquist, S. and Craig, E.A. (1988) Ann. Rev. Genet. 22: 631-677; each of which are herein incorporated by reference in their entireties). Recent evidence in cellular or organismal model systems of neurodegenerative diseases strongly support the notion that protein chaperones act both independently and in concert to ameliorate biochemical hallmarks or symptoms of the disease associated with unfolded or aggregated proteins. For example, in mammalian cell culture, mouse or Drosophila models of polyQ aggregation or alpha- synuclein toxicity, expression of the Hsp70 or Hsp40 chaperones can significantly suppress protein aggregation, increase protein solubility and turnover and ameliorate neuronal loss (see, e.g., Bailey, C. K., et al., (2002) Hum. MoI. Genet. 11(5): 515-523; Kitamura, A., et al., (2006) Nat. Cell Bio. 8(10): 1163- 1170; Pavan, K., et al., (2002) Science 295: 865-868; Chai, Y., et al., (1999) J. Neurosci. 19(23): 10338-10347; Muchowski, P.J, et al., (2000) Proc. Natl. Acad. Sci. USA 97: 7841- 7846; Jana, N.R., et al., (2000) Hum. MoI. Genet. 9: 2009-2018; Wyttenbach, A., et al., (2000) Proc. Natl. Acad. Sci. 97: 2898-2903; Adachi, H., et al., (2003) J. Neurosci. 23: 2203- 2211; Cummings, C.J., et al, (2001) Hum. MoI. Genet. 10:1511-1518; each of which are herein incorporated by reference in their entireties). Additional studies suggest that elevated expression of both Hsp70 and Hsp40 can synergize in the suppression of polyQ-mediated neuronal degeneration and that arimoclomal, an inducer of Hsp synthesis, significantly delays disease progression in a mouse model of ALS (see, e.g., Kieran, D., et al., (2004) Nature Medicine 10: 402-405; herein incorporated by reference in its entirety). From bacteria to human cells, Hsp synthesis is coordinately induced in response to stress conditions that result in protein unfolding, aggregation and proteolysis by stress-responsive transcription factors.

In cells from yeast to humans, the transcription of genes encoding Hsps is induced in response to stresses such as increased temperature through cώ-acting promoter elements called Heat Shock Elements (HSEs), composed of variations of the inverted repeated pentameric consensus sequence 5"-nGAAnnTTCnnGAAn-3' (SEQ ID NO:01) (see, e.g., Docket No. DUKE-30386/WO-l/ORD 17/66

Morimoto, R.I., Tissieres, A., and Georgopoulos, C. (1994) The Biology of Heat Shock Proteins and Molecular Chaperones, Cold Springs Harbor Laboratory Press, Cold Springs Harbor New York; Lindquist, S. and Craig, E.A. (1988) Ann. Rev. Genet. 22: 631-677; each of which are herein incorporated by reference in their entireties). In response to stress the Heat Shock Transcription Factor, HSF, binds as a homo-trimer to HSEs and activates target gene transcription. Indeed, HSFs and their cognate DNA binding site HSEs are two highly structurally and functionally conserved cis- and trans-acting regulatory factors (see, e.g., Wu, C. (1995) Ann. Rev. Cell Dev. Biol. 11 : 441-469; Pirkkala, L., et al, (2001) FASEB J. 15: 1118-1131; each of which are herein incorporated by reference in their entireties). The baker's yeast Sαcchαromyces cerevisiαe harbors a single gene encoding HSF that is essential for cell viability under all conditions tested (see, e.g., Sorger, P. K., and Pelham, H.R.B. (1988) Cell 54: 855-864; Wiederrecht, G., et al., (1988) Cell 54: 841-853; each of which are herein incorporated by reference in their entireties). Recent genome-wide expression and chromatin-immuoprecipitation experiments demonstrate that yeast HSF directly activates a broad range of genes encoding proteins that function as chaperones, in protein turnover and a variety of additional stress protection roles (see, e.g., Hahn, J.-S., et al., (2004) Molecular and Cellular Biology 24:5249-5256; herein incorporated by reference in its entirety).

In mammals, Drosophilα and C. elegαns HSFl responds to stress to activate transcription of genes encoding a family of protein chaperones (Wu, C. (1995) Ann. Rev. Cell Dev. Biol. 11 : 441-469; Pirkkala, L., Nykanen, et al, (2001) FASEB J. 15: 1118-1131; Hsu, A.L., (2003) Science 300: 1142-1145; Morley, J.F., and Morimoto, R.I. (2004) MoI. Bio. Cell 15: 657-664). While the precise mechanisms whereby HSFl from humans and other organisms sense and respond to stress have not been elucidated, a model that summarizes current understanding of this process in human cells is shown in Figure 1. HSFl activation is a multi-step process that occurs posttranslationally in response to elevated temperatures, the accumulation of unfolded proteins and other stressful conditions (see, e.g., Wu, C. (1995) Ann. Rev. Cell Dev. Biol. 11 : 441-469; Pirkkala, L., et al., (2001) FASEB J. 15: 1118-1131; Baler, R., et al., (1993) MoI. Cell. Biol. 13: 2486-2496; Sarge, K.D., et al., (1993) MoI. Cell. Biol. 13: 1392-1407; Zuo, J., et al., (1995) MoI. Cell. Biol. 15: 4319-4330; each of which are herein incorporated by reference in their entireties). In the absence of acute stress, HSFl is present largely in the cytoplasm as a monomer, and is thought to be associated with Hsp90, Docket No. DUKE-30386/WO-l/ORD 18/66

Hsp70 and other proteins (see, e.g., Zuo, J., et al, (1998) Cell 94: 471-480; AIi, A., et al, (1998) MoI. Cell. Biol. 18: 4949-4960; Guo, Y., et al., (2001) J. Biol. Chem. 276: 45791- 45799; each of which are herein incorporated by reference in their entireties). In vitro and in vivo experiments suggest that HSFl is retained in the momomeric state through intramolecular interactions between two coiled coil regions, Leucine Zipper 1-3 (LZ 1-3) and Leucine Zipper 4 (LZ4) (see, e.g., Rabindran, S.K., et al., (1993) Science 259: 230-234; Zuo, J., et al., (1994) MoI. Cell. Biol. 14: 7557-7568; each of which are herein incorporated by reference in their entireties). Indeed, point mutations in Leucine Zipper 4 (HSFllz4m) cause constitutive HSFl homo-trimerization in mammalian cells (see, e.g., Rabindran, S. K., et al., (1993) Science 259: 230-234; herein incorporated by reference in its entirety). In response to stress, HSFl is converted to a homo-trimer that is thought to be stabilized by inter-molecular coiled coil interactions and accumulates in the nucleus, where it engages in high affinity binding to HSEs within target gene promoters and activates target gene transcription. Heat shock induced Hsp target gene activation by HSFl is transient, and correspondingly, HSFl is ultimately converted back to the low affinity DNA binding monomeric form in the cytosol. HSFl is phosphorylated both under basal conditions where this modification is thought to maintain the protein in an inactive state and in response to stress, with this latter modification having functional consequences that are not well understood (see, e.g., Cotto, J. J., et al., (1996) J. Biol. Chem. 271 : 3355-3358; Guettouche, T., (2005) BMC Bochem. 6:1-14; herein incorporated by reference in its entirety).

