WO2008122038A1 - Regulating autophagy - Google Patents

Abstract

Autophagy is a cellular process by which cells canabalize non-essential cellular elements such as organelles to generate metabolites, or in some cases, to cause cell death. The present invention provides modulators of autophagy, which have been identified using a high-throughput phenotypic screen of over 3500 compounds. These modulators are useful in treating diseases ranging from proliferative diseases to neurodegenerative diseases to infectious diseases to protein misfolding states. Furthermore, the invention provides the treatment of proliferative disease such as cancer with a combination of autophagy inhibitors and protein kinase inhibitors.

Description

REGULATING AUTOPHAGY

Related Applications

[0001] The present invention claims priority under 35 U.S. C. § 119(e) to U.S. provisional patent application, USSN 60/909,640, filed April 2, 2007, which is incorporated herein by reference.

Government Support

[0002] This invention was made with U.S. Government support under contract number NOl -CO- 12400 awarded by the National Cancer Institute's Initiative for Chemical Genetics, and under grant number GM3862-7 awarded by the National Institute of General Medicine Sciences. The U.S. government may have certain rights in the invention.

Background of the Invention

[0003] The autophagy-lysosome and ubiquitin-proteasome pathways are the two major routes for protein and organelle clearance in eukaryotic cells. Proteasomes predominantly degrade short-lived nuclear and cytosolic proteins, which need to be unfolded to pass through the narrow pore of the proteasome barrel, precluding clearance of large membrane proteins and protein complexes (including oligomers and aggregates). Mammalian lysosomes, on the other hand, can degrade substrates like protein complexes and organelles. The bulk degradation of cytoplasmic proteins or organelles is largely mediated by macroautophagy, generally referred to as autophagy. Klionsky et al. "Autophagy as a regulated pathway of cellular degradation" Science 290:1717-20, 2000; incorporated herein by reference. [0004] Autophagy is the process by which cells canabalize cellular elements (e.g., proteins, organelles). Autophagy is the cell's major regulated mechanism for degrading long- lived proteins and the only known pathway for degrading organelles. Levine et ah, J. Clin. Invest. 115(10):2679-2688, October 2005; incorporated herein by reference. Autophagy occurs at a low basal levels in all cells due to cytoplasmic and organelle turnover. Autophagy is then upregulated when cells need to generate intracellular metabolites (e.g., during starvation or trophic factor withdrawal), to undergo architectural remodeling (e.g., during development), or to eliminate damaging cytoplasmic components (e.g., during oxidative stress, infection, accumulation of protein aggregates).

[0005] Autophagy involves the formation of double-membrance structures called autophagosomes/autophagic vacuoles (AVs), which fuse with lysosomes to form autolysosomes (also called autophagolysosomes) where their contents are then degraded by acidic lysosomal hydrolases. Autophagosomes are generated by elongation of small membrane structures known as autophagosome precursors.

[0006] Many external factors including hormones, growth factors, nutrients, oxygen concentration, temperature, cell density, etc. play a role in regulating autophagy. Several protein kinases regulate autophagy, the best characterised being the target of rapamycin (TOR) kinase. The target of rapamycin (TOR) kinase negatively regulates the autophagy pathway in organisms from yeast to man (for a review, see Lum et al., Nat. Rev. MoI. Cell Biol. 6:439-448, 2005; incorporated herein by reference). Downstream of TOR kinase, there are approximately seventeen gene products essential for autophagy and related pathways in yeast. Most of these genes have orthologs in higher eukaryotes (for a review, see Levine et al., Dev. Cell. 6:463-477, 2004; incorporated herein by reference). The TOR proteins constitute the central node of a nutrient-response signaling network controlling cell growth and cell size in eukaryotes. Jacinto et al. Nat. Rev. MoI. Cell. Biol. 4: 117-126, 2003; Wullshleger et al. Cell 124:471-484, 2006; each of which is incorporated herein by reference. However, TOR-independent signalling in the autophagy aparatus has also been described, for example, a TOR-dependent pathway where autophagy is induced by agents that lower inositol or inositol- 1,4,5-triphosphate (IP3) levels. Sarkar et al. "Lithium induces autophagy by inhibiting inositol monophosphatase" J. Cell Biol. 170: 1101-11, 1005; which is incorporated herein by reference.

[0007] Given the importance of autophagy in cell survival and cell death, the modulation of autophagy and/or the TOR pathway may be useful in treating diseases such as cancer, proliferative diseases, protein misfolding disorders, infectious diseases, and neurodegenerative diseases.

Summary of the Invention

[0008] The present invention stems from the recognition that modulators of autophagy may be useful in the treatment and/or prevention of a variety of diseases. Based on this discovery, the invention provides agents, particularly small molecules, that modulate autophagy. The agents may act by either inhibiting or promoting autophagy in a cell. That is, any agent that modulates the autophagy-lysosome pathway in a cell may be used in the treatment and/or prevention of disease. In certain embodiments, the agents may be used to treat diseases associated with autophagy such as cancer (e.g., leukemia, multiple myeloma), proliferative diseases, inflammatory diseases, autoimmune diseases, cardiovascular diseases (e.g., reperfusion injury, ischemic cardiac disease), infectious diseases (e.g., viral infections, bacterial infections), neurodegenerative diseases (e.g., Huntington's disease, Alzheimer's disease), and protein folding disorders (e.g., Alzheimer's disease, cystic fibrosis). The invention provides particular exemplary modulators of autophagy which were discovered based on a forward genetics approach. Using high-content, high-throughput, image-based screens, over 3,500 compounds were profiled for their ability to induce the phenotypic characteristics associated with autophagy (e.g., the accumulation of EGFP-LC3 positive autophagosomes in the cytosol). Although the mechanisms of action of most of these newly identified modulators of autophagy are unknown, at least some of them may modulate autophagy by affecting acetylation/deacetylation activity in the cell, such as histone deacetylase activity (e.g., HDAC6 inhibition). Therefore, in certain embodiments, the modulators of autophagy are also modulators of acetylation or deacetylation activity in the cell (e.g., HDAC inhibitors, tubulin deacetylase (TDAC) inhibitors). In certain embodiments, the modulators of autophagy act by affecting another target besides HDAC6. See U.S. Patent Application, USSN 11/386,959, filed March 22, 2006, which publised as US2006/0239909, on October 26, 2006, which is incorporated herein by reference.

[0009] Prior to the instant discovery, there were few known modulators of autophagy.

For example, chloroquine and methyladenine were known to inhibit autophagy, and rapamycin and lithium were known to induce or promote autophagy. The newly identified modulators of autophagy are useful for scientific investigations as wells as for therapeutic applications. For example, the identified modulators of autophagy may be used to design even better modulators of autophagy or to better understand the autophagy-lyosome pathway in cells. Also, the identified agents or derivatives thereof may be formulated for administration to a subject (e.g., a human) for the treatment of a disease. Several of the identified autophagy modulators (e.g. , fluoxetine, loperamide, doxorubicin, tamoxifen) are known drugs already approved for use in humans. In certain embodiments, the identifed agents have been approved by the U.S. Food and Drug Administration. The invention provides pharmaceutical compositions of the identified compounds and methods of using the identified compounds to treat diseases such as, proliferative diseases such as cancer, inflammatory diseases, autoimmune diseases, neurodegenerative diseases (e.g., Alzheimer's Disease, Parkinson's Disease), infectious diseases, cardiovascular diseases, and diseases caused by protein misfolding and/or mishandling. In certain embodiments, inhibitors of autophagy may be used to treat proliferative diseases such as cancer. In certain other embodiments, promoters of autophagy may be used to treat neurodegenerative disorders (e.g., Alzheimer's Disease), infectious diseases (e.g., bacterial or viral infections), or protein folding disorders.

[0010] The identified modulators of autophagy may also be combined with other pharmaceutical agents to provide combination therapies. The inhibition or promotion of autophagy may be combined with proteasome inhibition, kinase inhibition (e.g., receptor tyrosine kinase inhibition), growth factor pathway inhibition, or the inhibition of other cellular pathways. In certain embodiments, an autophagy modulator is used in combination with a proteasome inhibitor such as bortezomib in the treatment of cancer or other proliferative diseases. In other embodiments, an autophagy modulator is used in combination with a protein kinase inhibitor in the treatment of cancer or other proliferative diseases. In certain other embodiments, an autophagy modulator is used in combination with a growth factor pathway inhibitor in the treatment of cancer or other proliferative diseases. In certain embodiments, an autophagy modulator is used in combination with a therapeutic agent used to treat subjects with a neurodegenerative disease (e.g., acetylcholinesterase inhibitors, neurotransmitter agonists or antagonists). The agents of the combination therapy may be administered in combination or more likely separately. The invention not only provides methods of treating diseases with the inventive combinations but also compositions and kits that include the inventive combination of agents, that is, a modulator of autophagy and another agent.

[0011] In one particular aspect, the invention provides a novel approach to the treatment of cancer or other proliferative diseases. After growth factor withdrawal, cancer cells in tissue culture have been found to undergo autophagy to remain alive. Based on this finding, the inhibition of growth factor pathways would induce a comparable survival stimulus. Therefore, the inhibition of autophagy and the inhibition of growth factor pathways would provide a synergistic toxicity. In the present invention, an inhibitor of autophagy and an inhibitor of a growth factor pathway (e.g., a kinase inhibitor) are administered in combination to a subject with cancer or other proliferative disease. The invention provides methods of treatment using combinations including an inhibitor of autophagy and an inhibitor of a growth factor pathway. The inhibitors of growth factor pathways include kinase inhibitors such as erlotinib (TARCEV A^®), gefitinib (IRESSA^®), cetuximab, sorafenib (NEXAVAR), dasatinib, ZD6474 (ZACTIMA), lapatinib (TYKERB), STI571, imatinib (GLEEVEC), lestaurtinib (CEP-701), sunitinib maleate (SUTENT), panitumumab, EMD 72000, TheraCIM hR3, EKB-569, 2C4, AMG706, MP -412, XL647, XL 999, MLN518, PKC412, AMN107, AEE708, OSI-930, OSI-817, and AG-013736. The invention also provides pharmaceutical compositions and kits including a combination of an autophagy inhibitor and a kinase inhibitor.

[0012] In another aspect, the invention provides a novel approach to the treatment of cancer or other proliferative diseases using an autophagy modulator and a proteasome inhibitor. In certain embodiments, the autophagy modulator used in combination with a proteasome inhibitor is not an HDAC inhibitor. In certain particular embodiments, the autophagy modulator used in combination with a proteasome inhibitor is not an HDAC6 inhibitor. An inhibitor of autophagy is used in combination with a proteasome inhibitor to treat a subject with cancer or other proliferative disease. Exemplary proteasome inhibitors that may be used in combination with an autophagy modulator include, but are not limited to, bortezomib (VELCADE^®), peptide boronates, salinosporamide A (NPI-0052), lactacystin, epoxomicin (Ac(Me)-Ile-Ile-Thr-Leu-EX), MG- 132 (Z-Leu-Leu-Leu-al), PR- 171, PS-519, eponemycin, aclacinomycin A, CEP-1612, CVT-63417, PS-341 (pyrazylcarbonyl-Phe-Leu- boronate), PSI (Z-Ile-Glu(OtBu)-Ala-Leu-al), MG-262 (Z-Leu-Leu-Leu-bor), PS-273 (MNLB), omuralide (c/αsto-lactacystin-β-lactone), NLVS (Nip-Leu-Leu-Leu-vinyl sulfone), YLVS (Tyr-Leu-Leu-Leu-vs), dihydroeponemycin, DFLB (dansyl-Phe-Leu-boronate), ALLN (Ac-Leu-Leu-Nle-al), 3,4-dichloroisocoumarin, 4-(2-aminoethyl)-benzenesulfonyl fluoride, TMC-95A, gliotoxin, EGCG ((-)-epigallocatechin-3-gallate), and YUlOl (Ac- hFLFL-ex). The invention also provides pharmaceutical compositions and kits including a combination of an autophagy inhibitor and a proteasome inhibitor.

[0013] In another aspect, promoters of autophagy are used to treat certain diseases.

As shown herein, promoters of autophagy may be used to clear aggregate-prone, disease- causing proteins. Such proteins that have been shown to be cleared by autophagy include forms of tau (which have been shown to cause fronto-temporal dementia), proteins that cause spinocerebellar ataxia type 3, A53T and A30P α-synuclein (which have been shown to cause familial Parkinson's Disease), and mutant huntingtin (full length or exon 1) (which have been shown to cause Huntington's disease). Therefore, promoters of autophagy such as those identified herein may be administered to a subject (e.g., human) with a neurodegenerative disease, or at risk for developing a neurodegenerative disease, to promote autophagy and thereby increase the clearance of disease-causing proteins. Other diseases that may be treated using autophagy promoters include certain cardiac disease (e.g., ischemia/reperfusion injury) and infectious diseases. Infections wherein pathogens or pathogen proteins are degraded by autophagosomes and transferred to lysosomes for degradation are susceptible to treatment with promoters of autophagy. For example, tuberculosis, Group A Streptococcus infections, and viral infections (e.g., herpes simples virus type I) may be treated with promoters of autophagy.

[0014] The invention also provides agents that modulate the biological activity (e.g., the cytoxicity) of rapamycin, which is a known inducer of autophagy. Several of the enhancers and inhibitors of rapamycin' s activity are also inhibitors and enhancers of autophagy as described herein. These agents also have therapeutic as well as scientific applications. These agents may be used to design better modulators of the activity of rapamycin or other modulators of autophagy. In certain embodiments, these agents are administered in combination with rapamycin or other autophagy modulator such as those described herein.

[0015] In another aspect, the invention provides a system for identifying modulators of autophagy. In a certain embodiment, cells are cultured and treated with a test agent under particular conditions suitable for identifying inhibitors or promoters of autophagy. After a suitable length of time, the cells are analysed to look for the phenotypic characteristics associated with cells undergoing autophagy. The phenotypic characteristic may include formatin of EGFP-LC3 positive puncta. Inducers of autophagy such as rapamycin exhibit an increased number of puncta per cell. Modulators of autophagy may also be identified by increased average vesicle area. Other phenotypic changes that may be assessed include size of autophagosomes, number of autophagosomes, lysosome or autolysosome formation, rearrangement of subcellular membranes, and formation of intracellular vesicles. Other indicia of autophagy may also be used to identify modulators of autophagy.

Brief Description of the Drawing

[0016] Figure 1 shows the autophagy pathway and its role in cellular adaptation to nutrient deprivation (from Levine & Yuan, J. Clin. Invest. 115:2679-2688, 2005, incorporated herein by reference). Starvations or growth factor deprivation results in a decrease in intracellular nutrients and activation of nutrient-sensing signaling pathways that stimulate autophagy.

[0017] Figure 2 shows an exemplary high throughput, high content screen for small molecule modulators of autophagy based on EGFP-LC3 positive puncta. [0018] Figure 3 shows the development of the assay for identifying modulators of autophagy. Cells expressing EGFP-LC3 (green) are stained with Hoechst stain (blue). Automated detection of nuclei and vesicles results in a table of data with various phenotypic parameters measured for each cell in the sample. In the table, one row represent a cell. [0019] Figure 4 shows a negative control (DMSO) and a positive control (rapamycin,

2 μM) using the phenotypic -based screen.

[0020] Figure 5 is graph of percentage of cells with a greater than n number of puncta per cell. Data are shown for DMSO (negative control) and four concentrations of rapamycin. [0021] Figure 6 shows a scatter plot of percentage of cells with 7 or more puncta versus average vesicle size. The points corresponding to various modulators of autophagy are identified.

[0022] Figure 7 shows the well distibutions for DMSO (negative control) and rapamycin (positive control) versus percent of cells with greater than seven puncta. [0023] Figure 8A shows a scatter plot of percentage of cells with 7 or more puncta versus cell count from screening of the Kendall Bioactive Collection. Various modulators of autophagy are identified. Figure 8B shows a scatter plot of percentage of cells with 7 or more puncta versus average vesicle size from screening of the Prestwick Collection. [0024] Figure 9 shows a plot for vesicle average area versus concentration of chloroquine, rapamycin, and DMSO. [0025] Figure 10 are photographs of cells treated with bafilomycin A, carnitine, trimethobenzamide, and monensin.

[0026] Figure 11 is the single agent toxicity of IRESSA in lung cancer.

[0027] Figure 12 shows the increased toxicity of IRESSA in combination with chloroquine.

[0028] Figure 13 demonstrates the synergistic effect of combining chloroquine (a modulator of autophagy) and IRESSA (a kinase inhibitor).

[0029] Figure 14 shows the synergistic effect of combining IRESSA with a small molecule inhibitor of rapamycin (SMIR 20).