Experiments conducted during the course of development of embodiments for the present invention developed a specialized high throughput screen for identifying small molecules capable of activating the human Heat Shock Transcription Factor 1 (HSFl) protein from complex chemical libraries. In addition, experiments conducted during the course of developing embodiments for the present invention identified molecules capable of activating the human Heat Shock Transcription Factor 1. It was shown that these molecules activate human HSFl when added to cultured cells, and activate expression of heat shock proteins (e.g., HSP70, HSP25, and reduce aggregation of poly-glutamine proteins in neuronal cells). Accordingly, the present invention provides small molecules (e.g., compounds) capable of activating heat shock factors (e.g., facilitating HSFl homo-trimerization), activating heat shock factor (e.g., HSFl) target gene expression (e.g., Heat Shock genes) and protein Docket No. DUKE-30386/WO-l/ORD 19/66

expression (e.g., Heat Shock Proteins), methods for their discovery, and their therapeutic and/or research uses. Exemplary compositions and methods of the present invention are described in more detail in the following sections: I. HSF Activating Compound Screens; II. HSF Activating Compounds; III. Pharmaceutical Compositions; and IV. Therapeutic Applications.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, "Molecular cloning: a laboratory manual" Second Edition (Sambrook et al, 1989); "Oligonucleotide synthesis" (MJ. Gait, ed., 1984); "Animal cell culture" (R.I. Freshney, ed., 1987); the series "Methods in enzymology" (Academic Press, Inc.); "Handbook of experimental immunology" (D.M. Weir & CC. Blackwell, eds.); "Gene transfer vectors for mammalian cells" (J.M. Miller & M. P. Calos, eds., 1987); "Current protocols in molecular biology" (F.M. Ausubel et al, eds., 1987, and periodic updates); "PCR: the polymerase chain reaction" (Mullis et al, eds., 1994); and "Current protocols in immunology" (J.E. Coligan et al, eds., 1991), each of which is herein incorporated by reference in its entirety.

I. HSF Activating Compound Screens In some embodiments, the present invention provides screens for identifying activators of heat shock factor (e.g., activators capable of facilitating HSFl function), and for identifying activators of heat shock factor (e.g., HSFl) target gene expression (e.g., Heat Shock genes) and protein expression (e.g., Heat Shock Proteins). The screen is not limited to identifying activators of a particular heat shock factor. In some embodiments, the screens identify HSFl activators, HSF2 activators, and/or HSF4 activators. The present invention is not limited to identifying a particular type of heat shock factor activator. Examples of activators include, but are not limited to, small molecules (see, e.g., the compounds provided in Section II - Exemplary Compounds).

The present invention is not limited to a particular type of screen for identifying heat shock factor (e.g., HSFl) activators. In some embodiments, the present invention provides a yeast based screen. The present invention is not limited to use of a particular type of yeast. Docket No. DUKE-30386/WO-l/ORD 20/66

In some embodiments, the screen comprises genetically modified yeast. The screen is not limited to a particular type of genetically modified yeast. In some embodiments, the screens provide yeast that are genetically modified such that expression of yeast HSF is regulated. In some embodiments, the screens provide yeast that are genetically modified such that the yeast express human HSFl . While human HSFl and yeast HSF have similar structures, bind as homo-trimers to conserved HSEs and activate functionally common Hsp genes, expression of wild type human HSFl cannot suppress the viability defect associated with yeast HSF deletion (yhsfΔ) cells (see, e.g., Liu, X.D., et al, (1997) EMBO J. 16: 6466-6477; herein incorporated by reference in its entirety). Biochemical analysis of human HSFl demonstrated that human HSFl exists in yeast as a monomer and is not able to homo-trimerize under basal or stress conditions. Indeed, expression in yhsfΔ cells of the human HSFllz4m mutant, which is constitutively trimerized in culturized human cells, is able to rescue the yhsfΔ viability defect, bind to and activate stress-inducible target gene transcription such as from the yeast Hsp70 gene, and exist as a homo-trimer in yeast (see, e.g., Liu, X.D., et al., (1997) EMBO J. 16: 6466-6477; Liu, P.C.C., and Thiele, D.J. (1999) Journal of Biological Chemistry 274: 17219-17225; each of which are herein incorporated by reference in their entireties). The present invention is not limited to any mechanism of action. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nonetheless, it is contemplated that human HSFl fails to function in yeast due to a homotrimerization inability. In some embodiments, the screens of the present invention identify activators (e.g., compounds) capable of facilitating homotrimerization of HSFl.

The screens are not limited to a particular manner of genetically modifying yeast HSF expression. In some embodiments, genetically modified yeast HSF expression occurs through deleting the HSF gene open reading frame, thereby rendering a yhsfΔ strain that is inviable. In some embodiments, the yhsfΔ strains have a yeast HSF gene coupled with an inducible promoter (e.g., GALl-IO) thereby rendering growth of such yhsfΔ strains viable on a selectable medium (e.g., galactose). In some embodiments wherein yhsfΔ strains express a yeast HSF gene coupled with an GAL promoter, the strain is inviable at any temperature or under any condition tested when cells are shifted to a glucose medium thereby extinguishing yeast HSF espression. In some embodiments, the yhsfΔ strains expressing a yeast HSF gene coupled with a inducible promoter additionally harbor a plasmid configured for human HSFl Docket No. DUKE-30386/WO-l/ORD 21/66

(hHSFl) expression. In some embodiments, the plasmid configured for expression of hHSF is pRS424-GPD-hHSFl (wherein GPD is the constitutively expressed glucose phosphate dehydrogenase promoter).

In some embodiments, as shown in Figure 2, the yhsfΔ strains expressing a yeast HSF gene coupled with a inducible promoter configured for expression hHSFl are used for identifying activators of hHSFl (e.g., activators capable of facilitating HSFl homotrimerization), identifying activators of HSFl target gene expression (e.g., Heat Shock Proteins) and/or activation or inhibition of protein chaperone activity (e.g., increased protein folding, increased protein solubilization, protein degradation). This screen has several features, including but not limited to, (1) when yeast HSF expression is extinguished, only cells in which human HSFl has been activated are viable, providing a screen with a very low background; (2) this strain allows for positive selection of human HSFl activator molecules; (3) this strain in conjunction with an additional strain lacking hHSFl expression permits identification of molecules that act exclusively in a human HSFl -dependent manner rather than via the prevention of yeast HSF repression by glucose in yeast; and (4) the screen is amenable to automated liquid handling and optical density determination and is therefore high throughput in nature.

The screens are not limited to a particular method for identifying activators of hHSFl. In some embodiments, for example, a yhsfΔ strain expressing hHSFl is exposed to a small molecule under conditions inviable for growth absent homotrimerization or other means of activation of hHSFl . Growth in such conditions indicates that the small molecule is an activator of hHSFl (see, Figure 2). A lack of growth indicates that the small molecule is not an activator of hHSFl (see, Figure 2).

In some embodiments, the yeast strains used in the screens are genetically modified to maximize small molecule accumulation. The yeast strains are not limited to a particular manner of genetic modification to maximize small molecule accumulation. In some embodiments, genetic modification to maximize small molecule accumulation is accomplished through, for example, sequential deletion of the PDR5, SNQ2 and ERG6 genes in the yhsfΔ background. PDR5 and SNQ2 encode ATP binding cassette integral plasma membrane transport proteins which mediate multidrug resistance by exporting compounds with a broad range of structures and relatively low specificity (see, e.g., Emter, R., (2002) Docket No. DUKE-30386/WO-l/ORD 2.V66

FEBS Letters 521 : 57-61; herein incorporated by reference in its entirety). As such, yeast cells lacking Pdr5 and Snq2 accumulate organic molecules to a significantly higher steady state level than wild type strains. The ERG6 gene, encoding delta(24)-sterol C- methyltransferase, is a key enzyme in ergosterol biosynthesis. Erg6 mutants exhibit enhanced diffusion rates of lipophilic molecules across the plasma membrane (see, e.g., Emter, R., (2002) FEBS Letters 521 : 57-61; herein incorporated by reference in its entirety). In some embodiments, yeast strains having sequential deletion of PDR5, SNQ2 and ERG6 genes in the yhsfΔ background hyper-accumulate organic compounds.