[0030] Figure 15 shows the synergistic effect of combining GLEEVEC with an autophagy inhibitor such as chloroquine, and combining GLEEVEC with SMIR 20. [0031] Figure 16 shows the results of a small molecule screen for suppressors and enhancers of the cytostatic effects of rapamycin in the BY4742 strain. (A) Of 50,729 compounds screened in duplicate, 52 (0.001%) suppresors and 20 (0.0004%) enhancers were initially indentified, of which 21 suppressors and 12 enhancers were retested. (In cases where multiple structural analogs scored as primary assay positives, a single representative was chosen; exceptions are compounds with a lower-case letter in their name, e.g., SMIRl 9a). 427 candidate enhancers were subsequently found to be growth-inhibitory as single agents, and were eliminated from further consideration. (B) Table summarizing EC50 values (listed in descending order of potency) of 21 suppressors of rapamycin (SMIRs) (shown in green) and 12 enhancers of rapamycin (SMERs) (shown in red). Concentrations are listed in micromolar (μM). The EC50 of suppression was determined in 50 nM rapamycin; the EC50 of enhancement was determined in 20 nM rapamycin. EC50 values of asterisked compounds were determined in synthetic media; all other EC50 values were determined in rich media.

[0032] Figure 17 shows the potency and selectivity of 33 small-molecule modifiers of the cytostatic effects of rapamycin (rows) against a panel of 6 assay compounds (columns). Two-dimensional (2D-) heatmaps display negative log-transformed (green) and positive log-transformed (red) EC50 values derived from averaged duplicate OD₆oo absorbance measurements of a 2-fold dilution series of SMIRs (data shown in A) and of SMERs (data shown in B) treated with either 50 nM (used in A) and 20 nM (used in B) rapamycin or 555 nM cycloheximide (CHX) or 18.9 μM anisomycin or 16.6 μM nocodazole or 595 nM tunicamycin or 29 μM (used in A) and 14.5 μM (used in B) menadione. Black indicates no interaction between small-molecule modifiers and assay compounds; intense green corresponds to low half-maximal suppression; intense red corresponds to low half-maximal enhancement.

[0033] Figure 18 shows that SMERs 10, 18, and 28 enhance the clearance of mutant aggregate-prone proteins by autophagy in mammalian cell models of Huntington's and Parkinson's disease, independent of rapamycin. A. Chemical structures of SMERs 10, 18 and 28. B. A stable inducible PC12 cell line expressing A53T α-synuclein was induced with doxycycline for 48h, and expression of the trans gene was switched off for 24 hours, with DMSO (control), 47 μM SMERlO, 43 μM SMER18 or 47 μM SMER28 added in the switch- off period. Levels of A53T α-synuclein (α-syn) was analysed by immunoblotting with antibody against HA (i) and densitometry analysis relative to actin (ii). All the SMERs were used in the cell culture media at 1 :400 dilution of 5mg/ml stock solution (in DMSO). /Kθ.0001 (all SMERs). C. COS-7 cells transfected with EGFP-HDQ74 for 4h were treated with DMSO (control), 0.2 μM rapamycin (rap), 47 μM SMERlO, 43 μM SMERl 8 or 47 μM SMER28 for 48 hours. The effects of treatment on the percentage of EGFP-positive cells with EGFP-HDQ74 aggregates (i) or apoptotic morphology (cell death) (ii) were expressed as odds ratios and the control was taken as l. /?<0.0001 (rap and SMER28),p=0.013 (SMERlO), 77=0.019 (SMER18) (i);/><0.0001 (rap, SMER18 and SMER28), _jp=0.004 (SMERlO) (ii). Odds ratios were used to determine pooled estimates for the changes in aggregate formation or cell death, resulting from perturbations assessed in multiple independent experiments (Sarkar et al. Lithium induces autophagy by inhibiting inositol monophosphatase. J. Cell Biol. 170, 1101-11 (2005); Ravikumar et al. Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy. Hum. MoI. Genet. 11, 1107-17 (2002); Wyttenbach et al. Polyglutamine expansions cause decreased CREmediated transcription and early gene expression changes prior to cell death in an inducible cell model of Huntington's disease. Hum MoI. Genet. 10, 1829-45 (2001); each of which is incorporated herein by reference), as the effect of a perturbation tends to remain consistent in multiple experiments even if the basal levels of aggregation vary (e.g., due to cell confluency, which may vary slightly between experiments affecting transfection efficiency). D, E. Wild-type (Atg5^+/+; d) and knock-out (Atg5 ^; e) Atg5 mouse embryonic fibroblasts (MEFs) were transfected with EGFP-HDQ74 for 4h and treated with DMSO (control), 47 μM SMERlO, 43 μM SMERl 8 or 4 7μM SMER28 for 24 hours. The effects of treatment on the percentage of EGFP-positive cells with EGFP-HDQ74 aggregates were expressed as odds ratios and the control was taken as 1. ^=0.039 (SMERlO), /XO.OOOl (SMERl 8 and SMER28) (d); p=0.092 (SMERlO), ^=0.271 (SMER18), p=0.358 (SMER28) (e). F. COS-7 cells transfected with EGFP-LC3 construct for 4h were treated with DMSO (control), 0.2 μM rapamycin (rap), 47 μM SMERlO, 43 μM SMERl 8 or 47 μM SMER28 for 16h, and analysed by fluorescence microscopy. The effects of treatment on the percentage of EGFP-positive cells with >5 EGFP-LC3 vesicles are shown. pO.OOO 1 (all SMERs). G. HeLa cells stably expressing EGFP-LC3 were treated with DMSO (control), 47 μM SMERlO, 43 μM SMER 18 or 47 μM SMER28 for 24 hours. Confocal sections show cells containing EGFP-positive autophagic vesicles. Nuclei are stained with DAPI. Bar, 20 μM. H. HeLa cells stably expressing EGFP-LC3 were treated for 4 h with DMSO (control) or 200 nM bafilomycin Al (baf), or with 200 nM bafilomycin Al and 47 μM SMERlO, 43 μM SMER18 or 47 μM SMER28. Cells were left untreated or pre-treated with SMERs for 24 hours before adding bafilomycin Al. Levels of EGFP-LC3-II were determined by immunoblotting with antibody against EGFP (i) and densitometry analysis relative to actin (ii). /»=0.0259 (baf), /><0.0001 (SMERlO), ^=0.0003 (SMER18 and SMER28) vs control; ^=0.0025 (SMERlO), p=0.Q218 (SMERl 8), p=0.0195 (SMER28) vs bafilomycin Al. ***,p < 0.001; **, p < 0.01; *,p < 0.05; NS, Non-significant.

[0034] Figure 19 demonstrates that SMERs 10, 18 and 28 protect against neurodegeneration in Drosophila model of Huntingdon's disease. A-C. Flies treated with 100 μM SMERlO (a), 200 μM SMER18 (b) or 100 μM SMER28 (c) show a shift in the distribution of the number of rhabdomeres compared to flies treated with DMSO (control) alone (2 days after eclosion). Rhabdomere counts from all 3 independent experiments are included. «=600 ommatidia (SMERlO), «=1500 ommatidia (SMER18) and «=600 ommatidia (SMER28). Mann- Whitney test values /?<0.0001 (all SMERs). Student's Mest (1 tailed) ^=0.005 (SMERlO), ^=0.004 (SMERl 8), p=0.03 (SMER28) compared distributions of means of independent experiments. These SMER concentrations cause no overt toxicity to flies.

[0035] Figure 20 shows that rapamycin and SMERs have additive protective effects on the clearance and toxicity of mutant aggregate-prone proteins. A,B. COS-7 cells treated with DMSO (control), 47 μM SMERlO, 43 μM SMER18, 47 μM SMER28, or 0.2 μM rapamycin (rap) for 24 hours, were analysed for mTOR activity by immunoblotting for levels of phospho- and total p70S6K (a) and 4E-BP1 (b). Note that 4E-BP1 runs as a set of bands on gels, as phosphorylation slows its mobility-the bands with the slowest mobility are decreased with rapamycin. C. HeLa cells stably expressing Ub^G76V-EGFP reporter, treated with or without 10 μM lactacystin (lact), 47 μM SMERlO, 43 μM SMER18 or 47 μM SMER28 for 24 hours, were analysed for inhibition of proteasome activity by immunoblotting with antibody against EGFP. /7<0.0001 (lact), /7=0.873 (SMERlO), /7=0.8089 (SMERl 8), /?=0.8221 (SMER28). ***, p < 0.001; NS, Non-significant. [0036] Figure 21 further demonstrates that rapamycin and SMERs have additive protective effects on the clearance and toxicity of mutant aggregate-prone proteins. A-C. Clearance of A53T α-synuclein (α-syn) in stable PC 12 cells as in Figure 18b, treated with DMSO (control), or with 0.2 μM rapamycin alone, SMER alone [140 μM SMERlO (a), 43 μM SMERl 8 (b) or 47 μM SMER28 (c)] or both for 8 hours, was analysed by immunoblotting with antibody against HA (i) and densitometry analysis relative to actin (ii). The concentration of rapamycin is saturating for its effect on the clearance of A53T α- synuclein. /7=0.0025 (rap), /7=0.0018 (SMERlO), /7<0.0001 (SMER10+rap), /7<0.0001 (rap or SMERlO vs SMER10+rap) (a); /7=0.0069 (rap), /7=0.0498 (SMER18),/7<0.0001 (SMERl 8+rap), /7=0.0038 (rap vs SMERl 8+rap), /7=0.0007 (SMERl 8 vs SMERl 8+rap) (b); /XO.OOOl (rap, SMER28, rap vs SMER28+rap, SMER28 vs SMER28+rap) (c). D-F. The percentage of EGFP-positive cells with EGFP-HDQ74 aggregates (i) and cell death (ii) in COS-7 cells as in Figure 18c, treated with DMSO (control), or with 0.2 μM rapamycin alone, SMER alone [140 μM SMERlO (d), 43 μM SMER18 (e) or 47 μM SMER28 (f)] or both for 24h, were expressed as odds ratios, (i) For aggregation: /?=0.248 (rap), /7=0.217 (SMERlO), /><0.0001 (SMERl 0+rap),/7<0.001 (rap or SMERlO vs SMER10+rap) (d);/7=0.248 (rap), /7=0.543 (SMERl 8), /7<0.0001 (SMERl 8+rap), /7=0.008 (rap vs SMERl 8+rap), /7=0.002 (SMER18 vs SMER18+rap) (e);/7=0.248 (rap), /7=0.002 (SMER28),/7<0.0001 (SMER28+rap), /?<0.0001 (rap vs SMER28+rap), /7=0.012 (SMER28 vs SMER28+rap) (f). (ii) For cell death: /7=0.002 (rap),/7<0.0001 (SMERlO, SMER10+rap, rap or SMERlO vs SMER10+rap) (d); /7=0.002 (rap), /?=0.948 (SMER18),/7<0.0001 (SMERl 8+rap), /7=0.015 (rap vs SMERl 8+rap), /7<0.0001 (SMER18 vs SMER18+rap) (d);/7=0.002 (rap), /7<0.0001 (SMER28, SMER28+rap, rap or SMER28 vs SMER28+rap) (f). Note that we have treated cells for a shorter time in this experiment (24h), compared to Figure 18c (48 hours)-this probably accounts for the failure of the protective trends of rapamycin and some of the SMERs to reach significance for aggregation, on their own in this experiment. ***, p < 0.001; **, p < 0.01; *, p < 0.05; NS, Non-significant.

[0037] Figure 22 shows a screen of the chemical analogs of the autophagy- inducing

SMERs for their protective effects on the clearance and aggregation of mutant proteins. A-C. Clearance of A53T α-synuclein (α -syn) in stable PC12 cells as in Figure 18b, treated for 24 hours with either DMSO (control), or with 47 μM SMERlO and its analogs (SMERl 0a-c) (a), 43 μM SMERl 8 and its analogs (SMERl 8a-l) (b), or 47 μM SMER28 and its analogs (SMER28a-l) (c), was analysed by immunoblotting with anti-HA antibody (i) and densitometry analysis relative to actin (ii). All the analogs were used in the cell culture media at 1:400 dilution of 5mg/ml stock solution (in DMSO). /7=0.0058 (SMERlOa), /7=0.6736 (SMERlOb), /7=0.9507 (SMERlOc), /7=0.0481 (SMERlO) (a); /7=0.0006 (SMERl 8a), 77=0.0249 (SMERl 8b), />=0.0167 (SMER18c), /7=0.0117 (SMERl 8d), /7=0.0269 (SMER18e), /7=0.0165 (SMERl 8f), /7=0.0148 (SMERl 8g), /7=0.0011 (SMERl 8h), /7=0.7369 (SMER18i), 77=0.0012 (SMERl 8j), /7=0.1531 (SMERl 8k), /7=0.0006 (SMERl 81), /7=0.0001 (SMER18) (b);

(SMER28b), /7=0.0001 (SMER28d), /7=0.0048 (SMER28h), /7=0.0002 (SMER28i), /7=0.0162 (SMER28J), /7=0.0001 (SMER28k),/7<0.0001 (SMER28c, e-g, 1, SMER28) (c). D-F. The percentage of EGFP-positive cells with EGFP- HDQ74 aggregates in COS-7 cells as in Figure 18c, treated for 48 hours with either DMSO (control), or with 47 μM SMERlO and its analog (SMERlOa) (d), 43 μM SMERl 8 and its analogs (SMERl 8a, c-h, j, 1) (e), or 47 μM SMER28 and its analogs (SMER28a-l) (f), were expressed as odds ratios. All the analogs were used in the cell culture media at 1 :400 dilution of 5mg/ml stock solution (in DMSO). /7=0.003 (SMERlOa), /7=0.004 (SMERlO) (d); /7<0.0001 (SMER18a, c, d, f, h), /7=0.009 (SMERl 8e), /7=0.001 (SMER18g), /7=0.382 (SMERl 8j), /7=0.067 (SMERl 81), /7=0.031 (SMERl 8) (e); /7<0.0001 (SMER28a, c, e-g, i-1, SMER28), /7=0.574 (SMER28b), /7=0.041 (SMER28d), /7=0.002 (SMER28h) (f). ***, p < 0.001; **, p < 0.01; *, p < 0.05; NS, Non-significant.

[0038] Figure 23 shows the chemical structures of SMIRs. Twenty-one structurally non-redundant SMIRs that were identified from the primary assay positives. [0039] Figure 24 shows the chemical structures of SMERs. Twelve structurally non- redundant SMERs that were identified from the primary assay positives. [0040] Figure 25 shows that Protein-synthesis inhibitors that target the ribosome fail to suppress the cytostatic effects of rapamycin in yeast. A, B. Dose-response curves correspond to 2-fold dilutions of either anisomycin (data shown in A) or CHX (data shown in B) in the presence of 25 nM rapamycin (filled shapes) or vehicle (unfilled shapes). [0041] Figure 26 shows the results of a screen for the autophagy-inhibitory SMIRs in mammalian cell line. A,B. A stable inducible PC12 cell line expressing A53T α-synuclein mutant was induced with doxycycline for 48 hours, and expression of the trans gene was switched off for 24h, with DMSO (control), or 1:400 dilution of 5mg/ml SMIRs 1, 2, 7, 8b, 11, 12, 14-18, 19a, 19b, 20-23, 28, 29a, 29b, 30, 31, added in the switch-off period. The levels of A53T a-synuclein (α-syn) was analysed by immunoblotting with antibody against HA (A) and densitometry analysis relative to actin (B).

[0042] Figure 27 shows the results of a screen for the autophagy-inducing SMERs in mammalian cell line. A stable inducible PC12 cell line expressing A53T α-synuclein mutant was induced with doxycycline for 48 hours, and expression of the transgene was switched off for 24 hours, with DMSO (control), or 1:400 dilution of 5mg/ml SMERs 1-3, 6, 9-11, 13, 14, 16-24, 26, 28, added in the switch-off period. The levels of A53T α-synuclein (α-syn) was analysed by immunoblotting with antibody against HA (A) and densitometry analysis relative to actin (B).

[0043] Figure 28 shows increased mutant huntingtin aggregation in Atg5 knock-out mouse embryonic fibroblasts, compared to wild-type cells. Wild-type (Atg5^+/+) and knockout (Atg5 ^) Atg5 mouse embryonic fibroblasts were transfected with EGFP-HDQ74 construct for 4h and fixed at 48 hours post-transfection. The percentage of EGFP-positive cells with EGFP-HDQ74 aggregates were assessed and expressed as odds ratio. The control (EGFP-HDQ74 aggregation in Atg5^+/+ cells) was taken as l. /?<0.0001. ***,/?<0.001. [0044] Figure 29 shows the effect of SMERs 10, 18, and 28 on Beclin-1, Atg5, Atg7 and Atgl2. A. COS-7 cells treated with DMSO (control) or with 47 μM SMERlO, 43 μM SMERl 8 or 47 μM SMER28 for 24 hours, were analysed for Beclin-1 levels by immunoblotting with anti-Beclin- 1 antibody. B-D. HeLa cells treated with DMSO (control) or with 47 μM SMERlO, 43μM SMERl 8, or 47 μM SMER28 for 24 hours, were analysed for Atg5 (b), Atg7 (c) or Atgl2 (d) levels by immunoblotting with anti-Atg5 (b), anti-Atg7 (c) or anti-Atgl2 (d) antibody. [0045] Figure 30 shows saturating concentrations of SMERs 10, 18, and 28 for enhancing the clearance of A53T α-synuclein. A-C. Stable inducible PC 12 cell line expressing A53T α-synuclein mutant was induced with doxycycline for 48h, and expression of the transgene was switched off for 24h, with DMSO (control), or 1:400, 1 :200 or 1 : 100 dilutions of 5 mg/ml SMERs 10 (a), 18 (b) and 28 (c), added in the switch-off period. The levels of A53T α-synuclein (α-syn) was analysed by immunoblotting with anti-HA antibody. [0046] Figure 31 shows the structures of SMERs 10, 18 and 28 analogs. Chemical structures of three SMERlO analogs (SMER10a-c) (A), twelve SMER18 analogs (SMER18a- 1) (B), and twelve SMER 28 analogs (SMER28a-l) (C).