II. HSF Activating Compounds

Experiments conducted during the course of development of embodiments for the present invention identified HSF activating compounds (e.g., compounds capable of facilitating HSFl homotrimerization, compounds capable of activating HSFl target gene expression (e.g., Heat Shock Elements), compounds capable of activating HSFl protein function (e.g., Heat Shock Proteins), compounds capable of activating or inhibiting protein chaperone activity (e.g., increased protein folding, increased protein solubilization, protein degradation)). An understanding of the mechanism by which the compounds activate HSF proteins is not required to practice the present invention.

Exemplary HSFl activating compounds of the present invention are provided below. In certain embodiments, the composition comprises a compound described by the

following formula:

including salts, esters, and prodrugs thereof. The compound is not limited to particular definitions of Ri through Rg groups. In some embodiments, the Ri through Rg Docket No. DUKE-30386/WO-l/ORD 23/66 groups define a compound capable of HSF activation, identifiable using screening techniques described herein. In some embodiments, Ri is , H, or halogen (e.g., chlorine

). In some embodiments, Xi is

, or Xi is absent. In some embodiments, X₂ is S or C. In some embodiments, X₃ is S or C. In some embodiments, R₉ is -OCH₃. In some embodiments, R₂

embodiments, Docket No. DUKE-30386/WO-l/ORD 24/66

4 i_s U N _or C . In some embodiments, R₅ is S or C. In some embodiments, R₆ is H or O . In some embodiments, R₇ is H or O . In

some embodiments, Rg is or

In some embodiments, Rio is chlorine or CH₃. In some

embodiments, Rn is substituted or unsubstituted alkyl such as, for example,

or

Docket No. DUKE-30386/WO-l/ORD 25/66

In certain embodiments, the compound is

Docket No. DUKE-30386/WO-l/ORD 26/66

Docket No. DUKE-30386/WO-l/ORD 27/66

In some embodiments, the present invention provides HSF activating compounds described by any of the the compounds shown in Figure 7.

In certain embodiments, the present invention provides HSF activating compounds described by the following formulas: Docket No. DUKE-30386/WO-l/ORD 28/66

including salts, esters, and prodrugs thereof; and including both R and S enantiomeric forms and racemic mixtures thereof;

wherein Rl is

, H, or halogen (e.g., Chlorine);

wherein

wherein (i.e., R3 and R4 are C or N, but R3 and R4 are not the same); wherein R5 is S or C; wherein R6 is H or O ; wherein R7 is H or O ; Docket No. DUKE-30386/WO-l/ORD 29/66

wherein R8 is

wherein R9 is C

hlorine or CH3; and wherein RlO is • , or

In certain embodiments, the present invention provides the following HSFl activating compounds:

Docket No. DUKE-30386/WO-l/ORD 30/66

comprises a functional derivative of a compound capable of HSF activation (e.g.,

a functional derivative of HSFlA). In certain embodiments, the present invention provides any of the compounds described herein with further functionalization. For example, in some embodiments, the present invention provides any of the compounds functionalized with a biotin moiety (e.g., HSFlA-biotin)

( ). For example, such functionalized compounds can be used for identification of agents (e.g., protein(s)) with which they interact. Docket No. DUKE-30386/WO-l/ORD 31/66

Additional exemplary compounds are provided in the figures and experimental section, below.

III. Pharmaceutical compositions, formulations, and exemplary administration routes and dosing considerations

Exemplary embodiments of various contemplated medicaments and pharmaceutical compositions are provided below.

A. Preparing Medicaments It is contemplated that the compounds of the present invention are useful in the preparation of medicaments to treat a variety of conditions associated with misfolded proteins and/or deficient protein chaperone activity.

In addition, it is contemplated that the compounds are also useful for preparing medicaments for treating other disorders wherein the effectiveness of the compounds are known or predicted. Such disorders include, but are not limited to, neurological disorders. The methods and techniques for preparing medicaments of a compound of the present invention are well-known in the art. Exemplary pharmaceutical formulations and routes of delivery are described below.

One of skill in the art will appreciate that any one or more of the compounds described herein, including the many specific embodiments, are prepared by applying standard pharmaceutical manufacturing procedures. Such medicaments can be delivered to the subject by using delivery methods that are well-known in the pharmaceutical arts.

B. Exemplary pharmaceutical compositions and formulation In some embodiments of the present invention, the compositions are administered alone, while in some other embodiments, the compositions are preferably present in a pharmaceutical formulation comprising at least one active ingredient/agent, as defined above, together with a solid support or alternatively, together with one or more pharmaceutically acceptable carriers and optionally other therapeutic agents. Each carrier must be "acceptable" in the sense that it is compatible with the other ingredients of the formulation and not injurious to the subject. Docket No. DUKE-30386/WO-l/ORD 3-V66

Contemplated formulations include those suitable oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary administration. In some embodiments, formulations are conveniently presented in unit dosage form and are prepared by any method known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association (e.g. , mixing) the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, wherein each preferably contains a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. In other embodiments, the active ingredient is presented as a bolus, electuary, or paste, etc.

In some embodiments, tablets comprise at least one active ingredient and optionally one or more accessory agents/carriers are made by compressing or molding the respective agents. In some embodiments, compressed tablets are prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g., povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. , sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose)surface-active or dispersing agent. Molded tablets are made by molding in a suitable machine a mixture of the powdered compound (e.g., active ingredient) moistened with an inert liquid diluent. Tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or Docket No. DUKE-30386/WO-l/ORD 33/66

sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

Pharmaceutical compositions for topical administration according to the present invention are optionally formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. In alternatively embodiments, topical formulations comprise patches or dressings such as a bandage or adhesive plasters impregnated with active ingredient(s), and optionally one or more excipients or diluents. In some embodiments, the topical formulations include a compound(s) that enhances absorption or penetration of the active agent(s) through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide (DMSO) and related analogues.

If desired, the aqueous phase of a cream base includes, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane- 1, 3 -diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. In some embodiments, oily phase emulsions of this invention are constituted from known ingredients in a known manner. This phase typically comprises a lone emulsifier (otherwise known as an emulgent), it is also desirable in some embodiments for this phase to further comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier so as to act as a stabilizer. It some embodiments it is also preferable to include both an oil and a fat. Together, the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations. Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.

The choice of suitable oils or fats for the formulation is based on achieving the desired properties (e.g., cosmetic properties), since the solubility of the active compound/agent in most oils likely to be used in pharmaceutical emulsion formulations is very low. Thus creams should preferably be a non-greasy, non-staining and washable Docket No. DUKE-30386/WO-l/ORD 34/66

products with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used. Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the agent.

Formulations for rectal administration may be presented as a suppository with suitable base comprising, for example, cocoa butter or a salicylate.

Formulations suitable for vaginal administration may be presented as pessaries, creams, gels, pastes, foams or spray formulations containing in addition to the agent, such carriers as are known in the art to be appropriate.

Formulations suitable for nasal administration, wherein the carrier is a solid, include coarse powders having a particle size, for example, in the range of about 20 to about 500 microns which are administered in the manner in which snuff is taken, i.e., by rapid inhalation (e.g., forced) through the nasal passage from a container of the powder held close up to the nose. Other suitable formulations wherein the carrier is a liquid for administration include, but are not limited to, nasal sprays, drops, or aerosols by nebulizer, an include aqueous or oily solutions of the agents.

Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. In some embodiments, the formulations are presented/formulated in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, Docket No. DUKE-30386/WO-l/ORD 35/66

immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose or unit, daily subdose, as herein above -recited, or an appropriate fraction thereof, of an agent. It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavoring agents. It also is intended that the agents, compositions and methods of this invention be combined with other suitable compositions and therapies. Still other formulations optionally include food additives (suitable sweeteners, flavorings, colorings, etc.), phytonutrients {e.g., flax seed oil), minerals {e.g., Ca, Fe, K, etc.), vitamins, and other acceptable compositions {e.g., conjugated linoelic acid), extenders, and stabilizers, etc.

In some embodiments, the compounds of the present invention are provided in unsolvated form or are in non-aqueous solutions (e.g., ethanol). The compounds may be generated to allow such formulations through the production of specific crystalline polymorphs compatible with the formulations. In some embodiments, the compounds of the present invention are provided in conjunction with thermal therapy such as a thermal bath.

In certain embodiments, the present invention provides instructions for administering said compound to a subject. In certain embodiments, the present invention provides instructions for using the compositions contained in a kit for the treatment of conditions characterized by the dysregulation of apoptotic processes in a cell or tissue {e.g., providing dosing, route of administration, decision trees for treating physicians for correlating patient- specific characteristics with therapeutic courses of action). In certain embodiments, the present invention provides instructions for using the compositions contained in the kit to treat a variety of medical conditions associated with misfolded proteins or irregular HSFl activitiy (e.g., medical conditions involving misfolded proteins or irregular HSFl activity) (e.g., medical conditions involving irregular chaperone activity) (e.g., Alzheimer's disease, Parkinson's disease, Huntington disease, Amyotrophic Lateral Sclerosis, a prion-based disease, cataract, age-related cataract, glaucoma, macular degeneration, age-related macular degeneration, retinitis pigmentosa, cardiovascular disease and stroke, heat stroke, Docket No. DUKE-30386/WO-l/ORD 36/66

spinocerebellar ataxia, Machado Joseph disease, stress-related neuronal degeneration, aging, cancer, and type 2 diabetes mellitus.) In some embodiments, the methods are directed towards crystallins in age-related cataracts. In some embodiments, the methods are directed towards myocillin in glaucoma.

C. Exemplary administration routes and dosing considerations

Various delivery systems are known and can be used to administer therapeutic agents (e.g., exemplary compounds as described in Section III above) of the present invention, e.g., encapsulation in liposomes, microparticles, microcapsules, receptor-mediated endocytosis, and the like. Methods of delivery include, but are not limited to, intra-arterial, intramuscular, intravenous, intranasal, and oral routes. In specific embodiments, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, injection, or by means of a catheter. It is contemplated that the agents identified can be administered to subjects or individuals susceptible to or at risk of developing pathological growth of target cells and correlated conditions. When the agent is administered to a subject such as a mouse, a rat or a human patient, the agent can be added to a pharmaceutically acceptable carrier and systemically or topically administered to the subject. To determine patients that can be beneficially treated, a tissue sample is removed from the patient and the cells are assayed for sensitivity to the agent.

Therapeutic amounts are empirically determined and vary with the pathology being treated, the subject being treated and the efficacy and toxicity of the agent. When delivered to an animal, the method is useful to further confirm efficacy of the agent. In some embodiments, in vivo administration is effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations are carried out with the dose level and pattern being selected by the treating physician. Docket No. DUKE-30386/WO-l/ORD 37/66

Suitable dosage formulations and methods of administering the agents are readily determined by those of skill in the art. Preferably, the compounds are administered at about 0.01 mg/kg to about 200 mg/kg, more preferably at about 0.1 mg/kg to about 100 mg/kg, even more preferably at about 0.5 mg/kg to about 50 mg/kg. When the compounds described herein are co-administered with another agent (e.g., as sensitizing agents), the effective amount may be less than when the agent is used alone.

The pharmaceutical compositions can be administered orally, intranasally, parenterally or by inhalation therapy, and may take the form of tablets, lozenges, granules, capsules, pills, ampoules, suppositories or aerosol form. They may also take the form of suspensions, solutions and emulsions of the active ingredient in aqueous or nonaqueous diluents, syrups, granulates or powders. In addition to an agent of the present invention, the pharmaceutical compositions can also contain other pharmaceutically active compounds or a plurality of compounds of the invention.

More particularly, an agent of the present invention also referred to herein as the active ingredient, may be administered for therapy by any suitable route including, but not limited to, oral, rectal, nasal, topical (including, but not limited to, transdermal, aerosol, buccal and sublingual), vaginal, parental (including, but not limited to, subcutaneous, intramuscular, intravenous and intradermal) and pulmonary. It is also appreciated that the preferred route varies with the condition and age of the recipient, and the disease being treated.

Ideally, the agent should be administered to achieve peak concentrations of the active compound at sites of disease. This may be achieved, for example, by the intravenous injection of the agent, optionally in saline, or orally administered, for example, as a tablet, capsule or syrup containing the active ingredient. Desirable blood levels of the agent may be maintained by a continuous infusion to provide a therapeutic amount of the active ingredient within disease tissue. The use of operative combinations is contemplated to provide therapeutic combinations requiring a lower total dosage of each component antiviral agent than may be required when each individual therapeutic compound or drug is used alone, thereby reducing adverse effects. Docket No. DUKE-30386/WO-l/ORD 38/66

D. Exemplary co-administration routes and dosing considerations

The present invention also includes methods involving co-administration of the compounds described herein with one or more additional active agents. Indeed, it is a further aspect of this invention to provide methods for enhancing prior art therapies and/or pharmaceutical compositions by co-administering a compound of this invention. In coadministration procedures, the agents may be administered concurrently or sequentially. In one embodiment, the compounds described herein are administered prior to the other active agent(s). The pharmaceutical formulations and modes of administration may be any of those described above. In addition, the two or more co -administered chemical agents, biological agents or radiation may each be administered using different modes or different formulations.

The agent or agents to be co-administered depends on the type of condition being treated. For example, when the condition being treated is a neurological disorder (e.g., Huntington Disease), the additional agent can be an anticonvulsant medication. The additional agents to be co-administered can be any of the well-known agents in the art for a particular disorder, including, but not limited to, those that are currently in clinical use and/or experimental use.

IV. Therapeutic Application In certain embodiments, the present invention provides methods (e.g., therapeutic applications) for treating conditions associated with protein misfolding. The present invention is not limited to a particular type of method. In some embodiments, the methods for treating conditions associated with protein misfolding comprise a) providing: i. target cells having misfolded proteins; and ii. a composition (e.g., a composition comprising exemplary HSFl activating compounds as described in Section III above); and b) exposing the target cells to the composition under conditions such that the exposure results in enhanced HSFl activity. The methods are not limited to treating a particular condition associated with protein misfolding. In some embodiments, the condition associated with protein misfolding is a medical condition involving deficient chaperone activity. In some embodiments, the condition associated with protein misfolding is enhanced aging, Alzheimer's disease,

Parkinson's disease, Huntington disease, Amyotrophic Lateral Sclerosis, and prion-based Docket No. DUKE-30386/WO-l/ORD 39/66

disease (e.g., transmissible spongiform encephalopathy, Bovine spongiform encephalopathy, Creutzfeldt- Jakob disease, and Kuru). In some embodiments, the condition associated with protein misfolding is type 2 diabetes mellitus (see, e.g., Chung, J., et al., (2008) PNAS 105(5) 1739-1744; herein incorporated by refernece in its entirety). The methods are not limited to a particular type of target cells. In some embodiments, the target cells are neurological cells. In some embodiments, the target cells are within a living mammal (e.g., human, horse, dog, cat, pig, rat, mouse, ape, monkey).

Additionally, any one or more of these compounds can be used in combination with at least one other therapeutic agent (e.g., potassium channel openers, calcium channel blockers, sodium hydrogen exchanger inhibitors, anticonvulsant agents, antiarrhythmic agents, antiatherosclerotic agents, anticoagulants, antithrombotic agents, prothrombolytic agents, fibrinogen antagonists, diuretics, antihypertensive agents, ATPase inhibitors, mineralocorticoid receptor antagonists, phospodiesterase inhibitors, antidiabetic agents, antiinflammatory agents, antioxidants, angiogenesis modulators, antiosteoporosis agents, hormone replacement therapies, hormone receptor modulators, oral contraceptives, antiobesity agents, antidepressants, antianxiety agents, antipsychotic agents, antiproliferative agents, antitumor agents, antiulcer and gastroesophageal reflux disease agents, growth hormone agents and/or growth hormone secretagogues, thyroid mimetics, anti-infective agents, anti-spastic agents, antiviral agents, antibacterial agents, antifungal agents, cholesterol/lipid lowering agents and lipid profile therapies, and agents that mimic ischemic preconditioning and/or myocardial stunning, antiatherosclerotic agents, anticoagulants, antithrombotic agents, antihypertensive agents, antidiabetic agents, and antihypertensive agents selected from ACE inhibitors, AT-I receptor antagonists, ET receptor antagonists, dual ET/ All receptor antagonists, and vasopepsidase inhibitors, or an antiplatelet agent selected from GPIIb/IIIa blockers, P2Yi and P2Yi₂ antagonists, thromboxane receptor antagonists, aspirin) in along with a pharmaceutically-acceptable carrier or diluent in a pharmaceutical composition. Additional therapeutic agents for Huntington disease include, but are not limited to, anticonvulsant agents (e.g., valproic acid, clonazepam), antipsychotic agents (e.g., risperidone, haloperidol), rauwolfia alkaloids (e.g., reserpine), antidepressants (e.g., proxetine). Additional therapeutic agents for Parkinson's disease include, but are not limited to, dopamine prodrugs (e.g., levodopa/carbidopa), dopamine agonists (e.g., Docket No. DUKE-30386/WO-l/ORD 40/66

apomorphine, bromocriptine, pergolide, pramipexole, ropinirole, rotigotine), catechol-O- methyltransferase (COMT) inhibitors (e.g., tolcapone, entacapone, levodopa, carbidopa, entacapone), anticholinergics (e.g., trihexyphenidyl, benztropine mesylate), MAO-B inhibitors (e.g., selegiline, rasagiline), and N-methyl-D-aspartic acid inhibitors (e.g., amantadine). Additional therapeutic agents for Alzheimer's disease include, but are not limited to, centrally acting AChE inhibitors (e.g., rivastigmine), NMDA antagonists (e.g., memantine), and free-radical scavengers (e.g., tocopherol). Additional therapeutic agents for Amyotrophic Lateral Sclerosis include, but are not limited to, glutamate pathway antagonists (e.g., riluzole), antispastic agents (e.g., baclofen). Additional therapeutic agents for prion diseases include, but are not limited to, Congo red and its analogs, anthracyclines, amphotericin B and its analogs, sulfated polyanions, and tetrapyrroles. Additional therapeutic agents for type 2 diabetes mellitus include, but are not limited to, sulfonylurea agents (e.g., glipizide, glyburide, glimepiride), meglitinides (e.g., repaglinide, nateglinide), biguanides (e.g., metformin), thiazolidinediones (e.g., pioglitazone, rosiglitazone), dipeptidyl peptidase IV (DPP-4) inhibitors (e.g., sitagliptin), incretin mimetics (e.g., exenatide), amylin analogs (e.g., pramlintide acetate), and alpha-glucosidase inhibitors (e.g., acarbose, miglitol). Additional agents for glaucoma, cataract, retinitis pigmentosa, and/or macular degeneration include, but are not limited to, Levobunolol (Betagan), Timolol maleate/hemihydrate (Timoptic Timoptic XE, Betimol, Istalol), Carteolol (Cartrol, Ocupress), Betaxolol (Betoptic- S), Metipranolol hydrochloride (OptiPranolol), Levobetaxolol (Betaxon), Brimonidine

(Alphagan-P), Apraclonidine (Iopidine), Dipivefrin (AKPro, Propine), Epinephrine (Epifrin), Memantine (Namenda, Axura), Dorzolamide HCl (Trusopt), Brinzolamide (Azopt), Acetazolamide (Diamox), Methazolamide (Neptazane), Dorzolamide HCl/timolol maleate (Cosopt), Latanoprost (Xalatan), Bimatoprost (Lumigan), Travoprost ophthamic solution (Travatan), Unoprostone (Rescula), Pilocarpine (Pilocar, Pilagan, Pilogel, Ocusert),

Isosorbide (Ismotic), Mannitol (Osmitrol, Resectisol), Glycerin (Ophthalgan, Osmoglyn), Brimonidine/timolol (Combigan), Phenylephrine HCl (Neo-Synephrine), Prednisolone acetate (AK-Pred, Pred Forte), Dexamethasone (Ocu-Dex), Ciprofloxacin (Ciloxan), Erythromycin (E-Mycin), Nepafenac ophthalmic (Nevanac), Verteporfm (Visudyne), Pegaptanib (Macugen), Ranibizumab (Lucentis), Vitamin A (Aquasol A, DeI-Vi-A), Vitamin E (Aquasol E, Vitec), Ascorbic acid (Cebid, Ascorbicap, Cevalin, Cecon), Lutein or Docket No. DUKE-30386/WO-l/ORD 41/66

Zeaxanthin, Bilberry, Beta-carotene, Diltiazem (Cardizem, Dilacor, Tiamate), Acetazolamide (Diamox, Diamox Sequels), and Methazolamide (Neptazane)). Additional treatments for other diseases of protein misfolding include, but are not limited to, hyperthermia.

EXPERIMENTAL

The following examples are provided to demonstrate and further illustrate certain preferred embodiments of the present invention and are not to be construed as limiting the scope thereof.

Example I.

This example describes the optimization of growth conditions for the DTY512 strain. As demonstrated in Figure 3, the growth conditions for ayhsfA strain harboring 1) a yeast HSF gene coupled with a GAL promoter and 2) a pRS424-GPD-hHSFl plasmid for hHSFl expression (the DTY512 strain) were optimized from petri dishes to 96 well microtiter dish format. Cells were grown in Synthetic Complete medium lacking uracil and tryptophan to select for plasmid maintenance, in the presence of the non-inducing/non-repressing carbon source raffϊnose (2%). Galactose concentrations (0.01%) were empirically identified that induce sufficient levels of yeast HSF for robust yeast cell viability, while rendering cells sensitive to strong glucose repression of yeast HSF expression after the addition of 4% glucose. The screen cells are grown to midlog phase in selective synthetic complete medium with 2% raffinose and 0.01% galactose. The culture was then diluted to -5,000 cells/ml in the same growth medium in which 4% glucose was substituted for the galactose, to initiate glucose repression of yeast HSF expression. Cells (200 microliters) were seeded into 96 well microtiter dishes at -1,000 cells/well and compound or carrier solvent (dimethylsulfoxide, DMSO) distributed independently to each well using a Beckman Biomek FX liquid handling robot under sterile conditions. Plates were incubated at 30⁰C and optical density measured over time using an attached SpectraMax Plus Plate Reader. . As shown in Figure 3, when grown in galactose, yHSFl is expressed allowing for cell growth. However, upon shifting the cells to a glucose containing media, expression of yHSFl is repressed, allowing only cells expressing the constitutively active hHSFllz4m allele to grow. Activation of hHSFl by small molecules will also allow cell growth. Yeast culture growth curves were generated for each Docket No. DUKE-30386/WO-l/ORD 4^66

microtiter well and slopes calculated over the course of 96 hours. As shown in Figure 4, the growth of cells was quantitatively followed in each microtiter well, the background growth of cells expressing wild type human HSFl with either no addition or DMSO alone is very low and allowed for facile qualitative detection of positive candidates in the screen.