[0047] Figure 32 shows a Forward Chemical Genetic Study of Autophagy. (A)

Images of LN229 cells expressing GFP-LC3 treated with known chemical modulators of autophagy. Punctate fluoresence (arrows) indicates the accumulation of LC3 -positive autophagic vesicles (AV). (B) Sample analysis overlay identifies nuclei (green) and AV (red). (C) Treatment of cells with chloroquine, a known autophagy inhibitor, (50 μM) resulted in an increase in both the number of AV per cell and mean vesicle area. Treatment of cells with rapamycin, a known autophagy inducer, (10 μM) resulted in a significant increase in the number of AV per cell, with no effect on AV area. Each point represents one experimental well. Statistical significance determined by Student's t test. (D) LN229 EGFP- LC3 screening results; blue points represent DMSO controls, red experimental compounds. Compounds with Z-Score > 2 were considered putative autophagy modulators. (E) Selected autophagy modulators highlighted in table.

[0048] Figure 33 shows phenotype validation and dose response. (A, B) Variation in basal AV per cell in human cancer cell lines (mean +/- SD). (C) Treatment with hydroxychloroquine, but not rapamycin leads to a significant accumulation of AV in H1299 EGFP-LC3 cells. Statistical significance determined by Student's t test. (D) Screening in two cell lines yields 35 putative autophagy inhibitors. (E) H1299 GFP-LC3 screening results; blue points represent DMSO controls, red points represent experimental compounds. Highlighted compounds are identified in the table in Figure 34. (F) Structure-activity relationship of candidate compounds identifies three homologous bisindole maleimides compounds. (G) Dose-dependent accumulation of AV in H1299/GFP-LC3 cells treated with K252A, Go6976, and GF-109203X. [0049] Figure 34 is a table of autophagy modulators identified from H1299 GFP-LC3 screening..

[0050] Figure 35 focues on the autophagy inhibitors: K252A, Go6976, and GF-

109203X. (A) Immunoblot against LC3 in H1299 EGFP-LC3 cells treated with hydroxychloroquine (10 μM) and bis-indolyl maleimide compounds (1 μM, 10 μM). (B) K252A, Go6976, and GF-109230X reduce the rate of protein degradation relative to DMSO control. Statistical significance determined by Student's t test. (** p < 0.01, *** p < 0.001). (C) Electron micrographs of H 1299 GFP-LC3 cells treated with either DMSO or K252A. Scale Bar = 2 μM. (D) Quantitation of electron micrographs (mean +/- SD). Statistical significance determined by Student's t test.

[0051] Figure 36 shows the structure-activity relationship of K252A analogs. (A)

Rank-ordered activity of K252a analogs (all 1 μM) in H1299 GFP/LC3 cells (mean +/- SD). (B) Chemical structures of K252a analogs #9 and #11. (C) Treatment with K252A and analog #11, but not analog #9, lead to dose-dependent increase in AV in H1299 GFP-LC3 cells (mean +/- SD).

[0052] Figure 37. (A) Rank-ordered activity of a panel of clinically relevant kinase inhibitors (all 6.3 μM) in H1299 GFP-LC3 cells (mean +/- SD). (B) Chemical structures of sunitinib, UCNOl, PKC412, and ruboxistaurin. Sunitinib, UCNOl, PKC412, and ruboxistaurin were found to be autophagy inhibitors. (C) Increased accumulation of AV is dose-dependent (mean +/- SD). (D) Immunoblot against LC3 in H1299 GFP-LC3 cells treated with clinically relevant kinase inhibitors (all 6.3 μM) in presence and absence of bafilomycin (400 nM). (E) Electron micrographs of H 1299 GFP-LC3 cells treated with UCNOl. (F) Quantitation of electron micrographs (mean +/- SD). Statistical significance determined by Student's t test.

[0053] Figure 38. (A) Immunoblot against LC3 in RPMI cells (a multiple myeloma cell line) treated with clinically relevant kinase inhibitors, sunitinib, UCNOl, PKC412, and ruboxistaurin (all 6.3 μM). (B) Electron micrographs of RPMI cells treated with DMSO, K252A, or UCNOl. (C) Quantitation of electron micrographs (mean +/- SD). Statistical significance determined by Student's t test. (D) Autophagy inhibitors demonstrate selective toxicity to the mm. Is and RPMI-8826 cells lines compared to H1299 (mean +/- SD). [0054] Figure 39 is table listing all LN229 EGFP-LC3 assay positives with > 7 (Z- score > 2). [0055] Figure 40. (A) Quantitation of number of AV in LN229 GFP-LC3 cells. (B)

Histograms of 3500 compound screened in LN229 cells DMSO controls (blue), experimental compounds (red).

[0056] Figure 41. Mean vesicle area in H 1299 GFP-LC3 cells treated with either

DMSO, rapamycin, or hydroxychloroquine.

[0057] Figure 42. Images of H1299 GFP-LC3 cells treated with K252A, sunitinib,

UCNOl, ruboxistaruin, PKC412, and rapamycin.

Definitions

[0058] Before further description of the invention, and in order that the invention may be more readily understood, certain terms are defined and collected here for convenience. [0059] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito: 1999, the entire contents of which are incorporated herein by reference.

[0060] Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and ϊrαws-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.

[0061] Isomeric mixtures containing any of a variety of isomer ratios may be utilized in accordance with the present invention. For example, where only two isomers are combined, mixtures containing 50:50, 60:40, 70:30, 80:20, 90: 10, 95:5, 96:4, 97:3, 98:2, 99: 1, or 100:0 isomer ratios are all contemplated by the present invention. Those of ordinary skill in the art will readily appreciate that analogous ratios are contemplated for more complex isomer mixtures. [0062] If, for instance, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers. [0063] One of ordinary skill in the art will appreciate that the synthetic methods, as described herein, utilize a variety of protecting groups. By the term "protecting group", as used herein, it is meant that a particular functional moiety, e.g., O, S, or N, is temporarily blocked so that a reaction can be carried out selectively at another reactive site in a multifunctional compound. In preferred embodiments, a protecting group reacts selectively in good yield to give a protected substrate that is stable to the projected reactions; the protecting group should be selectively removable in good yield by readily available, preferably non-toxic reagents that do not attack the other functional groups; the protecting group forms an easily separable derivative (more preferably without the generation of new stereogenic centers); and the protecting group has a minimum of additional functionality to avoid further sites of reaction. As detailed herein, oxygen, sulfur, nitrogen, and carbon protecting groups may be utilized. Hydroxyl protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), ϊ-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p- methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), ϊ-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2- methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2- (trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3- bromotetrahydropyranyl, tetrahydrothiopyranyl, 1 -methoxycyclohexyl, A- methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, A- methoxytetrahydrothiopyranyl S,S-dioxide, l-[(2-chloro-4-methyl)phenyl]-4- methoxypiperidin-4-yl (CTMP), l,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1 -ethoxyethyl, 1- (2-chloroethoxy)ethyl, 1 -methyl- 1-methoxy ethyl, 1 -methyl- 1-benzyloxy ethyl, 1 -methyl- 1- benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2 -(phenyls elenyl)ethyl, t- butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, />-nitrobenzyl, />-halobenzyl, 2,6-dichlorobenzyl, p- cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl, p,p '-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α- naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p- methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'- bromophenacyloxyphenyl)diphenylmethyl, 4,4',4"-tris(4,5- dichlorophthalimidophenyl)methyl, 4,4',4"-tris(levulinoyloxyphenyl)methyl, 4,4',4"- tris(benzoyloxyphenyl)methyl, 3-(imidazol-l-yl)bis(4',4"-dimethoxyphenyl)methyl, 1,1- bis(4-methoxyphenyl)-l'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10- oxo)anthryl, l,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, ϊ-butyldimethylsilyl (TBDMS), t- butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), ϊ-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, /?-chloroplienoxyacetate, 3-phenylpropionate, A- oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, /»-phenylbenzoate, 2,4,6- trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl /»-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-l-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o- (dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, A- (methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4- methylphenoxyacetate, 2,6-dichloro-4-(l,l,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(l,l- dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2- methyl-2-butenoate, ø-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'- tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts). For protecting 1,2- or 1,3-diols, the protecting groups include methylene acetal, ethylidene acetal, l-£-butylethylidene ketal, 1 -phenylethylidene ketal, (4- methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p- methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene ortho ester, 1 -methoxyethylidene ortho ester, 1 -ethoxyethylidine ortho ester, 1,2-dimethoxyethylidene ortho ester, α-methoxybenzylidene ortho ester, 1-(N,N- dimethylamino)ethylidene derivative, α-(N,N'-dimethylamino)benzylidene derivative, 2- oxacyclopentylidene ortho ester, di-ϊ-butylsilylene group (DTBS), 1,3-(1, 1,3,3- tetraisopropyldisiloxanylidene) derivative (TIPDS), tetra-?-butoxydisiloxane-l,3-diylidene derivative (TBDS), cyclic carbonates, cyclic boronates, ethyl boronate, and phenyl boronate. Amino-protecting groups include methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10, 10-dioxo-lO, 10, 10, 10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1- (l-adamantyl)-l-methylethyl carbamate (Adpoc), l,l-dimethyl-2-haloethyl carbamate, 1,1- dimethyl-2,2-dibromoethyl carbamate (OB-t-BOC), l,l-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1 -methyl- l-(4-bipheny Iy l)ethyl carbamate (Bpoc), l-(3,5-di-t- butylphenyl)- 1 -methylethyl carbamate (ϊ-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Py oc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, ?-butyl carbamate (BOC), 1- adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1- isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Νoc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), />-methoxybenzyl carbamate (Moz), /»-nitobenzyl carbamate, p- bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, A- methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p- toluenesulfonyl)ethyl carbamate, [2-(l,3-dithianyl)]methyl carbamate (Dmoc), A- methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2- phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1- dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p- (dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)- 6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(ø- nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl derivative, N'-p- toluenesulfonylaminocarbonyl derivative, N'-phenylaminothiocarbonyl derivative, ?-amyl carbamate, «S-benzyl thiocarbamate, />-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p- decyloxybenzyl carbamate, 2,2-dimethoxycarbonylvinyl carbamate, o-(N,N- dimethylcarboxamido)benzyl carbamate, 1 , 1 -dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p '-methoxyphenylazo)benzyl carbamate, 1 -methylcyclobutyl carbamate, 1- methylcyclohexyl carbamate, 1 -methyl- 1 -cyclopropylmethyl carbamate, l-methyl-l-(3,5- dimethoxyphenyl)ethyl carbamate, 1 -methyl- 1 -(p-phenylazophenyl)ethyl carbamate, 1- methyl-1-phenylethyl carbamate, 1 -methyl- l-(4-pyridyl)ethyl carbamate, phenyl carbamate, />-(phenylazo)benzyl carbamate, 2,4,6-tri-?-butylphenyl carbamate, A- (trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3- phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxycarbonylamino)acetamide, 3-(p- hydroxyphenyl)propanamide, 3 -(o-nitrophenyl)propanamide, 2-methyl-2-(o- nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, A- chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2- one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5- dimethylpyrrole, N-l,l,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5- substituted l,3-dimethyl-l,3,5-triazacyclohexan-2-one, 5-substituted l,3-dibenzyl-l,3,5- triazacyclohexan-2-one, 1 -substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(l-isopropyl-4- nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4- methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N- [(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7- dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N'- oxide, N-l,l-dimethylthiomethyleneamine, N-benzylideneamine, N-p- methoxybenzylideneamine, N-diphenylmethyleneamine, N- [(2- pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, NN'- isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5- chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N- cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-l-cyclohexenyl)amine, N-borane derivative, N-diphenylborinic acid derivative, N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Νps), 2,4- dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4- methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3 -nitropyridinesulfenamide (Νpys), />-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4- methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6- dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3 ,5,6-tetramethyl-4- methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6- trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β- trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'- dimethoxynaphthylmethyl)benzenesulfonamide (DΝMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.. Exemplary protecting groups are detailed herein, however, it will be appreciated that the present invention is not intended to be limited to these protecting groups; rather, a variety of additional equivalent protecting groups can be readily identified using the above criteria and utilized in the method of the present invention. Additionally, a variety of protecting groups are described in Protective Groups in Organic Synthesis, Third Ed. Greene, T. W. and Wuts, P. G., Eds., John Wiley & Sons, New York: 1999, the entire contents of which are hereby incorporated by reference. [0064] It will be appreciated that the compounds, as described herein, may be substituted with any number of substituents or functional moieties. In general, the term "substituted" whether preceded by the term "optionally" or not, and substituents contained in formulas of this invention, refer to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. Furthermore, this invention is not intended to be limited in any manner by the permissible substituents of organic compounds. Combinations of substituents and variables envisioned by this invention are preferably those that result in the formation of stable compounds useful in the treatment, for example, of infectious diseases or proliferative disorders. The term "stable", as used herein, preferably refers to compounds which possess stability sufficient to allow manufacture and which maintain the integrity of the compound for a sufficient period of time to be detected and preferably for a sufficient period of time to be useful for the purposes detailed herein. [0065] The term "aliphatic", as used herein, includes both saturated and unsaturated, straight chain (i.e., unbranched), branched, acyclic, cyclic, or poly cyclic aliphatic hydrocarbons, which are optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, "aliphatic" is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term "alkyl" includes straight, branched and cyclic alkyl groups. An analogous convention applies to other generic terms such as "alkenyl", "alkynyl", and the like. Furthermore, as used herein, the terms "alkyl", "alkenyl", "alkynyl", and the like encompass both substituted and unsubstituted groups. In certain embodiments, as used herein, "lower alkyl" is used to indicate those alkyl groups (cyclic, acyclic, substituted, unsubstituted, branched or unbranched) having 1-6 carbon atoms.

[0066] In certain embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1 -4 carbon atoms. Illustrative aliphatic groups thus include, but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, -CH₂-cyclopropyl, vinyl, allyl, n-butyl, sec- butyl, isobutyl, tert-butyl, cyclobutyl, -CH₂-cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert- pentyl, cyclopentyl, -CH^-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl, -Ctt-cyclohexyl moieties and the like, which again, may bear one or more substituents. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, l-methyl-2-buten-l- yl, and the like. Representative alkynyl groups include, but are not limited to, ethynyl, 2- propynyl (propargyl), 1-propynyl, and the like.

[0067] The term "alkoxy", or "thioalkyl" as used herein refers to an alkyl group, as previously defined, attached to the parent molecule through an oxygen atom or through a sulfur atom. In certain embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-20 alipahtic carbon atoms. In certain other embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups contain 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups contain 1 -4 aliphatic carbon atoms. Examples of alkoxy, include but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy, and n-hexoxy. Examples of thioalkyl include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

[0068] The term "alkylamino" refers to a group having the structure -NHR', wherein

R is aliphatic, as defined herein. In certain embodiments, the aliphatic group contains 1-20 aliphatic carbon atoms. In certain other embodiments, the aliphatic group contains 1-10 aliphatic carbon atoms. In yet other embodiments, the aliphatic group employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the aliphatic group contains 1-6 aliphatic carbon atoms. In yet other embodiments, the aliphatic group contains 1-4 aliphatic carbon atoms. Examples of alkylamino groups include, but are not limited to, methylamino, ethylamino, n-propylamino, iso-propylamino, cyclopropylamino, n- butylamino, tert-butylamino, neopentylamino, n-pentylamino, hexylamino, cyclohexylamino, and the like.

[0069] The term "dialkylamino" refers to a group having the structure -NRR', wherein

R and R are each an aliphatic group, as defined herein. R and R' may be the same or different in an dialkyamino moiety. In certain embodiments, the aliphatic groups contains 1- 20 aliphatic carbon atoms. In certain other embodiments, the aliphatic groups contains 1-10 aliphatic carbon atoms. In yet other embodiments, the aliphatic groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the aliphatic groups contains 1-6 aliphatic carbon atoms. In yet other embodiments, the aliphatic groups contains 1-4 aliphatic carbon atoms. Examples of dialkylamino groups include, but are not limited to, dimethylamino, methyl ethylamino, diethylamino, methylpropylamino, di(n-propyl)amino, di(iso-propyl)amino, di(cyclopropyl)amino, di(n-butyl)amino, di(tert-butyl)amino, di(neopentyl)amino, di(n-pentyl)amino, di(hexyl)amino, di(cyclohexyl)amino, and the like. In certain embodiments, R and R' are linked to form a cyclic structure. The resulting cyclic structure may be aromatic or non-aromatic. Examples of cyclic diaminoalkyl groups include, but are not limted to, aziridinyl, pyrrolidinyl, piperidinyl, morpholinyl, pyrrolyl, imidazolyl, 1,3,4-trianolyl, and tetrazolyl.