Example II.

This example describes validation of candidate HSFl activating compounds. As shown in Figure 3 appropriate growth conditions, galactose induction and glucose repression parameters for application of the yeast-based human HSFl activator screen to a high throughput 96-well format was identified. This screen evaluated a combinatorial chemistry library of -10,500 diverse compounds built from approximately 75 unique scaffold structures in the PPD library (PPD Discovery Research, Research Triangle Park, NC). While this was a modest-sized screen, ~50 distinct library components were identified that allowed modest to robust yeast cell growth within 48-96 hours post-seeding at a concentration of 10 micromolar. Figure 4A shows a small sample of five compounds that stimulated yeast cell growth with different efficacy, the DMSO solvent control and one compound that was negative from this screen. Subsequent analysis of 32 of the more potent compounds demonstrated that the efficacy of all of these molecules in the yeast-based screen was dependent on human HSFl. The structural features of three unique compounds were evaluated, denoted HSFl-A, HSFl-B and HSFl-C, that were among the most effective in stimulating cell growth in the humanized yeast-based screen. Figure 4B shows two non-

Docket No. DUKE-30386/WO-l/ORD 43/66

functional derivatives (HSF1AD2

and HSFlAD3

that were incapable of stimulating yeast cell growth in comparison to HSFlA. Experiments conducted during the course of developing embodiments for the present invention determined that HSFlADl is a functional derivative Docket No. DUKE-30386/WO-l/ORD 44/66

of HSFlA (e.g., HSFlADl

is capable of stimulating yeast cell growth similar to HSFlA).

As shown in Figure 5 the three chemically distinct compounds HSFlA, HSFlB, and HSFlC exhibited common structural features including the conservation of aryl moieties typical of pyrazol benzamides, as well as polarized bonds to oxygen near the center of each molecule. Figure 6 shows synthetic routes for HSFlA, HSFlB, and HSFlC. Figure 7 shows additional compounds, including HSFl-A, HSFl-B, and HSF-IC, identified as HSFl activating compounds identified from the PPD library. It should be understood that derivates of these compounds may also be used in the compositions and methods described herein (see, e.g., HSFlADl).

Additional screens were conducted with compounds from the LOPAC chemical library and the Prestwick chemical library, which resulted in the identification of additional HSFl activating compounds. Figure 8 shows HSFl activating compounds identified thorugh screens conducted with compounds from the LOPAC and Prestwick chemical libraries. It should be understood that derivates of these compounds may also be used in the compositions and methods described herein.

Example III.

This example describes materials and methods for Examples IV-VII. Cell Culture. Wild-type and hsfl -/- mouse embryonic fibroblast (MEF) cells were grown in DMEM + 10%FBS (37⁰C, 5% CO₂). PC-12 cells were grown in DMEM + 5% FBS/10%horse serum (37⁰C, 10% CO₂). For MEF cells, at the time of the experiment 6X10⁵ Docket No. DUKE-30386/WO-l/ORD 45/66

cells were seeded into each well of a 6-well plate in serum containing media. The cells were incubated under those conditions for an additional 12 hr upon which time the cells were washed twice in IX PBS and the growth media was changed to OptiMEM media (Invitrogen) without serum. For PC- 12 cell, 5X10⁵ cells were seeded into each well of a 6-well plate, in 2 ml OptiMEM media without serum and incubated under those conditions for an additional 12 hrs. After 12 hr, HSFlA or DMSO was added to either the MEF or PC-12 cells and incubated for 15 hr at 37⁰C.

Western Blotting. After 15 hr treatment, the cells were washed twice in IX PBS and harvested by scraping. The cells were lysed using cell lysis buffer (25mM Tris, 15OmM NaCl, 1% Triton X-IOO, 0.1% SDS, ImM EDTA) and soluble proteins were quantified using BCA assay. Equal amounts of total protein was separated on 10%-20% gradient gel, transferred to a nitrocellulose membrane and analyzed for expression of HSP70, mHSP25, HSFl, Q74-GFP and SODl using anti-HSP70 (SC-24, Santa Cruz), anti-niHSP25 (SPA-801, Stressgen), anti-HSFl (Bethyl), anti-GFP (SC-8334) and anti-SODl (SOD-100, Stressgen) antibodies respectively. Secondary antibodies were either anti-mouse or anti-rabbit- HRP conjugated antibodies from GE Healthcare Life Sciences. Proteins were visualized using the Pico chemio luminescence kit (Pierce).

Example IV. This example demonstrates that HSFlA activates the expression of the HSP70 and

HSP25 heat shock protein (chaperone) genes in Mouse Embryonic Fibroblast (MEF) cells. As shown in Figure 9, MEF cells were treated with the indicated concentrations of HSFlA (in micromolar) or the DMSO solvent control in OptiMEM media without serum for 15 hours or heat shocked (HS) in OptiMEM media without serum for 2 hours at 42⁰C and allowed to recover for 15 hr at 37⁰C. Total cellular protein extracts were prepared, fractionated by SDS- polyacrylamide gel electrophoresis, transferred to a solid membrane and the Hsp70, Hsp25 and SODl proteins detected by probing with their respective specific antibodies followed by standard immunoblotting techniques to identify the proteins. Docket No. DUKE-30386/WO-l/ORD 46/66

Example V.

This example demonstrates HSFlA dependent activation of HSP70 is dependent on the presence of the gene encoding HSFl. As shown in Figure 10, wild-type and hsfl -/- MEF cells were treated with the indicated concentrations of HSFlA or the DMSO solvent as control in OptiMEM media without serum for 15 hr or heat shocked (HS) for 2 hr in

OptiMEM media without serum at 42⁰C and allowed to recover for 15 hr at 37⁰C. The Hsp70, HSFl and SODl proteins were detected by immunoblotting as described in Figure 9.

Example VI. This example demonstrates that HSFlA can act synergistically with heat shock to activate expression of the HSP70 protein chaperone. As shown in Figure 11, MEF cells were treated with the indicated concentrations of HSFlA or the solvent DMSO as control in OptiMEM media without serum for 15 hr at 37⁰C, or with 30 micromolar HSFlA for lhr at 37⁰C, followed by a 1 hr heat shock at a sub-optimal heat shock temperature of 4O⁰C followed by a 15 hr recovery at 37⁰C. Independently, cells were treated at the optimal heat shock temperature of 42⁰C for two hours followed by a 15 hr recovery. The HSP70 and SODl proteins were detected by immunoblotting as described in Figure 9. Note that 30 micromolar HSFlA treatment synergizes with a sub-optimal heat shock temperature to result in higher levels of expression of the HSP70 protein chaperone.

Example VII.