[0070] Some examples of substituents of the above-described aliphatic (and other) moieties of compounds of the invention include, but are not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; -OH; -NO₂; - CN; -CF₃; -CH₂CF₃; -CHCl₂; -CH₂OH; -CH₂CH₂OH; -CH₂NH₂; -CH₂SO₂CH₃; -C(O)R_x; - CO₂(R_x); -CON(R_X)₂; -OC(O)R_x; -OCO₂R_x; -OCON(R_X)₂; -N(R_x)₂; -S(O)₂R_x; -NR_x(CO)R_x wherein each occurrence Of R_x independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples that are described herein. [0071] In general, the terms "aryl" and "heteroaryl", as used herein, refer to stable mono- or polycyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated moieties having preferably 3-14 carbon atoms, each of which may be substituted or unsubstituted. Substituents include, but are not limited to, any of the previously mentioned substitutents, i.e., the substituents recited for aliphatic moieties, or for other moieties as disclosed herein, resulting in the formation of a stable compound. In certain embodiments of the present invention, "aryl" refers to a mono- or bicyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like. In certain embodiments of the present invention, the term "heteroaryl", as used herein, refers to a cyclic aromatic radical having from five to ten ring atoms of which one ring atom is selected from S, O, and N; zero, one, or two ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.

[0072] It will be appreciated that aryl and heteroaryl groups can be unsubstituted or substituted, wherein substitution includes replacement of one, two, three, or more of the hydrogen atoms thereon independently with any one or more of the following moieties including, but not limited to: aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F; -Cl; -Br; -I; -OH; -NO₂; -CN; -CF₃; -CH₂CF₃; -CHCl₂; - CH₂OH; -CH₂CH₂OH; -CH₂NH₂; -CH₂SO₂CH₃; -C(O)R_x; -CO₂(R_x); -CON(R_X)₂; -OC(O)R_x; -OCO₂R_x; -0C0N(R_x)₂; -N(R_x)₂; -S(O)₂R_x; -NR_x(CO)R_x, wherein each occurrence of R_x independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substitutents are illustrated by the specific embodiments shown in the Examples that are described herein.

[0073] The term "heteroaliphatic", as used herein, refers to aliphatic moieties that contain one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moieties may be branched, unbranched, cyclic or acyclic and include saturated and unsaturated heterocycles such as morpholino, pyrrolidinyl, etc. In certain embodiments, heteroaliphatic moieties are substituted by independent replacement of one or more of the hydrogen atoms thereon with one or more moieties including, but not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F; - Cl; -Br; -I; -OH; -NO₂; -CN; -CF₃; -CH₂CF₃; -CHCl₂; -CH₂OH; -CH₂CH₂OH; -CH₂NH₂; - CH₂SO₂CH₃; -C(O)R_x; -CO₂(R_x); -CON(R_X)₂; -OC(O)R_x; -OCO₂R_x; -0C0N(R_x)₂; -N(R_X)₂; - S(O)₂R_x; -NR_x(CO)R_x, wherein each occurrence Of R_x independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substitutents are illustrated by the specific embodiments shown in the Examples that are described herein. [0074] The terms "halo" and "halogen" as used herein refer to an atom selected from fluorine, chlorine, bromine, and iodine.

[0075] The term "heterocyclic", as used herein, refers to a non-aromatic 5-, 6-, or 7- membered ring or a polycyclic group, including, but not limited to a bi- or tri-cyclic group comprising fused six-membered rings having between one and three heteroatoms independently selected from oxygen, sulfur and nitrogen, wherein (i) each 5-membered ring has O to 1 double bonds and each 6-membered ring has O to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms may be optionally be oxidized, (iii) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above heterocyclic rings may be fused to a benzene ring. Representative heterocycles include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl. In certain embodiments, a "substituted heterocycloalkyl or heterocycle" group is utilized and as used herein, refers to a heterocycloalkyl or heterocycle group, as defined above, substituted by the independent replacement of one, two or three of the hydrogen atoms thereon with but are not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F; -Cl; -Br; -I; -OH; -NO₂; -CN; -CF₃; -CH₂CF₃; -CHCl₂; - CH₂OH; -CH₂CH₂OH; -CH₂NH₂; -CH₂SO₂CH₃; -C(O)R_x; -CO₂(R_x); -C0N(R_x)₂; -OC(O)R_x; -OCO₂R_x; -0C0N(R_x)₂; -N(R_x)₂; -S(O)₂R_x; -NR_x(CO)R_x, wherein each occurrence of R_x independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substitutents are illustrated by the specific embodiments shown in the Examples which are described herein.

[0076] The term "independently selected" is used herein to indicate that the R groups can be identical or different.

[0077] Definitions of non-chemical terms used throughout the specification include:

[0078] As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human, at any stage of development. In some embodiments, "animal" refers to a non-human animal, at any stage of development. In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and/or worms. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or clone.

[0079] The term "compound" or "chemical compound" as used herein can include organometallic compounds, organic compounds, metals, transitional metal complexes, and small molecules. In certain preferred embodiments, polynucleotides are excluded from the definition of compounds. In other preferred embodiments, polynucleotides and peptides are excluded from the definition of compounds. In a particularly preferred embodiment, the term compounds refers to small molecules (e.g., preferably, non-peptidic and non-oligomeric) and excludes peptides, polynucleotides, transition metal complexes, metals, polymers, and organometallic compounds. [0080] The phrase, "pharmaceutically acceptable form", as used herein, denotes any pharmaceutically acceptable salt, ester, salt of such ester, stereoisomer (e.g., enantiomer), isomer, tautomer, protected form, or any other adduct or derivative which, upon administration to a patient, is capable of providing (directly or indirectly) a compound as otherwise described herein, or a metabolite or residue thereof. Pharmaceutically acceptable forms thus include among others pro-drugs. A pro-drug is a derivative of a compound, usually with significantly reduced pharmacological activity, which contains an additional moiety, which is susceptible to removal in vivo yielding the parent molecule as the pharmacologically active species. An example of a pro-drug is an ester, which is cleaved in vivo to yield a compound of interest. Pro-drugs of a variety of compounds, and materials and methods for derivatizing the parent compounds to create the pro-drugs, are known and may be adapted to the present invention. The biological activity of pro-drugs may also be altered by appending a functionality onto the compound, which may be catalyzed by an enzyme. Also, included are oxidation and reduction reactions, including enzyme-catalyzed oxidation and reduction reactions. Certain exemplary pharmaceutical compositions and pharmaceutically acceptable forms are discussed in more detail herein below. [0081] As used herein, the term "small molecule" refers to a non-peptidic, non- oligomeric organic compound either synthesized in the laboratory or found in nature. Small molecules, as used herein, can refer to compounds that are "natural product-like", however, the term "small molecule" is not limited to "natural product-like" compounds. Rather, a small molecule is typically characterized in that it contains several carbon-carbon bonds, and has a molecular weight of less than 1500 g/mol, although this characterization is not intended to be limiting for the purposes of the present invention. In certain other preferred embodiments, natural-product-like small molecules are utilized. In certain embodiments, the molecular weight of the small molecule is less than 1000 g/mol.

[0082] The term "administration" or "administering" includes routes of introducing the compound of the invention(s) to a subject to perform their intended function. Examples of routes of administration that may be used include injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal), oral, inhalation, rectal and transdermal. The pharmaceutical preparations may be given in forms suitable for each administration route. For example, these preparations are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc., administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administration is preferred. The injection can be bolus or can be a continuous infusion. Depending on the route of administration, the compound of the invention can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally effect its ability to perform its intended function. The compound of the invention can be administered alone, or in conjunction with either another agent as described above or with a pharmaceutically-acceptable carrier, or both. The compounds of the invention can be administered prior to the administration of the other agent, simultaneously with the agent, or after the administration of the agent. Furthermore, the compound of the invention can also be administered in a pro-form which is converted into its active metabolite, or more active metabolite in vivo.

[0083] The language "biological activities" of a compound of the invention includes all activities elicited by compound of the inventions in a responsive cell. It includes genomic and non-genomic activities elicited by these compounds. In certain embodiments, biological activities refers to phenotypic changes. In certain embodiments, biological activity refers to cytotoxicity, inhibition of autophagy, stimulation of autophagy, inhibition of deacetylation or acetylation activity, or stimulation of deacetylation or acetylation activity. [0084] The term "effective amount" includes an amount effective, at dosages and for periods of time necessary, to achieve the desired result, e.g., sufficient to treat cancer, to treat a protein degradation disorder, to treat an infection, to treat a cardiovascular disease, or to treat or prevent a neurodegenerative disease. An effective amount of compound of the invention may vary according to factors such as the disease state, age, health, and weight of the subject, and the ability of the compound of the invention to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. An effective amount is also one in which any toxic or detrimental effects (e.g., side effects) of the compound of the invention are outweighed by the therapeutically beneficial effects. [0085] A therapeutically effective amount of compound of the invention (i.e., an effective dosage) may range from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health, and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a compound of the invention can include a single treatment or, preferably, can include a series of treatments. In one example, a subject is treated with a compound of the invention in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of a compound of the invention used for treatment may increase or decrease over the course of a particular treatment.

[0086] "Therapeutic agent" as used herein refers to a small molecule, peptide, protein, enzyme antibody, nucleic acid, etc. that is effective to treat or is suspected of being effective to treat a disease (e.g., a proliferative disease, a neurodegenerative disease, infectious disease, cardiovascular disease, a protein misfolding state, protein mishandling state, etc.). [0087] The term "modulate" refers to increases or decreases in the activity (e.g., autophagy, activity of rapamycin) of a cell in response to exposure to a compound described herein, e.g., the inhibition of autophagy in at least a sub-population of cells in an animal such that a desired end result is achieved, e.g., a therapeutic result. In preferred embodiments, this phrase is intended to include cellular element degradation by autophagy. [0088] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0089] The term "prodrug" includes compounds with moieties that can be metabolized in vivo. Generally, the prodrugs are metabolized in vivo by esterases or by other mechanisms to active drugs. Prodrugs may only become active upon such reaction under biological conditions, or they may have activity in their unreacted forms. Examples of prodrugs and their uses are well known in the art (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. ScL 66: 1-19; incorporated herein by reference). The prodrugs can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent. Hydroxyl groups can be converted into esters via treatment with a carboxylic acid. Examples of prodrug moieties include substituted and unsubstituted, branched or unbranched lower alkyl ester moieties, (e.g., propionoic acid esters), lower alkenyl esters, di-lower alkyl-amino lower-alkyl esters (e.g., dimethylaminoethyl ester), acylamino lower alkyl esters (e.g., acetyloxymethyl ester), acyloxy lower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl-lower alkyl esters (e.g., benzyl ester), substituted (e.g., with methyl, halo, or methoxy substituents) aryl and aryl-lower alkyl esters, amides, lower-alkyl amides, di-lower alkyl amides, and hydroxy amides. Other examples of prodrugs include derivatives of compounds of any of the formulae disclosed herein that comprise -NO, -NO₂, -ONO, or -ONO₂ moieties. Preferred prodrug moieties are acyl esters. Prodrugs which are converted to active forms through other mechanisms in vivo are also included. The compounds of the invention may be synthesized as pro-drugs that are metabolized by the subject into the compound of the invention.

[0090] The term "subject" and "patient" are used interchangeably herein and include organisms which are capable of suffering from a protein degradation disorder or who could otherwise benefit from the administration of a compound of the invention, such as human and non-human animals. Preferred human animals include human patients suffering from or prone to suffering from a proliferative disease, a cardiovascular disease, infectious disease, neurodegenerative disease, or protein degradation disorder or associated state, as described herein. The term "non-human animals" of the invention includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, and non-mammals, such as non-human primates, e.g., sheep, dog, cow, chickens, amphibians, reptiles, etc. Susceptible to a disease is meant to include subjects at risk of developing a disease.

Detailed Description of Certain Embodiments of the Invention

[0091] The present invention stems from the recognition that a variety of diseases can be treated using agents that modulate autophagy. Autophagy is a process by which cells cannibalize cellular elements to generate metabolites, or, in some instances, to cause cell death. Autophagy is both a mechanism for cell survival as well as a mechanism for cell death.. Based on the importance of autophagy in the life of a cell, a screen was performed of over 3,500 compounds to identify compounds that induce the characteristic phenotype of autophagy. In this screen, both inhibitors and enhancers of autophagy were identified. Several of the identified compounds are known drugs already approved and formulated for administration to humans. The identified compounds, as well as future compounds identified by the inventive screening system, derivatives of the identified compounds, or other compounds found to be modulators of autophagy, may be used alone or in combination with other drugs to treat proliferative diseases such as cancer, inflammatory diseases, autoimmune dieseases, neurodegenerative diseases, cardiovascular diseases, infectious diseases, and diseases characterized by protein misfolding and/or mishandling. The present invention also provides pharmaceutical compositions and kits including combinations with other therapeutic agents, and methods of using such compositions.

[0092] In another aspect, the invention provides agents, particularly small molecules, that have been identified to modulate or modify the biological activity of rapamycin or other modulators of autophagy. Rapamycin is a known inducer of autophagy. In certain embodiments, these enhancers and inhibitors of rapamycin' s activity are also useful as inhibitors and enhancers of autophagy by themselves. These compounds may be used alone or in combination with other agents (e.g., kinases inhibitors, rapamycin, proteasome inhibitors, growth factor pathway inhibitors, autophagy modulators, etc.) to treat proliferative disease such as cancer, neurodegenerative diseases, inflammatory diseases, autoimmune diseases, cardiovascular diseases, infectious diseases, and diseases characterized by protein misfolding and/or mishandling. In certain embodiments, the compounds are used in combination with rapamycin. In certain embodiments, the compounds are used in combination with the autophagy modulators described herein or analogs of the autophagy modulators described herein. Furthermore, analogs of some of the identified enhancers of rapamycin' s activity have been either obtained or prepared and tested for their ability to modify the biological activity of rapamycin. These analogs as described herein are provided by the present invention. The invention also provides pharmaceutical compositions and kits including combinations with other therapeutic agents and methods of using such compositions.

Modulators of Autophagy

[0093] Modulators of autophagy were identified using a phenotype-based screen as generally described in U.S. Patent applications, USSN 60/379,296, filed May 10, 2002; and USSN 10/435,827, filed May 12, 2003, published on March 9, 2006 as US 2006/0050946; each of which is incorporated herein by reference. Over 3,500 compounds were screened for their ability to induce the characteristic phenotype of autophagy (i.e., the accumulation of EGFP-LC3 positive autophagosomes in the cytosol) in human glioblastoma LN-229 cells. Many of the compounds were subsequently screened in H1299 EGFP-LC3 cells. The screen was used to identify both inhibitors and promoters of autophagy.

[0094] Based on data from this screen, the following compounds were identified as late inhibitors of autophagosome-lysosome fusion: cefamandole, monensin, astemizole, spiramycin, (lS,9R)-beta-hydrastine, carnitine, tomatine, K252A, atranorin, tetrandrine, amlodipine, benzyl isothiocyanate, pristimerin, homochlorcyclizine (e.g., homochlorcyclizine dihydrochloride), and fluoxetine (e.g., fluoxetine hydrochloride). Bafilomycin Al, wiskostatin, monensin, quinacrine, nocodazole, vinblastine, colchicine, puromycin, bepridil, spiramycin, migericin, 2-methylcinngel, amiprilose, carnitine, tyrphostin 9, salinomycin, PPl, lavendustin A, ZL3VS, astemizole, GO6976, RWJ-60475-(AM)3, D609, mefenamic acid, cytochalasin D, E6 berbamine, beta-peltatin, aesculin, GF-109203D, benzyl isothiocyanate, monensin, podophyllotoxin, thimerosal, maprotiline hydrochloride, vinblastine, norethindrone, and gramacidin were also indentified as inhibitors of autophagy. The kinase inhibitors sunitinib, UCNOl, PKC412, and ruboxistaurin were also identified as inhibitors of autophagy. Therefore, the identified compounds or pharmaceutically acceptable forms thereof may be useful in treating diseases where the inhibition of autophagy would be beneficial (e.g., in the treatment of cancer). These compounds represent a diverse class of structurally dissimilar compounds that have been found to inhibit autophagy. Either these compounds or derivatives of these compounds may be used to inhibit autophagy in a cell. [0095] Based on a screen of the Prestwick collection of off-patent FDA-approved drugs, three compounds with a bis-indolyl maleimide core were identified as inhibitors of autophagy — K252A, Go6976, and GF-109203X. The structures of these three hits are shown in Figure 33. Analogs of these three hits that are provide by the present invention or are useful in accordance with the present invention include compounds of one of the formula:

wherein each V is independently -CH₂-, -(C=O)-, or -CH(OH)-; each occurrence of a dashed line independently represents a single bond, double bond, or the absence of a bond;

Ri is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -C(=0)R_A; -C0₂R_A; -SOR_A; -SO₂R_A; -N(RA)₂; -NHC(0)R_A; or - C(R_A)₃; wherein each occurrence of R_A is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₂ is is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -C(=O)R_B; -CO₂R_B; -SOR_B; -SO₂R_B; -N(R_B)₂; -NHC(O)R₈; or - C(R_B)₃; wherein each occurrence of R_B is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; and pharmaceutically acceptable forms thereof. [0096] In certain embodiments, one V is -CH₂-; and the other V is -C(=O)-. In certain embodiments, one V is -CH₂-; and the other V is -CH(OH)-. In certain embodiments, one V is -CH(OH)-; and the other V is -C(=0)-. In certain embodiments, both V are - C(=O)-. In certain embodiments, both V are -CH₂-. In certain embodiments, both V are - CH(OH)-.

[0097] In certain embodiments, Ri is hydrogen. In certain embodiments, Ri is optionally substituted aliphatic. In certain embodiments, Ri is C₁-Ce alkyl. In certain embodiments, Ri is substituted, acyclic aliphatic. In certain embodiments, Ri is optionally substituted heteroaliphatic. In certain embodiments, Ri is substituted, acyclic heteroaliphatic. In certain embodiments, Ri is optional substituted aryl. In certain embodiments, Ri is optionally substituted heteroaryl. In certain embodiments, Ri is acyl. In certain embodiments, Ri is -C(=O)R_A.

[0098] In certain embodiments, R₂ is hydrogen. In certain embodiments, R₂ is optionally substituted aliphatic. In certain embodiments, R₂ is C₁-Ce alkyl. In certain embodiments, R2 is substituted, acyclic aliphatic. In certain embodiments, R2 is optionally substituted heteroaliphatic. In certain embodiments, R₂ is substituted, acyclic heteroaliphatic. In certain embodiments, R₂ is optional substituted aryl. In certain embodiments, R₂ is optionally substituted heteroaryl. In certain embodiments, R₂ is acyl. In certain embodiments, R2 is -C(=O)Rβ.

[0099] In certain embodiments, Ri and R₂ are taken together to form an optionally substituted heteroyclic moiety. In certain embodiments, Ri and R₂ are taken together to form a substituted heteroyclic moiety. In certain embodiments, Ri and R₂ are taken together to form an optionally substituted heteroyclic, bicyclic moiety. In certain embodiments, Ri and R2 are taken together to form an optionally substituted 6-membered heteroyclic moiety. In certain embodiments, Ri and R₂ are taken together to form an optionally substituted 7- membered heteroyclic moiety. In certain embodiments, Ri and R₂ are taken together to form an optionally substituted 8-membered heteroyclic moiety. [00100] In certain embodiments, the compound is of the formula:

wherein R₁, R₂, and V are defined herein. [00101] In certain embodiments, the compound is of the formula:

wherein R₁, R₂, and V are defined herein. [00102] In certain embodiments, the compound is of the formula:

wherein R₁, R₂, and V are defined herein.

[00103] In certain embodiments, the compound is of the formula:

wherein R₁, R₂, and V are defined herein.

[00104] In certain embodiments, the compound is of the formula:

wherein R₁, R₂, and V are defined herein.

[00105] In certain embodiments, the compound is of the formula:

wherein each of R₃-R₇ is independently hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR_A; -C(=O)R_A; -CO₂R_A; -CN; -SCN; - SR_A; -SOR_A; -SO₂RA; -NO₂; -N(RA)₂; -NHC(0)R_A; or -C(RA)₃; wherein each occurrence of R_A is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety. In certain embodiments, R₃ is hydrogen. In certain embodiments, R₃ is C₁-Ce alkyl. In certain embodiments, R₃ is -CH₃. In certain embodiments, R₄ is acyl. In certain embodiments, R₄ is -C(=O)CH₃. In certain embodiments, R₄ is -OH. In certain embodiments, R₅ is -OH. In certain embodiments, Re is hydrogen. In certain embodiments, R₇ is hydrogen. In certain embodiments, both Re and R7 are hydrogen.

[00106] Based on data from this screen, the following compounds were identified as inducer of autophagy: pimozide, trifluoperazine, and loperamide. Therefore, the identified compounds or pharmaceutically acceptable forms thereof may be useful in treating diseases where the promotion of autophagy would be beneficial (e.g., neurodegenerative diseases). These compounds represent a diverse class of structurally dissimilar compounds that have been found to induce autophagy. Either there compounds or derivatives of these compounds may be used to promote autophagy in a cell. [00107] Other modulators (i.e., both promoters and inhibitors) of autophagy identified using the screen include LY-83583, pimozide, gramicidin, manoalide, doxorubicin (e.g., doxorubicin hydrochloride), daunorubicin (e.g., daunorubicin hydrochloride), rhodomyrtoxin B, isogedunin, solanine alpha (solanidine), ellipticine, amiprilose, gentian violet, wiskostatin, manumycin A, tetrandrine, trimethobenzamide, tamoxifen, (e.g., tamoxifen citrate), RWJ- 60475 -(AM)3, amphotericin B, hexetidine, maprotiline (e.g., maprotiline hydrochloride), D609, GO6976, nigericin, methyl benzethonium chloride, nocodazole, GF-109203X, FK- 506, PPl, strophanthidinic acid lactone, mitoxantrone (e.g., mitoxantrone dihydrochloride), tyrothricin, puromycin, chukrasin, tyrphostin 9, norethindrone, colchicine, vinblastine, metixene (e.g., metixene hydrochloride), clemastine (e.g., clemastine fumarate), thioridazine (e.g., thioridazine hydrochloride), creatinine, phorbol 12-myristate 13-acetate, ZL3VS, and triflupromazine (e.g., triflupromazine hydrochloride). The identified autophagy modulators or pharmaceutically acceptable forms thereof may be used in the treatment of diseases where the modulation of autophagy would be beneficial. These compounds represent a diverse class of structurally dissimilar compounds that have been found to modulate autophagy. Either there compounds or derivatives of these compounds may be used to modulate autophagy in a cell.

[00108] Examples of diseases that may be treated using modulators of autophagy include proliferative diseases, neurodegenerative diseases, inflammatory diseases, autoimmune diseases, infectious diseases, cardiovascular diseases, or diseases characterized by protein misfolding and/or mishandling. In certain embodiments, inhibitors of autophagy are used to treat proliferative diseases such as cancer, inflammatory disease, or autoimmune where it is desired to halt the growth of unwanted cells. In other embodiments, promoters of autophagy are typically used to treat neurodegenerative diseases, cardiac disease (e.g., ischemia/reperfusion injury), or infectious diseases, where the increased clearance of unwanted proteins is desired.

Enhancers and Inhibitors ofRapamycin

[00109] In a different screen but also based on phenotypic screening, a library of compounds was screened to identify compounds capable of modifying the biological activity (i.e., cytostatic effect) of rapamycin in yeast. Rapamycin is a known inducer of autophagy. Based on the screen in yeast, inhibitors and enhancers of rapamycin's activity were identified. The twenty-one identified small- molecule inhibitors of rapamycin (SMIRs) are shown in Figure 23, and the twelve identified small-molecule enhancers of rapamycin (SMERs) are shown in Figure 24. The twenty-one SMIRs represent eighteen distinct structural classes; and the twelve SMERs represent eleven distinct structural classes. Two of the SMIRs are known bioactive compounds: D609 is a potassium xanthate derived compound and a potential glutathione mimetic, and LY83583 is a guanylate cyclase inhibitor and, specifically, a modulator of the yeast mitochondrial GTPase, Guflp. The present invention not only provides the SMIRs and SMERs as shown in Figures 23-24 but also provides derivatives of these compounds, in particular, those with the ability to modify the activity of rapamycin.

[00110] Furthermore, as part of a structure-activity relationship (SAR) analysis undertaken with respect to three of the SMERs, three analogs of SMERlO, twelve analogs of SMERl 8, and twelve analogs of SMER28 were tested for their ability to modify rapamycin's activity. These analogs of SMERlO, SMER18, and SMER28 are shown in Figure 31. Analogs of SMERlO, SMER18, and SMER28 provided by the present invention or useful in accordance with the present invention are described below.

[00111] SMERlO is an aminopyrimidone. Three analogs of SMERlO are available commercially and were tested for their ability to modify the activity of rapamycin. The pyrimidone functionality of SMERlO seems to be important for the compound's autophagy- inducing activity. SMERlOa (as shown in Figure 31) has the amino group at postion 3 removed creating a hypoxanthine. SMERlOa is slightly more active than the parent compound SMERlO. SMERlOb (as shown in Figure 31) has a bulky substitution of a phenyl group at position 2. SMERlOc (as shown in Figure 31) has a bulky substitution of a fused tetrazole. Such bulky substitutions at this side of the molecule nearly abolish activity of these compounds.

[00112] Based on the yeast screen and subsequent SAR studies, the present invention provides analogs of SMERlO. Certain SMERlO analogs have the ability to modify rapamycin's biological activity. In certain embodiments, the SMERlO analogs enhance rapamycin's biological activity. Analogs of SMERlO that are provide by the present invention or are useful in accordance with the present invention include compounds of one of the formulae:

wherein

Ri is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR_A; -C(=O)R_A; -CO₂R_A; -CN; -SCN; -SR_A; -SOR_A; -SO₂R_A; -NO₂; -N(R_A)₂; -NHC(0)R_A; or -C(R_A)3; wherein each occurrence of R_A is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₂ is is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR_B; -C(=O)R_B; -CO₂R_B; -CN; -SCN; -SR_B; -SOR_B; -SO₂R_B; -NO₂; -N(R_B)₂; -NHC(0)R_B; or -C(R_B)3; wherein each occurrence of R_A is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

*--'' is a substituted or unsubstituted heterocyclic or heteroaryl moiety; and pharmaceutically acceptable forms thereof. In certain embodiments, Ri is hydrogen. In other embodiments, Ri is C₁-Ce alkyl. In certain embodiments, Ri is substituted or unsubstituted aryl or heteroaryl. In certain embodiments, Ri is substituted or unsubstituted phenyl. In certain embodiments, Ri is unsubstituted phenyl. In certain embodiments, R₂ is hydrogen. In certain embodiments, R₂ is C₁-Ce alkyl. In certain embodiments, R₂ is acyl. In certain embodiments, R2 is -OR_B. In certain embodiments, R2 is -N(R_B)₂. In certain embodiments,

R₂ is -NHRβ. In certain embodiments, R₂ is -NH₂. In certain embodiments, — *' is a

substituted or unsubstituted heteroaryl moiety. In certain embodiments, — '' is a substituted or unsubstituted, five- or six-membered heteroaryl moiety. In certain

embodiments, ---'' is a substituted or unsubstituted pyrrole. In certain embodiments,

is a substituted or unsubstituted imidazole. In certain embodiments, -—'' is a

substituted or unsubstituted triazole. In certain embodiments, -—'' is a substituted or

unsubstituted tetrazole. In certain embodiments, -—'' is ^N . In certain embodiments, the SMERlO analog is of the formula:

wherein R₂ is hydrogen or Ci -Ce alkyl; and Ri is a defined above. In certain particular embodiments, R₂ is hydrogen.

[00113] SMERl 8 is a vinylogous amide. Twelve commercially avaible analogs of

SMERl 8 were obtained and tested for their ability to modify the biological activity of rapamycin. The analogs were used to assess vrious substitutions on the two terminal aromatic rings. Chaning the hydroxyl group from the meta position to the para position as in SMERl 8g or to the ortho position as in SMERl 8f (as shown in Figure 31) reduces but does not abolish activity. Furthermore, removal of the hydroxyl group as in SMERl 8i does abolish activity. Therefore hydroxyl group at the meta position seems to be important for the biological activity of the compound. Removal of the vinyl space as in SMERl 8d reduces but does not completely abolish activity.

[00114] Based on the yeast screen and subsequent SAR studies, the present invention provides analogs of SMERl 8. Certain SMERl 8 analogs have the ability to modify rapamycin's biological activity. In certain embodiments, the SMERl 8 analogs enhance rapamycin's biological activity. Analogs of SMERl 8 that are provide by the present invention or are useful in accordance with the present invention include compounds of one of the formulae:

wherein

Ri is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR_A; -C(=O)R_A; -CO₂R_A; -CN; -SCN; -SR_A; -SOR_A; -SO₂R_A; -NO₂; -N(R_A)₂; -NHC(O)R_A; or -C(R_A)3; wherein each occurrence of R_A is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₂ is is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR_B; -C(=O)R_B; -CO₂R_B; -CN; -SCN; -SR_B; -SOR_B; -SO₂R₈; -NO₂; -N(R_B)₂; -NHC(O)R_B; or -C(R_B)3; wherein each occurrence of R_B is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R3 is is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR_C; -C(=O)R_C; -CO₂R_C; -CN; -SCN; -SR_C; -SOR_C; -SO₂Rc; -NO₂; -N(Rc)₂; -NHC(O)Rc; or -C(Rc)3; wherein each occurrence of Rc is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; each occurrence of n is independently an integer between O and 5, inclusive; and pharmaceutically acceptable forms thereof. In certain embodiments, Ri is hydrogen. In certain embodiments, Ri is Ci-Cβ alkyl. In certain particular embodiments, Ri is methyl. In certain particular embodiments, Ri is ethyl. In certain embodiments, Ri is propyl. In certain embodiments, R₂ is -OR_B. In certain embodiments, R₂ is -OH. In certain embodiments, R₂ is halogen. In certain embodiments, R₂ is fluoro. In certain embodiments, R₂ is chloro. In certain embodiments, R₂ is bromo. In certain embodiments, R3 is -ORc. In certain embodiments, R3 is -OH. In certain embodiments, R3 is halogen. In certain embodiments, R3 is fluoro. In certain embodiments, R3 is chloro. In certain embodiments, R3 is bromo. In certain embodiments, n is O. In other embodiments, n is 1. In still other embodiments, n is 2. In yet other embodiments, n is 3. In certain embodiments, the SMERl 8 analog is of the formula:

wherein Ri, R₂, R₃ ,and n are defined as above. In certain embodiments, the SMERl 8 analog is of the formula:

wherein R₁, R₂, and R3 are defined as above. In certain embodiments, the SMERl 8 analog is of the formula:

wherein R₁, R₂, and R3 are defined as above. In certain embodiments, the SMERl 8 analog is of the formula:

wherein R₁, R₂, and R3 are defined as above. In certain embodiments, the SMERl 8 analog is of the formula:

wherein R₁, R₂, and R3 are defined as above. In certain embodiments, the SMERl 8 analog is of the formula:

wherein R₁, R₂, and R3 are defined as above. In certain embodiments, the SMERl 8 analog is of the formula:

wherein R₁, R₂, and R3 are defined as above. In certain embodiments, the SMERl 8 analog is of the formula:

wherein R₁, R₂, and R₃ are defined as above. In certain embodiments, the SMERl 8 analog is of the formula:

wherein R₁, R₂, and n are defined as above. In certain embodiments, the SMERl 8 analog is of the formula:

wherein Ri and R₂ are defined as above. In certain embodiments, the SMERl 8 analog is of the formula:

wherein Ri and R₂ are defined as above. In certain embodiments, the SMERl 8 analog is of the formula:

wherein Ri and R₂ are defined as above. In certain embodiments, the SMERl 8 analog is of the formula:

wherein Ri and R₂ are defined as above. In certain embodiments, the SMERl 8 analog is of the formula:

wherein Ri and R₂ are defined as above. In certain embodiments, the SMERl 8 analog is of the formula:

wherein Ri and R₂ are defined as above. In certain embodiments, the SMERl 8 analog is of the formula:

wherein Ri and R₂ are defined as above.

[00115] SMER28 is a substituted quinazoline. Twelve structural analogs (see Figure

31) were prepared as described in Example 1 below and were tested for their ability to modify the activity of rapamycin. The majority of individual substitutions were well tolerated. Mutliple concurrent substitutions were not generally tolerated. None of the analogs, however, turned out to be more potent than the parent compound. The desbromo analog (SMER28b) retained most of the parent compound's activity. Also, the reduced version (SMER28f) also exhibited activity. The greatest loss of activity occurred when the bromine was replace with a hydroxyl group as in SMER28J.

[00116] Based on the yeast screen and subsequent SAR studies, the present invention provides analogs of SMER28. Certain SMER28 analogs have the ability to modify rapamycin's biological activity. In certain embodiments, the SMER28 analogs enhance rapamycin's biological activity. Analogs of SMER28 that are provide by the present invention or are useful in accordance with the present invention include compounds of the formulae:

wherein

Ri is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR_A; -C(=O)R_A; -CO₂R_A; -CN; -SCN; -SR_A; -SOR_A; -SO₂R_A; -NO₂; -N(R_A)₂; -NHC(0)R_A; or -C(R_A)3; wherein each occurrence of R_A is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₂ is is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -OR_B; -C(=O)R_B; -CO₂R_B; -CN; -SCN; -SR_B; -SOR_B; -SO₂R_B; -NO₂; -N(R_B)₂; -NHC(0)R_B; or -C(R_B)3; wherein each occurrence of R_B is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; n is an integer between O and 4, inclusive; and pharmaceutically acceptable forms thereof. In certain embodiments, Ri is -OR_A. In certain embodiments, Ri is -SR_A. In certain embodiments, Ri is -NHR_A. In certain embodiments, R_A is C₁-Ce aliphatic. In certain embodiments, R_A is C₂-C_O alkenyl. In certain embodiments, R_A is vinyl. In certain embodiments, R_A is allyl. In certain embodiments, Ri is -OR_A, wherein R_A is allyl. In certain embodiments, Ri is -NHR_A, wherein R_A is allyl. In certain embodiments, R_A is benzyl. In certain embodiments, R₂ is halogen. In certain embodiments, R₂ is fluoro. In certain embodiments, R₂ is chloro. In certain embodiments, R₂ is bromo. In certain embodiments, R₂ is -0R_B. In certain embodiments, R₂ is -OH. In certain embodiments, n is 0. In certain embodiments, n is 1. In certain embodiments, n is 2. In certain embodiments, the analog of SMER28 is of the formula:

wherein Ri and R₂ are defined as above.