This example demonstrates that HSFlA promotes expression of HSP70 and reduces the aggregation of poly-glutamine (polyQ) proteins in rat neuronal precursor (PC-12) cells. As shown in Figure 12 A, PC-12 cells were treated with the indicated concentrations of HSFlA or the DMSO solvent as control in OptiMEM media without serum for 15 hr or heat shocked in OptiMEM media without serum for 2 hr at 42⁰C and allowed to recover for 15 hr at 37⁰C (HS). HSP70 and SODl proteins were detected by immunoblotting as described in Figure 9. Figure 12B shows PC-12 cells, stably expressing the Huntington Q74-GFP fusion protein were treated with 20 micromolar HSFlA or the solvent DMSO (0.5%) as control in OptiMEM media without serum for 15 hr to stimulate HSP70 expression. After 15 hr incubation, expression of the Q74-GFP protein was induced via the addition of 1 microgram Docket No. DUKE-30386/WO-l/ORD 47/66

per milliliter doxycyclin. The cells were incubated in the presence of doxycyclin and HSFlA for an additional 48 hrs after which the cells were harvested and soluble proteins (S) were isolated using cell lysis buffer. Insoluble proteins (P) were pelleted by centrifugation. The pellet was washed once in 1 milliliter cell lysis buffer and solubilized via a mixture urea buffer (cell lysis buffer + 5M Urea) and Laemmli buffer (1 :2 ratio). The polyglutamine-GFP protein (Q74-GFP) was detected by immunoblotting with anti-GFP antibody in a manner similar to that described in Figure 9.

Example VIII. An example of a neurodegenerative disease that results from protein misfolding is called Huntington's disease, a progressive neurodegenerative disease that leads to neuronal cell death. This disease is due to the aggregation of a protein called Huntingtin, where those patients with this disease gene express a Huntingtin protein that harbors a genetically inherited expansion of the amino acid glutamine (abbreviated Q). The polyQ Huntingtin protein mis-folds and aggregates, thereby causing neuron death and the disease symptoms. A fly model of polyQ protein-mediated neuronal degeneration has been used to test if HSFlA- mediated elevation in protein chaperone production can ameliorate eye cell degeneration in a fly in which polyQ protein is strongly expressed in the eye. Figure 13 shows that HSFlA functions to ameliorate eye degeneration in a fruit fly model of poly glutamine disease. As shown in Figure 13, UAS-M JDtrQ78 flies were crossed to gmr-GAL4 flies in the chronic presence of food supplemented with DMSO, 200 μM HSFlA or 5 μM 17-AAG (positive control). Reductions in eye morphological defects and de-pigmentation, caused by polyQ-protein expression, were observed with HSFlA and 17-AAG treatment. Control flies were UAS-MJDtrQ78 flies lacking the Gal4 transcription factor.

Example IX.

Many chemical activators of mammalian HSFl have, as their mechanism the binding and inhibition of the Hsp90 protein, a protein that represses HSFl activity. Figure 14 shows that the novel humanized yeast HSFl activation screen identifies molecules that act independently of Hsp90. As shown in Figure 14, HSFlA did not bind to the Hsp90 protein nor did it compete for binding with a known HSFl activating chemical, geldanamycin, that Docket No. DUKE-30386/WO-l/ORD 48/66

activates by binding and inhibiting Hsp90. These data distinguish HSFlA from other known HSFl activating molecules.

As shown in Figure 14 A, purified Hsp90 (AssayDesigns) was incubated with either 17- AAG, a known Hsp90 binding chemical, or HSFlA at the indicated concentration for 30 min at 4⁰C. 1 μM Geldanamycin, a known Hsp90 binding chemical, was coupled to the affinity matrix biotin (Geldanamycin-biotin) and was added for 1 hr at 4⁰C to displace 17- AAG and HSFlA from Hsp90. Geldanmycin-biotin bound Hsp90 was captured by the addition of neutravidin-agarose beads (Pierce) at 4⁰C for 30 min. Hsp90 was eluted from beads by heating to 95⁰C for 5 min and analyzed by immunob lotting utilizing and anti-Hsp90 antibody. As shown in Figure 14B, purified Hsp90 was incubated with either 10 μM

Geldanamycin-biotin (GD-B) or 100 μM HSFlA-biotin for 60 min at 4⁰C and bound Hsp90α was captured by the addition of neutravidin-agarose beads at 4⁰C for 30 min.

All publications and patents mentioned in the above specification are herein incorporated by reference for all purposes. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims

Docket No. DUKE-30386/WO-l/ORD 49/66

We claim:

A composition comprising a compound capable of facilitating HSFl activation, wherein said compound is described by the following formula:

including salts, esters, and prodrugs thereof; including both R and S enantiomeric forms and racemic mixtures thereof;

wherein Ri is selected from the group consisting of

, -OH, H, and halogen (e.g., Chlorine);

wherein Xi is

, or Xi is absent; wherein X₂ is S or C; wherein X3 is S or C; Docket No. DUKE-30386/WO-l/ORD 50/66

wherein

and

wherein R₅ is selected from the group consisting of S and C; wherein R₆ is selected from the group consisting of H and O ; wherein R₇ is selected from the group consisting of H and O ; Docket No. DUKE-30386/WO-l/ORD 51/66

wherein R₈ is selected from the group consisting of

and

wherein Rio is selected from the group consisting of Chlorine and CH3; and

wherein Rn is selected from the group consisting

and

2. The composition of claim 1, wherein said compound is selected from the group consisting of:

Docket No. DUKE-30386/WO-l/ORD 5-V66

Docket No. DUKE-30386/WO-l/ORD 53/66

Docket No. DUKE-30386/WO-l/ORD 54/66

3. A method for treating a condition associated with misfolded proteins, comprising: a) providing a subject having irregular HSFl activity, and a composition comprising a compound described by the following

formula:

including salts, esters, and prodrugs thereof; including both R and S enantiomeric forms and racemic mixtures thereof;

wherein Ri is selected from the group consisting of

^" , -OH, H, and halogen (e.g., Chlorine);

wherein Xi is

, or Xi is absent; wherein X₂ is S or C; wherein X₃ is S or C; Docket No. DUKE-30386/WO-l/ORD 55/66

wherein

and

wherein R₅ is selected from the group consisting of S and C; wherein R₆ is selected from the group consisting of H and O ; wherein R₇ is selected from the group consisting of H and O ; Docket No. DUKE-30386/WO-l/ORD 56/66

wherein R₈ is selected from the group consisting

wherein Rio is selected from the group consisting of Chlorine and CH3; and

wherein Rn is selected from the group consisting of

and

b) administering to said subject said composition.

4. The method of claim 3, wherein said compound is capable of inducing HSFl activation.

5. The method of claim 3, wherein said condition associated with protein misfolding is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington disease, Amyotrophic Lateral Sclerosis, a prion-based disease, type 2 diabetes mellitus, cataract and age-related cataract, glaucoma, macular degeneration and age-related macular degeneration, cardiovascular disease and stroke, heat stroke, spinocerebellar ataxias, Machado Joseph disease, stress-related neuronal degeneration, aging and cancer, and retinitis pigmentosa. Docket No. DUKE-30386/WO-l/ORD 57/66

6. The method of claim 3, wherein said composition is co-administered with one or more therapeutic agents selected from the group consisting of anticonvulsant agents, antipsychotic agents, rauwolfϊa alkaloids, antidepressants, dopamine prodrugs, dopamine agonists, catechol-O-methyltransferase (COMT) inhibitors, anticholinergics, MAO-B inhibitors, N- methyl-D-aspartic acid inhibitors, AChE inhibitors, NMDA antagonists, free-radical scavengers, glutamate pathway antagonists, antispastic agents, Congo red and its analogs, anthracyclines, amphotericin B and its analogs, sulfated polyanions, tetrapyrroles, sulfonylurea agents, meglitinides, biguanides, thiazolidinediones, dipeptidyl peptidase IV (DPP-4) inhibitors, incretin mimetics, amylin analogs, alpha-glucosidase inhibitors, Levobunolol (Betagan), Timolol maleate/hemihydrate (Timoptic Timoptic XE, Betimol, Istalol), Carteolol (Cartrol, Ocupress), Betaxolol (Betoptic-S), Metipranolol hydrochloride (OptiPranolol), Levobetaxolol (Betaxon), Brimonidine (Alphagan-P), Apraclonidine (Iopidine), Dipivefrin (AKPro, Propine), Epinephrine (Epifrin), Memantine (Namenda, Axura), Dorzolamide HCl (Trusopt), Brinzolamide (Azopt), Acetazolamide (Diamox), Methazolamide (Neptazane), Dorzolamide HCl/timolol maleate (Cosopt), Latanoprost