[00117] The compounds, including the autophagy modulators, SMIRs, and SMERs described herein, or analogs thereof may be used as therapeutic agents in the treatment of various diseases. Diseases that may be treated using the identified compounds are proliferative diseases, inflammatory diseases, autoimmune diseases, infectious diseases, cardiovascular diseases, neurodegenerative diseases, and diseases associated with protein misfolding and/or mishandling. The compounds may be used alone to treat a diseases or used in conjunction with another agent. A therapeutically effective amount of the compound is typically administered to a subject in need thereof. The subject may be any animal. In certain embodiments, the animal is a vertebrate. In certain embodiments, the animal is a mammal. In certain embodiments, the animal is a human. In certain embodiments, the animal is a domesticated animal such as a dog, cat, horse, etc.

[00118] In certain embodiments, autophagy modulators, SMIRs, SMERs, or analogs thereof are used to treat proliferative diseases. Exemplary proliferative diseases include, but are not limited to, any type of cancer, benign neoplasms, and diabetic retinopathy. Inflammatory diseases and autoimmune disease are also considered to be proliferative diseases in certain instances. In certain embodiments, the identified compounds or derivatives thereof are used to treat multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's disease, chronic or acute leukemia, lymphoma, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), breast cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, leukemia, lymphoma, skin cancer, brain cancer, cervical cancer, stomach cancer, bone cancer, pancreatic cancer, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, rectal cancer, cancer of the anal region, colon cancer, carcinoma of the Fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the Vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, cancer of the bladder, cancer of the kidney, cancer of the ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system, primary CNS lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, etc. In certain embodiments, the cancer is metastatic. In certain embodiments, the cancer is resistant or refractory to other treatment regimens. For example, the cancer may be resistant to existing treatments for the disease. [00119] Proliferative diseases are typically treated with agents that inhibit autophagy.

However, in certain embodiments, proliferative diseases may also be treated with modulators of autophagy or promoters of autophagy. In certain embodiments, proliferative diseases are treated with the inhibitors of autophagy described herein. In certain particular embodiments, the inhibitor of autophagy used to treat a proliferative disease is cefamandole, monensin, astemizole, spiramycin, (lS,9R)-beta-hydrastine, carnitine, tomatine, K252A, atranorin, tetrandrine, amlodipine, benzyl isothiocyanate, pristimerin, homochlocyclizine (e.g., homochlorocyclizine dihydrochloride), or fluoxetine (e.g., fluoxetine hydrochloride). In certain embodiments, an analog of one of these identified inhibitors of autophagy is used in the treatment of a proliferative disease. In certain embodiments, the proliferative disease is treated with a modulator of autophagy selected from the group consisting of LY-83583, pimozide, gramicidin, manoalide, doxorubicin (e.g., doxorubicin hydrochloride), daunorubicin (e.g., daunorubicin hydrochloride), rhodomyrtoxin B, isogedunin, solanine alpha (solanidine), ellipticine, amiprilose, gentian violet, wiskostatin, manumycin A, tetrandrine, trimethobenzamide, tamoxifen, (e.g., tamoxifen citrate), RWJ-60475-(AM)3, amphotericin B, hexetidine, maprotiline (e.g., maprotiline hydrochloride), D609, GO6976, nigericin, methyl benzethonium chloride, nocodazole, GF-109203X, FK-506, PPl, strophanthidinic acid lactone, mitoxantrone (e.g., mitoxantrone dihydrochloride), tyrothricin, puromycin, chukrasin, tyrphostin 9, norethindrone, colchicine, vinblastine, metixene (e.g., metixene hydrochloride), clemastine (e.g., clemastine fumarate), thioridazine (e.g., thioridazine hydrochloride), creatinine, phorbol 12-myristate 13-acetate, ZL3VS, and triflupromazine (e.g., triflupromazine hydrochloride). In certain embodiments, an analog of one of these identified modulators is used in the treatment of proliferative disease. In certain embodiments, proliferative diseases are treated with derivatives of the modulators of autophagy described herein. In certain embodiments proliferative diseases are treated with a SMER or SMIR. The SMER or SMIR may be used in conjunction with rapamycin. In other embodiments, proliferative diseases are treated with inhibitors of autophagy identified using the screen described herein. In still other embodiments, proliferative diseases are treated with compounds that are similar to, are analogs of, or are derived from the compounds described herein. For example, the inhibitors described herein may be used as a lead compound to develop other modulators of autophagy. In certain embodiments, a modulator of autophagy is combined with another agent such as a cytotoxic agent, kinase inhibitor, proteasome inhibitor, inhibitor of a growth factor pathway, or other anti-neoplastic agent. [00120] In another aspect, autophagy modulators, SMIRs, SMERs, or analogs thereof are used to treat neurodegenerative diseases. Any neurodegenerative disease may be treated using these compounds. Exemplary neurodegenerative diseases that may be treated using the compoundsdescribed herein include Alexander disease, Alper's disease, Alzheimer's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, Batten disease (Spielmeyer-Vogt-Sjogren- Batten disease), Canavan disease, Cockayne disease, corticobasal degeneration, Creutzfeldt- Jakob disease, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe disease, Lewy body disease, Machado- Joseph disease (spinocerebellar ataxia type 3), multiple sclerosis, multiple system atrophy, Parkinson's disease, Pelizaeus-Merzbacher disease, Pick's disease, primary lateral sclerosis, Refsum's disease, Sandhoff disease, Schilder's disease, spinocerebellar ataxia, spinal muscular atrophy, Steele-Richardson- Olszewski disease, tabes dorsalis, dementia, etc. In certain embodiments, the neurodegenerative disease is spinocerebellar ataxia. In certain embodiments, the neurodegenerative disease is a dementia (e.g., fronto-temporal dementia). In certain embodiments, the neurodegenerative disease is Alzheimer's disease. In certain embodiments, the neurodegenerative disease is Parkinson's disease. In certain embodiments, the neurodegenerative disease is Huntington's disease. In certain embodiments, the neurodegenerative disease is amyotrophic lateral sclerosis (ALS). In certain embodiments, a compound described herein is administered to a subject in order to prevent a neurodegenerative disease.

[00121] Infectious diseases may also be treated with autophagy modulators, SMIRs,

SMERs, or analogs thereof. Any infectious diseases may be treated using these compounds. The infectious disease may be caused by a virus, bacteria, mycobacteria, mycoplasma, spirochete, fungus, parasite, amoeba, helminth, or sporozoan. In certain embodiments, the disease is a bacterial infection. In other embodiments, the disease is a viral infection. In certain embodiments, the disease is tuberculosis, which is cause by Mycobacterium tuberculosis. In certain embodiments, the infectious disease is cause by a Group A Streptococcus. In certain embodiments, the disease is viral disease. In certain particular embodiments, the viral infection is caused by a herpes virus (e.g., herpes simplex virus type

I)-

[00122] The compounds described herein such as autophagy modulators, SMIRs,

SMERs, and analogs thereof may also be used to treat diseases that are associated with protein misfolding and/or mishanlding. Diseases associated with protein misfolding and/or mishandling include Wilson's disease, spinocerebellar ataxia, prion disease, Parkinson's disease, Huntington's disease, familial amytrophic lateral sclerosis, amyloidosis, Alzheimer's disease, Alexander's disease, alcoholic liver disease, cystic fibrosis, Pick's disease, and Lewy body dementia. In certain embodiments, such a disease is prevented using the administration of a compound described herein.

[00123] The compounds described herein such as autophagy modulators, SMIRs,

SMERs, and analogs thereof may also be used to treat cardiac diseases. In certain embodiments, the cardiac disease is ischemic cardiac diseases. In certain embodiments, the cardiac disease is cardiac disease due to reperfusion injury. In certain embodiments, a compound described herein of analog thereof is administered to a subject in order to prevent reperfusion injury. For example, a subject suffering from ischemic heart disease may be administered an autophagy enhancer in order to prevent reperfusion injury once the ischemia is relieved.

[00124] Neurodegenerative diseases, infectious diseases, cardiac diseases, and diseases characterized by protein misfolding and/or mishandling are typically treated with agents that promote autophagy. In certain embodiments, these diseases are treated with the inducers of autophagy described herein. In certain embodiments, these diseases are treated with a modulator of autophagy selected from the group consisting of LY-83583, pimozide, gramicidin, manoalide, doxorubicin (e.g., doxorubicin hydrochloride), daunorubicin (e.g., daunorubicin hydrochloride), rhodomyrtoxin B, isogedunin, solanine alpha (solanidine), ellipticine, amiprilose, gentian violet, wiskostatin, manumycin A, tetrandrine, trimethobenzamide, tamoxifen, (e.g., tamoxifen citrate), RWJ-60475-(AM)3, amphotericin B, hexetidine, maprotiline (e.g., maprotiline hydrochloride), D609, GO6976, nigericin, methyl benzethonium chloride, nocodazole, GF-109203X, FK-506, PPl, strophanthidinic acid lactone, mitoxantrone (e.g., mitoxantrone dihydrochloride), tyrothricin, puromycin, chukrasin, tyrphostin 9, norethindrone, colchicine, vinblastine, metixene (e.g., metixene hydrochloride), clemastine (e.g., clemastine fumarate), thioridazine (e.g., thioridazine hydrochloride), creatinine, phorbol 12-myristate 13-acetate, ZL3VS, and triflupromazine (e.g., triflupromazine hydrochloride). In certain embodiments, these diseases are treated with derivatives of the modulators of autophagy described herein. In other embodiments, the diseases are treated with inducers of autophagy identified using the screen described herein. In still other embodiments, these diseases are treated with compounds that are similar to or are derived from the compounds described herein, in particular, derivatives or analogs of the autophagy inducers described herein. For example, the inhibitors described herein may be used as a lead compound to identify or prepare other inducers of autophagy. In certain embodiments, an inducer of autophagy is used in conjunction with another agent typically used to treat the disease.

[00125] Determination of a therapeutically effective amount or a prophylactically effective amount of the compound utlized in accordance with the present invention, can be readily made by the physician or veterinarian (the "attending clinician"), as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. The dosages may be varied depending upon the requirements of the subject in the judgment of the attending clinician; the severity of the condition being treated and the particular compound being employed. In determining the therapeutically effective amount or dose, and the prophylactically effective amount or dose, a number of factors are considered by the attending clinician, including, but not limited to: the specific disease state; pharmacodynamic characteristics of the particular agent and its mode and route of administration; the desired time course of treatment; the species of mammal; its size, age, and general health; the specific disease involved; the degree of or involvement or the severity of the disease; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the kind of concurrent treatment (i.e., the interaction of the compound of the invention with other co-administered therapeutic agents); and other relevant circumstances.

[00126] Treatment can be initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage may be increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired. A therapeutically effective amount and a prophylactically effective amount of a compound is expected to vary from about 0.1 milligram per kilogram of body weight per day (mg/kg/day) to about 100 mg/kg/day. In certain embodiments, the daily dosage ranges from 0.1 mg/kg/day to 10 mg/kg/day. The dosages given herein are dose equivalents with respect to the active ingredient.

[00127] The administration of the therapeutically effective amount of a compound may be by any route of administering known in the pharmaceutical arts. The compound may be administered orally, parenterally, intravenously, transdermally, submuscosally, inhalationally, rectally, vaginally, subcutaneously, intramuscularly, intrathecally, etc. In certain embodiments, the compound is administered orally. In certain embodiments, the compound is administered parenterally. In certain embodiments, the compound is administered intravenously.

[00128] Compounds determined to be effective for the prevention or treatment of proliferative diseases, autoimmune diseases, inflammatory diseeases, cardiac diseases, infectious diseases, neurodegenerative diseases, or protein misfolding disorders in animals, e.g., dogs, chickens, and rodents, may also be useful in treatment of the disorders in humans. Those skilled in the art of treating these diseases in humans will know, based upon the data obtained in animal studies, the dosage and route of administration of the compound to humans. In general, the dosage and route of administration in humans is expected to be similar to that in other animals.

[00129] The identification of those patients who are in need of prophylactic treatment for proliferative disease states is well within the ability and knowledge of one skilled in the art. Certain of the methods for identification of patients which are at risk of developing proliferative disease states which can be treated by the subject method are appreciated in the medical arts, such as family history, the presence of risk factors associated with the development of that disease state in the subject patient. A clinician skilled in the art can readily identify such candidate patients, by the use of, for example, clinical tests, physical examination, and medical/family history.

[00130] Methods of modulating autophagy in a cell comprise contacting cells or subjects with a modulator of autophagy as described herein. The contacting may be by addition of the inhibitor to a fluid surrounding the cells, for example, to the growth media in which the cells are living or existing. The contacting may also be by directly contacting the modulator to the cells. Alternately, the contacting may be by passage of the modulator through a subject, for example, after administration, depending on the route of administration, the inhibitor may travel through the digestive tract or the blood stream or may be applied or administered directly to cells in need of the autophagy modulation.

Pharmaceutical Compositions

[00131] This invention also provides a pharmaceutical preparation comprising at least one of the compounds described herein, or a pharmaceutically acceptable derivative thereof, which compounds module autophagy or modulate the biological activity of rapamycin or other autophagy inhibitor. In certain embodiments, the compounds are cytotoxic (e.g., the compound inhibit autophagy) and are useful in the treatment of proliferative diseases. In other embodiments, the compounds stimulate autophagy and are useful in treating diseases such as neurodegenerative diseases or diseases associated with protein misfolding or mishandling. In other embodiments, the compounds show cytostatic or cytotoxic activity against neoplastic cells such as cancer cells. In yet other embodiments, the compounds inhibit the growth of or kill rapidly dividing cells such as stimulated inflammatory cells. [00132] As discussed above, the present invention provides novel compounds that modulate autophagy, and thus the inventive compounds are useful for the treatment of a variety of medical conditions including cancer, benign neoplasms, autoimmune diseases, inflammatory diseases, diabetic retinopathy, neurodegengerative diseases, cardiovascular diseases, infectious diseases, or diseases associated with protein misfolding. Accordingly, in another aspect of the present invention, pharmaceutical compositions are provided, wherein these compositions comprise any one of the compounds as described herein, and optionally comprise a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises an activator of autophagy. Exemplary activators of autophagy include SMERlO. In other embodiments, the pharmaceutical composition comprises an inhibitor of autophagy. Exemplary inhibitors of autophagy include cefamandole, monensin, astemizole, spiramycin, (lS,9R)-beta-hydrastine, carnitine, tomatine, K252A, atranorin, tetrandrine, amlopidine, benzyl isothiocyanate, pristimerin, homochlorocyclizine, fluoxetine, LY-83583, pimozide, gramicidin, manoalide, doxorubicin, daunorubicin, rhodomyrtoxin B, isogedunin, solanine alpha (solanidine), elliticine, amiprilose, gentian violet, wiskostatin, manumycin A, tetrandrine, trimethobenzamide, tamoxifen, RWJ-60475-(AM)3, amphotericin B, hexetidine, maprotiline, D609, GO6976, nigericin, methyl benzethonium chloride, nocodazole, GF-109203X, FK-506, PPl, strophanthidinic acid lactone, mitoxantrone, tyrothricin, puromycin, chukrasin, tyrphostin 9, norethindrone, colchicine, vinblastine, metixene, clemastine, thioridazine, creatinine, phorbol 12-myristate 13 -acetate, ZL3VS, and triflupromazine. In certain embodiments, the autophagy inhibitors is a late inhibitor of autophagosome-lysosome fusion (e.g., cefamandole, monensin, astemizole, spiramycin, (lS,9R)-beta-hydrastine, carnitine, tomatine, K252A, atranorin, tetrandrine, amlopidine, benzyl isothiocyanate, pristimerin, homochlorocyclizine, and fluoxetine) [00133] In certain embodiments, these compositions optionally further comprise one or more additional therapeutic agents, e.g., another another anti-proliferative agent. In certain embodiments, the other agent is an inhibitor of a growth factor pathway. The inhibitor of a growth factor pathway may be a small molecule, a protein (e.g., an antibody or antibody fragment), a peptide, or a polynucleotide. In certain embodiments, the inhibitor of the growth factor pathway is a small molecule. In other embodiments, the inhibitor of the growth factor pathway is a protein. In certain embodiments, the inhibitor of the growth factor pathway is an antibody or a fragment thereof. In certain particular embodiments, the inhibitor of the growth factor pathway is a humanized antibody or antibody fragment. In certain particular embodiments, the other agent is a kinase inhibitor In certain particular embodiments, the kinase inhibitor is a receptor tyrosine kinase inhibitor. In certain particular embodiments, the composition comprises another agent such as erlotinib (T ARCEV A^®), gefitinib (IRESSA^®), cetuximab, sorafenib (NEXAVAR), dasatinib, ZD6474 (ZACTIMA), lapatinib (TYKERB), STI571, imatinib (GLEEVEC^®), lestaurtinib (CEP-701), sunitinib maleate (SUTENT), panitumumab, EMD 72000, TheraCIM hR3, EKB-569, 2C4, AMG706, MP-412, XL647, XL 999, MLN518, PKC412, AMN 107, AEE708, OSI-930, OSI-817, and AG-013736. In certain embodiments, the additional agent is gefitinib (IRESSA^®). In other embodiments, the additional agent is imatinib (GLEEVEC^®). In yet other embodiments, the additional agent is erlotinib (TARCEV A^®). In yet other embodiments, the additional agent is cetuximab. In yet other embodiments, the additional agent is dasatinib. In other embodiments, the additional agent is ZD6474 (ZACTIMA). In other embodiments, the additional agent is lapatinib (TYKERB). In still other embodiments, the additional agent is lestaurtinib (CEP-701). In still other embodiments, the additional agent is sunitinib maleate (SUTENT). [00134] In certain embodiments, the invention provides a combination of an autophagy inhibitor and a kinase inhibitor for the treatment of a proliferative disease. In certain particular embodiments, the combination pharmaceutical composition comprises an autophagy inhibitor selected from the group consisting of cefamandole, monensin, astemizole, spiramycin, (lS,9R)-beta-hydrastine, carnitine, tomatine, K252A, atranorin, tetrandrine, amlopidine, benzyl isothiocyanate, pristimerin, homochlorocyclizine, fluoxetine, LY-83583, pimozide, gramicidin, manoalide, doxorubicin, daunorubicin, rhodomyrtoxin B, isogedunin, solanine alpha (solanidine), elliticine, amiprilose, gentian violet, wiskostatin, manumycin A, tetrandrine, trimethobenzamide, tamoxifen, RWJ-60475-(AM)3, amphotericin B, hexetidine, maprotiline, D609, GO6976, nigericin, methyl benzethonium chloride, nocodazole, GF-109203X, FK-506, PPl, strophanthidinic acid lactone, mitoxantrone, tyrothricin, puromycin, chukrasin, tyrphostin 9, norethindrone, colchicine, vinblastine, metixene, clemastine, thioridazine, creatinine, phorbol 12-myristate 13 -acetate, ZL3VS, and triflupromazine; and a kinase inhibitor selected from the group consisting of erlotinib (TARCEVA^®), gefitinib (IRESSA^®), cetuximab, sorafenib (NEXAVAR), dasatinib, ZD6474 (ZACTIMA), lapatinib (TYKERB), STI571, imatinib (GLEEVEC^®), lestaurtinib (CEP-701), sunitinib maleate (SUTENT), panitumumab, EMD 72000, TheraCIM hR3, EKB-569, 2C4, AMG706, MP-412, XL647, XL 999, MLN518, PKC412, AMN107, AEE708, OSI-930, OSI- 817, and AG-013736.

[00135] In certain embodiments, the inventive compositions further comprise a proteasome inhibitor. The agents may be packaged separaterly or together in the same composition. In certain embodiments, the proteasome inhibitor is selected from the group consisting of Exemplary proteasome inhibitors that may be used in combination with an autophagy modulator include, but are not limited to, bortezomib (VELCADE^®), peptide boronates, salinosporamide A (NPI-0052), lactacystin, epoxomicin (Ac(Me)-Ile-Ile-Thr-Leu- EX), MG-132 (Z-Leu-Leu-Leu-al), PR-171, PS-519, eponemycin, aclacinomycin A, CEP- 1612, CVT-63417, PS-341 (pyrazylcarbonyl-Phe-Leu-boronate), PSI (Z-IIe-GIu(OtBu)-AIa- Leu-al), MG-262 (Z-Leu-Leu-Leu-bor), PS-273 (MNLB), omuralide (c/αsto-lactacystin-β- lactone), NLVS (Nip-Leu-Leu-Leu-vinyl sulfone), YLVS (Tyr-Leu-Leu-Leu-vs), dihydroeponemycin, DFLB (dansyl-Phe-Leu-boronate), ALLN (Ac-Leu-Leu-Nle-al), 3,4- dichloroisocoumarin, 4-(2-aminoethyl)-benzenesulfonyl fluoride, TMC-95A, gliotoxin, EGCG ((-)-epigallocatechin-3-gallate), and YUlOl (Ac-hFLFL-ex). In certain embodiments, the proteasome inhibitor is bortezomib (VELCAD E^®). In certain particular embodiments, the composition includes an autophagy inhibitor and a proteasome inhibitor. In certain particular embodiments, the combination pharmaceutical composition comprises an autophagy inhibitor selected from the group consisting of cefamandole, monensin, astemizole, spiramycin, (lS,9R)-beta-hydrastine, carnitine, tomatine, K252A, atranorin, tetrandrine, amlopidine, benzyl isothiocyanate, pristimerin, homochlorocyclizine, fluoxetine, LY-83583, pimozide, gramicidin, manoalide, doxorubicin, daunorubicin, rhodomyrtoxin B, isogedunin, solanine alpha (solanidine), elliticine, amiprilose, gentian violet, wiskostatin, manumycin A, tetrandrine, trimethobenzamide, tamoxifen, RWJ-60475-(AM)3, amphotericin B, hexetidine, maprotiline, D609, GO6976, nigericin, methyl benzethonium chloride, nocodazole, GF- 109203X, FK-506, PPl, strophanthidinic acid lactone, mitoxantrone, tyrothricin, puromycin, chukrasin, tyrphostin 9, norethindrone, colchicine, vinblastine, metixene, clemastine, thioridazine, creatinine, phorbol 12-myristate 13-acetate, ZL3VS, and triflupromazine; and a proteasome inhibitor selected from the group consisting of bortezomib (VELCADE^®), peptide boronates, salinosporamide A (NPI-0052), lactacystin, epoxomicin (Ac(Me)-IIe-IIe- Thr-Leu-EX), MG-132 (Z-Leu-Leu-Leu-al), PR-171, PS-519, eponemycin, aclacinomycin A, CEP- 1612, CVT-63417, PS-341 (pyrazylcarbonyl-Phe-Leu-boronate), PSI (Z-IIe-GIu(OtBu)- Ala-Leu-al), MG-262 (Z-Leu-Leu-Leu-bor), PS-273 (MNLB), omuralide (c/αsto-lactacystin- β-lactone), NLVS (Nip-Leu-Leu-Leu-vinyl sulfone), YLVS (Tyr-Leu-Leu-Leu-vs), dihydroeponemycin, DFLB (dansyl-Phe-Leu-boronate), ALLN (Ac-Leu-Leu-Nle-al), 3,4- dichloroisocoumarin, 4-(2-aminoethyl)-benzenesulfonyl fluoride, TMC-95A, gliotoxin, EGCG ((-)-epigallocatechin-3-gallate), and YUlOl (Ac-hFLFL-ex). [00136] It will also be appreciated that certain of the compounds of the present invention can exist in free form for treatment, or where appropriate, as a pharmaceutically acceptable derivative thereof. According to the present invention, a pharmaceutically acceptable derivative includes, but is not limited to, pharmaceutically acceptable salts, esters, salts of such esters, or any other adduct or derivative which upon administration to a patient in need is capable of providing, directly or indirectly, a compound as otherwise described herein, or a metabolite or residue thereof, e.g., a prodrug.

[00137] As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences , 66: 1-19, 1977; incorporated herein by reference. The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base functionality with a suitable organic or inorganic acid. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hernisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate, and aryl sulfonate.

[00138] Additionally, as used herein, the term "pharmaceutically acceptable ester" refers to esters which hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety advantageously has not more than 6 carbon atoms. Examples of particular esters include formates, acetates, propionates, butyrates, acrylates, and ethylsuccinates. In certain embodiments, the esters are cleaved by enzymes such as esterases. [00139] Furthermore, the term "pharmaceutically acceptable prodrugs" as used herein refers to those prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention. The term "prodrug" refers to compounds that are rapidly transformed in vivo to yield the parent compound of the above formula, for example by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference.

[00140] As described above, the pharmaceutical compositions of the present invention additionally comprise a pharmaceutically acceptable carrier, which, as used herein, includes any and all solvents, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical Sciences, Fifteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1975) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the anti-cancer compounds of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; Cremophor; Solutol; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

Uses of Compounds and Pharmaceutical Compositions

[00141] The invention further provides a method of treating proliferative disorders, inflammatory disease, autoimmune diseases, infectious diseases, cardiovascular diseases, neurodegenerative disorders, and disease associated with protein misfolding and/or mishandling. The method involves the administration of a therapeutically effective amount of a compound described herein or a pharmaceutically acceptable form thereof to a subject (including, but not limited to a human, vertebrate, mammal, domesticated animal, or animal) in need thereof.

[00142] The compounds and pharmaceutical compositions of the present invention may be used in treating or preventing any disease or conditions including proliferative diseases (e.g., cancer, benign neoplasms), inflammatory disease (e.g., autoimmune diseases), neurodegenerative disorders (e.g., Parkinson's disease), and diseases associated with protein misfolding or mishandling (e.g., cystic fibrosis). The compounds and pharmaceutical compositions may be administered to animals, preferably mammals (e.g., domesticated animals, cats, dogs, mice, rats), and more preferably humans. Any method of administration may be used to deliver the compound of pharmaceutical compositions to the animal. In certain embodiments, the compound or pharmaceutical composition is administered orally. In other embodiments, the compound or pharmaceutical composition is administered parenterally.

[00143] In yet another aspect, according to the methods of treatment of the present invention, cells are killed, or their growth is inhibited by contacting the cells with a compound, as described herein. Thus, in still another aspect of the invention, a method for the treatment of a proliferative disease is provided comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an autophagy modulator (e.g., an autophagy inhibitor) to a subject in need thereof, in such amounts and for such time as is necessary to achieve the desired result. In certain embodiments of the present invention a "therapeutically effective amount" of the inventive compound or pharmaceutical composition is that amount effective for killing or inhibiting the growth of the unwanted or malignant cells. The compounds and compositions, according to the method of the present invention, may be administered using any amount and any route of administration effective for killing or inhibiting the growth of these cells. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular compound, its mode of administration, its mode of activity, and the like. The compounds of the invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

[00144] In yet another aspect, according to the methods of treatment of the present invention, the processing of misfolded or aggregated proteins in cells is increased by contacting the cells with a compound as described herein. In yet another aspect, according to the methods of treatment of the present invention, the degradation of the proteins of infectious organisms is increased by contacting the cells with a compound as described herein. In still another aspect of the invention, a method for the treatment of a neurodegenerative disease or a disease associated with protein misfolding and/or mishandling is provided comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an autophagy modulator to a subject in need thereof, in such amounts and for such time as is necessary to achieve the desired result. In certain embodiments of the present invention, a "therapeutically effective amount" of the inventive compound or pharmaceutical composition is that amount effective for upregulating the degradation of misfolded proteins in the cell or proteins of an infectious organism. The compounds and compositions, according to the method of the present invention, may be administered using any amount and any route of administration effective for upregulating autophagy in the cell. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular compound, its mode of administration, its mode of activity, and the like. The compounds of the invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

[00145] Furthermore, after formulation with an appropriate pharmaceutically acceptable carrier in a desired dosage, the pharmaceutical compositions of this invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), bucally, as an oral or nasal spray, or the like, depending on the severity of the infection being treated. In certain embodiments, the compounds of the invention may be administered orally or parenterally at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect. The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).

[00146] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the compounds of the invention are mixed with solubilizing agents such an Cremophor, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and combinations thereof. [00147] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[00148] The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[00149] In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide- polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

[00150] Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non- irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

[00151] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar— agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents. [00152] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like.

[00153] The active compounds can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

[00154] Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, and eye drops are also contemplated as being within the scope of this invention. Additionally, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel. [00155] It will also be appreciated that the compounds and pharmaceutical compositions of the present invention can be employed in combination therapies, that is, the compounds and pharmaceutical compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, an inventive compound may be administered concurrently with another anticancer agent), or they may achieve different effects (e.g., control of any adverse effects).

[00156] In still another aspect, the present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention, and in certain embodiments, includes an additional approved therapeutic agent for use as a combination therapy. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

Screening

[00157] The present invention also provides a system for screening chemical compounds to identify modulators of autophagy. Given the usefulness of compounds that modulate autophagy, a screening system has been developed for use in identifying compounds that modulate autophagy. The screening system may be used to identify inhibitors as well as stimulators of autophagy. In certain embodiments, the screening system is performed in a high-throughput format allowing for the screening of tens, hundreds, or thousands of compounds at once.

[00158] The screening methods is based on phenotypic changes in cells treated with autophagy modulators. Cells are treated with a test compound under suitable conditions to inhibit autophagy. The cells are then visualized by microscopy, and images of the treated cells are acquired. Optionally, the cells are stained before visualization. For example, in certain embodiments, the cells are stained with EGFP-LC3, which localized to autophagic membranes, to identify EGFP-LC3 positive autophagosomes. In other embodiments, the cells are stained with DAPI to visualize nuclei. The cells may be stained with more than one stain to visualize various biomolecules or organelles in the cells. The acquired images are then processed to determine if the cells exhibit one or more of the phenotypic characteristics of autophagy. Examples of phenotypic characteristics associated with autophagy include number of EGFP-LC3 positive autophagosomes, total vesicle size, average vesicle size, total vesicle intensity, number of vesicles, imaging EGFP-LC3, or other indicia of autophagy. Compounds that lead to changes in phenotypic characteristics associated with autophagy as compared to a control are identified as either inhibitors or promoters of autophagy. In certain embodiments, one or more characteristics of autophagy is affected befor a test compound is identified as a modulator of autophagy. In certain embodiments, two or more characteristics of autophagy are altered. In other embodiments, three or more characteristics are changed. Novel compounds identified by the inventive screen are considered to be part of the invention. Such compounds may be formulated as described herein and used to treat proliferative diseases, neurodegenerative disease, or protein misfolding diseases. [00159] A similar screening system may also be used to identify compounds that modulate the activity of an autophagy modulator. In certain embodiments, the autophagy modulator is rapamycin, a known autophagy inhibitor. Cells are treated in combination with a test compound and a known modulator of autophagy. Phenotypic characteristics of the treated cells are compared to the characteristics of cells treated with the autophagy modulator alone. Compounds that affect at least one phenotypic characteristic as compared to the control are identified as enhancers or inhibitors of the autophagy modulator. Example 1 below describes such a screen to identify modulators of rapamycin' s biological activity. [00160] The present invention also provides kits for used in practicing the inventive screening methods. The kits may include all or a portion of the reagents needed to screen a library of compounds. In certain embodiments, the kits includes all or some of the following: cell line, multi-well plates, cell culture plates, media, buffer, autophagy modulator (e.g., inhibitors and/or promoters), stains, software, and instructions. The kits may be packaged with enough materials to screen at least 10, 50, 100, 200, 300, 400, 500, 1000, or 2000 compounds. Preferably the components of the kit are conveniently packaged for use by a researcher. [00161] These and other aspects of the present invention will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain particular embodiments of the invention but are not intended to limit its scope, as defined by the claims.

Examples

Example 1-Small-molecule enhancers of rapamycin-induced TOR inhibition promote autophagy and reduce toxicity in Huntington's disease models

Introduction

[00162] Autophagy is an important process modulating the penetrance of a range of human diseases caused by toxic, aggregate-prone, intracytosolic proteins, which become inaccessible to the proteasome when they oligomerise. These diseases include Huntington's disease (HD), an autosomal-dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion (>35 repeats), which encodes an abnormally long polyglutamine (polyQ) tract in the N-terminus of the huntingtin protein (Ravikumar et al. Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy. Hum. MoL Genet. 11, 1107-17 (2002); Ravikumar et al. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat. Genet. 36, 585-95 (2004); Rubinsztein, Lessons from animal models of Huntington's disease. Trends Genet. 18, 202-9 (2002); each of which is incorporated herein by reference). Mutant huntingtin toxicity is thought to be exposed after it is cleaved to form N-terminal fragments comprising the first 100-150 residues with the expanded polyQ tract, which are also the toxic species found in aggregates. Thus, HD pathogenesis is frequently modelled with exon 1 fragments containing expanded polyQ repeats which cause aggregate formation and toxicity in cell models and in vivo (Rubinsztein,Lessons from animal models of Huntington's disease. Trends Genet. 18, 202-9 (2002); incorporated herein by reference).