(Xalatan), Bimatoprost (Lumigan), Travoprost ophthamic solution (Travatan), Unoprostone (Rescula), Pilocarpine (Pilocar, Pilagan, Pilogel, Ocusert), Isosorbide (Ismotic), Mannitol (Osmitrol, Resectisol), Glycerin (Ophthalgan, Osmoglyn), Brimonidine/timolol (Combigan), Phenylephrine HCl (Neo-Synephrine), Prednisolone acetate (AK-Pred, Pred Forte), Dexamethasone (Ocu-Dex), Ciprofloxacin (Ciloxan), Erythromycin (E-Mycin), Nepafenac ophthalmic (Nevanac), Verteporfm (Visudyne), Pegaptanib (Macugen), Ranibizumab (Lucentis), Vitamin A (Aquasol A, DeI-Vi-A), Vitamin E (Aquasol E, Vitec), Ascorbic acid (Cebid, Ascorbicap, Cevalin, Cecon), Lutein or Zeaxanthin, Bilberry, Beta-carotene, Diltiazem (Cardizem, Dilacor, Tiamate), Acetazolamide (Diamox, Diamox Sequels), and Methazolamide (Neptazane)).

7. The method of claim 3, wherein said subject is selected from the group consisting of a human being, a mouse, a rat, a cat, a dog, a monkey, and an ape. Docket No. DUKE-30386/WO-l/ORD 58/66

8. A method for identifying HSFl activating agents, comprising: a) providing a yeast strain expressing human HSF, wherein said yeast strain comprises a yeast HSF gene coupled with an inducibile-repressible promoter; b) growing said yeast strain on an inducing medium; c) exposing said yeast strain to a candidate compound; d) switching said yeast strain to a repressing growth medium; e) assessing the growth of said yeast strain; and f) characterizing said candidate compound as a human HSFl activating agent if said yeast strain grows on said non-inducing medium.

9. The method of claim 8, wherein said human HSF is expressed via a pRS424-GPD- hHSFl plasmid.

10. The method of claim 8, wherein said inducing medium is a galactose medium, wherein said repressing medium is a glucose medium.

11. A method for treating a condition associated with protein misfolding, comprising: a) providing a subject having misfolded proteins, and a composition comprising a therapeutic agent that promotes HSFl activation; and b) administering to said subject said composition.

12. The method of claim 11 , wherein said condition associated with misfolded proteins is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington disease, Amyotrophic Lateral Sclerosis, a prion-based disease, type 2 diabetes mellitus, cataract and age-related cataract, glaucoma, macular degeneration and age-related macular degeneration, cardiovascular disease and stroke, heat stroke, spinocerebellar ataxias, Machado Joseph disease, stress-related neuronal degeneration, aging and cancer, and retinitis pigmentosa. Docket No. DUKE-30386/WO-l/ORD 59/66

13. The method of claim 11 , wherein said composition is co-administered with one or more therapeutic agents selected from the group consisting of anticonvulsant agents, antipsychotic agents, rauwolfia alkaloids, antidepressants, dopamine prodrugs, dopamine agonists, catechol-O-methyltransferase (COMT) inhibitors, anticholinergics, MAO-B inhibitors, N-methyl-D-aspartic acid inhibitors, AChE inhibitors, NMDA antagonists, free- radical scavengers, glutamate pathway antagonists, antispastic agents, Congo red and its analogs, anthracyclines, amphotericin B and its analogs, sulfated polyanions, tetrapyrroles, sulfonylurea agents, meglitinides, biguanides, thiazolidinediones, dipeptidyl peptidase IV (DPP-4) inhibitors, incretin mimetics, amylin analogs, alpha-glucosidase inhibitors, Levobunolol (Betagan), Timolol maleate/hemihydrate (Timoptic Timoptic XE, Betimol, Istalol), Carteolol (Cartrol, Ocupress), Betaxolol (Betoptic-S), Metipranolol hydrochloride (OptiPranolol), Levobetaxolol (Betaxon), Brimonidine (Alphagan-P), Apraclonidine (Iopidine), Dipivefrin (AKPro, Propine), Epinephrine (Epifrin), Memantine (Namenda, Axura), Dorzolamide HCl (Trusopt), Brinzolamide (Azopt), Acetazolamide (Diamox), Methazolamide (Neptazane), Dorzolamide HCl/timolol maleate (Cosopt), Latanoprost

(Xalatan), Bimatoprost (Lumigan), Travoprost ophthamic solution (Travatan), Unoprostone (Rescula), Pilocarpine (Pilocar, Pilagan, Pilogel, Ocusert), Isosorbide (Ismotic), Mannitol (Osmitrol, Resectisol), Glycerin (Ophthalgan, Osmoglyn), Brimonidine/timolol (Combigan), Phenylephrine HCl (Neo-Synephrine), Prednisolone acetate (AK-Pred, Pred Forte), Dexamethasone (Ocu-Dex), Ciprofloxacin (Ciloxan), Erythromycin (E-Mycin), Nepafenac ophthalmic (Nevanac), Verteporfm (Visudyne), Pegaptanib (Macugen), Ranibizumab (Lucentis), Vitamin A (Aquasol A, DeI-Vi-A), Vitamin E (Aquasol E, Vitec), Ascorbic acid (Cebid, Ascorbicap, Cevalin, Cecon), Lutein or Zeaxanthin, Bilberry, Beta-carotene, Diltiazem (Cardizem, Dilacor, Tiamate), Acetazolamide (Diamox, Diamox Sequels), and Methazolamide (Neptazane))

14. The method of claim 11 , wherein said subject is selected from the group consisting of a human being, a mouse, a rat, a cat, a dog, a monkey, and an ape. Docket No. DUKE-30386/WO-l/ORD 60/66

15. The method of claim 11 , wherein said therapeutic agent comprises a compound

described by the following formula:

including salts, esters, and prodrugs thereof; including both R and S enantiomeric forms and racemic mixtures thereof;

wherein Ri is selected from the group consisting of

-OH, H, and halogen (e.g., Chlorine);

wherein Xi is

, or Xi is absent; wherein X₂ is S or C; wherein X3 is S or C;

Docket No. DUKE-30386/WO-l/ORD 61/66

wherein

and

wherein R₅ is selected from the group consisting of S and C; wherein R₆ is selected from the group consisting of H and O ; wherein R₇ is selected from the group consisting of H and O ; Docket No. DUKE-30386/WO-l/ORD 6-V66

wherein R₈ is selected from the group consisting of

and

1

wherein Rio is selected from the group consisting of Chlorine and CH3; and wherein Rn is selected from the group consisting

and

Docket No. DUKE-30386/WO-l/ORD 63/66

16. The method of claim 11, wherein said therapeutic agent comprises a compound selected from the group consisting of:

Docket No. DUKE-30386/WO-l/ORD 64/66

Docket No. DUKE-30386/WO-l/ORD 65/66