[00163] In addition to mutant huntingtin, autophagy also regulates the clearance of other aggregate-prone disease-causing proteins, like those causing spinocerebellar ataxia type 3, forms of tau (causing fronto-temporal dementias) and the A53T and A3 OP α-synuclein mutants (which cause familial Parkinson's disease (PD)) (Sarkar et al. Lithium induces autophagy by inhibiting inositol monophosphatase. J. Cell Biol. 170, 1101-11 (2005); Ravikumar et al.. Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy. Hum. MoI. Genet. 11, 1107-17 (2002); Ravikumar et al. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat. Genet. 36, 585-95 (2004); Berger et al. Rapamycin alleviates toxicity of different aggregate-prone proteins. Hum. MoI. Genet. 15, 433-42 (2006); Webb et al. Alpha-Synuclein is degraded by both autophagy and the proteasome. J. Biol. Chem. 278, 25009-13 (2003); each of which is incorporated herein by reference). Autophagy induction reduces mutant huntingtin levels and protects against its toxicity in cell, Drosophila and mouse models (Ravikumar et al.. Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy. Hum. MoI. Genet. 11, 1107-17 (2002); Ravikumar et al. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat. Genet. 36, 585-95 (2004); each of which is incorporated herein by reference). Similar effects are seen with other polyQ-containing proteins and tau in cells and flies (Berger et al. Rapamycin alleviates toxicity of different aggregate-prone proteins. Hum. MoI. Genet. 15, 433-42 (2006); incorporated herein by reference). Another class of diseases that may be treatable by autophagy upregulation are certain bacterial and viral infections, where the pathogens can be engulfed by autophagosomes and transferred to lysosomes for degradation. These include Mycobacterium tuberculosis (that causes tuberculosis), Group A Streptococcus , and viruses like herpes simplex virus type I (Nakagawa et al. Autophagy defends cells against invading group A Streptococcus. Science 306, 1037-40 (2004); Ogawa et al. Escape of intracellular Shigella from autophagy. Science 307, 727-31 (2005); Talloczy et al. PKR- Dependent Autophagic Degradation of Herpes Simplex Virus Type I. Autophagy 2, 24-9 (2006); each of which is incorporated herein by reference).

[00164] Currently, the only confirmed pharmacological strategy for upregulating autophagy in mammalian brains is treatment with rapamycin, a specific TOR inhibitor (Ravikumar et al. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat. Genet. 36, 585-95 (2004); incorporated herein by reference). However, TOR controls several cellular processes besides autophagy in organisms from yeast to man, including repression of ribosome biogenesis and protein translation, and transcriptional induction of compensatory metabolic pathways (Sarbassov et al. Growing roles for the mTOR pathway. Curr. Opin. Cell Biol. 17, 596-603 (2005); incorporated herein by reference). These probably contribute to the complications seen with long-term rapamycin use, like immunosuppression, which is not compatible with therapy for infectious diseases. Accordingly, safer ways of inducing autophagy are needed, for example, either by identifying mTOR substrates regulating autophagy, identifying compounds that enhance the activity of rapamycin, or identifying compounds acting via mTOR- independent pathways.

[00165] Novel compounds that activate mammalian autophagy have been identified using a primary yeast-based screen. Three small molecules that enhance the cytostatic effects of rapamycin in yeast also induce autophagy independently of rapamycin in mammalian cells. These small molecules enhance the clearance of mutant aggregate-prone proteins and reduce mutant huntingtin toxicity in both cell and Drosophila models of HD. Follow-up screens of structural analogs of these compounds identified additional autophagy inducers that may have potential for the treatment of HD and related neurodegenerative disorders.

Results and Discussion

Identification of small-molecule enhancers and suppressors of the cytostatic effects of rapamycin in a yeast-based screen

[00166] Novel enhancers of mammalian autophagy have been identified by starting with a small-molecule screen in yeast (Huang et al. Finding new components of the target of rapamycin (TOR) signaling network through chemical genetics and proteome chips. Proc. Natl. Acad. ScL USA 101, 16594-9 (2004); incorporated herein by reference). We reasoned that a small-molecule screen would uncover enhancers and suppressors of the physiological state induced by rapamycin in yeast, and that the activities of at least some of these modifiers would be conserved in mammalian systems. We observed suppressors of the cytostatic effects of rapamycin in yeast -small-molecule inhibitors of rapamycin (SMIRs)-in a temporal window that spans 48-72 hours; we discerned both fast- and slow-acting SMIRs, but hereafter do not distinguish between them. Later, at 96 hours, we observed enhancers of the cytostatic effects of rapamycin-small-molecule enhancers of rapamycin (SMERs)-in wells containing yeast that failed to exit rapamycin-induced growth arrest. [00167] We retested 72 total primary assay positives, from which we identified a structurally non-redundant set of 21 SMIRs and 12 SMERs (Figure 16a; structures appear in Figures 23 and 24). The 21 SMIRs comprise 18 distinct structural classes; the 12 SMERs comprise 11 structural classes. Interestingly, two SMIRs are known bioactive compounds: D609 is a potassium xanthate derivative and a potential glutathione mimetic (Sultana et al. Protective effect of the xanthate, D609, on Alzheimer's amyloid β-peptide (l-42)-induced oxidative stress in primary neuronal cells. Free Radical Research 38, 449-458 (2004); incorporated herein by reference); LY-83583 has been historically described as a guanylate cyclase inhibitor (Mulsch et al. LY 83583 interferes with the release of endothelium-derived factor and inhibits soluble guanylate cyclase. J. Pharmacol. Exp. Ther. 247 ^', 283-288 (1988); incorporated herein by reference), and more recently, as a modulator of the yeast mitochondrial GTPase, Guflp (Butcher et al. Microarray-based method for monitoring yeast overexpression strains reveals small-molecule targets in TOR pathway. Nat. Chem. Biol. 2, 103-109 (2006); incorporated herein by reference). We determined the half-maximal effective concentration (EC₅₀) of suppression and enhancement of the cytostatic effects of rapamycin by each SMIR and SMER, respectively (Figure 16b). The EC50 of suppression spans two orders of magnitude, from >50 μM to as low as 0.37 μM. Four SMIRs displayed sub-micromolar suppression of rapamycin (described, where appropriate, by their core heterocycle): D609; SMIR28, a thiourea; SMIR30, a dihydroquinoline; SMIR32, a quinazoline. The EC50 of enhancement spanned a smaller range from 50 μM to 1.4 μM, with SMERl 7, a piperazine, being the strongest enhancer. The overall suppression and enhancement profile was neither strain- nor species-specific, as all tested SMIRs and SMERs exhibited comparable activity in another S. cerevisiae strain (RMl 1-1 a), as well as in laboratory strains of Saccharomyces bay anus, Saccharomyces mikatae, and Saccharomyces paradoxus. Dose responses were performed in rich media (YPD), and in almost every instance, suppression or enhancement is insensitive to changes in carbon or nitrogen source in the culture media, with a few exceptions (Figure 16b).

Potency and selectivity of the small-molecule modifiers of the cytostatic effects of rapamycin [00168] Most modifiers displayed modest activity (10 μM-50 μM) (Figure 16b), which may be explained either by weak to modest small-molecule modulation of TOR- relevant targets, or by modest to strong small-molecule modulation of TOR- irrelevant targets, e.g., xenobiotic-response genes. In order to distinguish between these two possibilities, we assessed the selectivity of each small-molecule modifier against small-molecule perturbagens (SMPs) other than rapamycin, including ones that either target processes related or unrelated to those affected by rapamycin. Our goal was to eliminate SMIRs and SMERs that exhibit a lack of selectivity towards the cellular actions of other SMPs; however, a useful by-product of this analysis is the discovery of potentially selective small-molecule modifiers of the growth inhibition induced by SMPs other than rapamycin. We included the protein-synthesis inhibitors cycloheximide (CHX) and anisomycin (Jacklet, Neuronal circadian rhythm: phase shifting by a protein synthesis inhibitor. Science 198, 69-71 (1977); incorporated herein by reference); the microtubule depolymerizer nocodazole; the protein-glycosylation inhibitor tunicamycin; the oxidant menadione.

[00169] The most non-selective SMIRs are 19a and 19b, two structurally related thiophenes; these compounds suppressed 6 of 6 assayed compounds (Figure 17a) but enhanced the ergosterol-biosynthesis inhibitors ketoconazole and flutrimazole, which suggests that SMIR19a and SMIR19b promote xenobiotic efflux by altering membrane permeability. No other SMIRs and none of the SMERs suppressed the anti-proliferative effects of menadione, an inducer of oxidative stress, which is a pathway not directly controlled by TOR. Interestingly, four SMIRs (7, 15, 16 and 18) enhanced the antiproliferative effects of anisomycin, while seven of twelve SMERs (3, 6, 10, 14, 20, 22 and 23) suppressed the antiproliferative effects of both anisomycin and CHX (Figure 17a and 17b). We hypothesize that the subset of SMIRs that enhances the growth inhibition induced by protein-synthesis inhibitors does so by modulating regulatory targets upstream of ribosomes. This hypothesis is consistent with our observation that neither CHX nor anisomycin, which both inhibit protein synthesis at the ribosome, is a SMIR, i.e., suppresses the cytostatic effects of rapamycin in yeast at sub-lethal concentrations (Figures 25a and 25b).

Secondary screen of SMERs and SMIRs as autophagy modulators in mammalian cells [00170] Although these SMIRs and SMERs were discovered as being modulators of the effects of rapamycin on yeast growth, we tested if they were also potential modulators of mammalian autophagy. We screened all the yeast primary assay positives in mammalian cells (including hits that were excluded from the above analyses in yeast due to their poor potency) in the absence of rapamycin for their potential to induce clearance of the autophagy substrate A53T α-synuclein. We used a stable doxycycline-inducible PC 12 cell line expressing A53T mutant α-synuclein, where the transgene expression is first induced by adding doxycycline and then switched off by removing doxycycline from the medium (Webb et al. Alpha-Synuclein is degraded by both autophagy and the proteasome. J. Biol. Chem. 278, 25009-13 (2003); incorporated herein by reference). If the transgene expression level is measured at 24 hours after switching off expression after an initial induction period of 48 hours, one can assess if specific agents alter the clearance of the transgene product, as the amount of transgene product decays when synthesis is stopped (Webb et al. Alpha-Synuclein is degraded by both autophagy and the proteasome. J. Biol. Chem. 278, 25009-13 (2003); incorporated herein by reference). Interestingly, we observed that 13 SMIRs (14-18, 19a, 19b, 22, 23, 29a, 29b, 30, 31) slowed the clearance of this autophagy substrate, while 4 SMERs (10, 16, 18 and 28) enhanced its clearance (Figures 26a and 27a). We categorised SMIRs as possible inhibitors of autophagy if they increased the levels of A53T α-synuclein by 40-50% or more. Likewise, we categorised SMERs as possible enhancers of autophagy if they reduced the levels of this mutant protein to 50% or lower than the control (Figures 26b and 27b).

SMERs 10, 18, and 28 reduce mutant huntingtin aggregation and toxicity by autophagy [00171] We then concentrated on the autophagy-inducing SMERs, which may have the greatest immediate utility as drug leads for neurodegenerative diseases. We confirmed that SMERs 10, 18, and 28 significantly enhanced A53T α-synuclein clearance in stable PC12 cells independent of rapamycin treatment (Figures 18a and 18b). We next studied the effect of these SMERs on mutant huntingtin, another aggregate-prone protein cleared by autophagy (3; 4; each of which is incorporated herein by reference). SMERs 10, 18 and 28 reduced aggregation and cell death caused by EGFP-tagged huntingtin exon 1 with 74 polyQ repeats (EGFP HDQ74) in COS-7 cells (Figure 18c). We excluded SMER 16 (subsequently redesignated SMIR 33 because upon additional retesting it was found to a suppressor of the cytostatic effects of rapamycin) from our subsequent experiments as it was toxic in COS-7 and other cell lines at the concentration that enhanced the clearance of A53T α-synuclein in PC12 cells. No overt toxicity was observed with SMERs 10, 18, and 28. [00172] To confirm that this reduction of EGFP-HDQ74 aggregation is through autophagy, we used autophagy -competent mouse embryonic fibroblasts (MEFs) (Atg5^+/+) or matched MEFs lacking the essential autophagy gene Atg5 (Atg5 ^) (17; incorporated herein by reference). EGFP-HDQ74 aggregation was significantly increased in untreated Atg5 (autophagy-deficienct) cells compared to untreated Atg5^+/+ cells, as mutant huntingtin is an autophagy substrate (Figure 28). When these cells were treated with SMERs 10, 18, and 28, the EGFP-HDQ74 aggregation was significantly reduced in Atg5^+/+ cells, but not in Atg5^~7~ cells (Figures 18d and 18e). Thus, these SMERs can only reduce mutant huntingtin aggregation in autophagy-competent cells.

SMERs 10, 18 and 28 induce autophagy in mammalian cells

[00173] We first assessed the effect of these SMERs on autophagy by transfecting

COS-7 cells with the microtubule-associated protein 1 light chain 3 (LC3) fused to EGFP (EGFP-LC3). LC3 (and EGFP-LC3) localizes only to autophagic membranes but not on other membrane structures and serves as a specific marker for autophagosomes (Kabeya et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. Embo J. 19, 5720-8 (2000); incorporated herein by reference). As EGFP-LC3 overexpression does not affect autophagic activity, the number of EGFP-LC3 vesicles has frequently been used to assess autophagosome number, and therefore to make inferences about autophagic activity (Mizushima, Methods for monitoring autophagy. Int. J. Biochem. Cell Biol. 36, 2491-502 (2004); incorporated herein by reference). SMERs 10, 18, and 28 significantly increased the proportion of cells with EGFP-LC3 vesicles, compared to control (DMSO-treated) cells. Rapamycin, as expected, also increased the proportion of cells containing EGFP-LC3 vesicles (Figure 18f).

[00174] We next tested for autophagy induction by the SMERs in an EGFP-LC3 expressing stable HeLa cell line (Bampton et al. The dynamics of autophagy visualised in live cells: from autophagosome formation to fusion with endo/lysosomes. Autophagy 1, 23- 36 (2005); incorporated herein by reference). Treatment of these cells with SMERs 10, 18 and 28 led to overt EGFP-LC3 vesicle formation compared to the control cells (Figure 18g). (Note that some cell types, e.g., HeLa cells, have more autophagosomes per cell than other cell types, e.g., COS-7 cells).

[00175] Endogenous LC3 is processed post-translationally into LC3-I, which is cytosolic. LC3-I is in turn converted to LC3-II, which associates with autophagosome membranes (Kabeya et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. Embo J. 19, 5720-8 (2000); incorporated herein by reference). Accumulation of LC3-II can occur due to increased upstream autophagosome formation, but also if there is impaired downstream autophagosome-lysosome fusion. In order to distinguish between those two possibilities, we assayed LC3-II in the presence of bafilomycin Al, which blocks downstream autophagosome-lysosome fusion (Yamamoto et al. Bafilomycin Al prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct. Fund. 23, 33-42 (1998); incorporated herein by reference). Bafilomycin Al increased EGFP-LC3- II levels in stable HeLa cells, as expected {Figure 18K). The dose of bafilomycin Al used is saturating for LC3-II levels in this assay and no further increases in LC3-II are observed when we treat cells with bafilomycin Al and agents that block autophagosome-lysosome fusion via independent mechanisms (like the dynein inhibitor, erythro-9-[3-(2- hydroxynonyl)] adenine (EHNA) (Ekstrom et al. Inhibition of fast axonal transport by erythro-9-[3-(2- hydroxynonyl)]adenine. J. Neurochem. 43, 1342-5 (1984); incorporated herein by reference)). SMERs 10, 18 and 28 significantly increased EGFP-LC3-II levels in presence of bafilomycin Al, compared to bafilomycin Al alone, strongly arguing that the increased autophagosomes induced by these SMERs are the result of their modulating regulatory elements located upstream of autophagosome-lysosome fusion, i.e., at the level of autophagosome formation (Figure 18h).

SMERs 10, 18 and 28 protect against neurodegeneration in Drosophila model of Huntington 's disease

[00176] We tested the therapeutic potential of the three autophagy-enhancing SMERs in vivo using a Drosophila model of HD expressing the first 171 residues of mutant huntingtin with 120 polyQ repeats in photoreceptors, using the pseudopupil technique (see Methods). The compound eyes in flies consist of several hundred ommatidia, each containing eight photoreceptor neurons with light-gathering parts called rhabdomeres, seven of which can be visualised using the pseudopupil technique. This method assesses the number of visible rhabdomeres by light microscopy and has been widely used to quantify the toxicity of proteins with long polyQs in the fly eye (Berger et al. Rapamycin alleviates toxicity of different aggregate-prone proteins. Hum. MoI. Genet. 15, 433-42 (2006); Jackson et al. Polyglutamine-expanded human huntingtin transgenes induce degeneration of Drosophila photoreceptor neurons. Neuron 21, 633-42 (1998); Marsh et al. Drosophila in the study of neurodegenerative disease. Neuron 52, 169-178 (2006); each of which is incorporated herein by reference). The number of visible rhabdomeres in each ommatidium decreases over time in the mutant Drosophila expressing mutant huntingtin with 120 polyQ repeats in photoreceptors, compared to the wild-type flies or transgenic flies expressing otherwise identical huntingtin with 23 polyQ (wild-type) repeats (where there is no degeneration). SMERs 10, 18, and 28 protected against neurodegeneration in Drosophila expressing mutant huntingtin, compared to flies treated with the vehicle (DMSO) (Figures 19a-19c). Thus, these SMERs protect against polyglutamine toxicity in vivo in neurons.

Effects of autophagy-inducing SMERs are mTOR-independent

[00177] We next tested whether the effects of the autophagy-inducing SMERs acted primarily via the pathway that is negatively regulated by mTOR. mTOR kinase activity can be inferred by the levels of phosphorylation of its substrates, ribosomal S6 protein kinase (S6K1, also known as p70S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E- BPl) at Thr389 and Thr37/46, respectively (Schmelzle et al. TOR, a central controller of cell growth. Cell 103, 253-62 (2000); incorporated herein by reference). While rapamycin dramatically reduced the amounts of total p70S6K and 4E-BP1 that were phosphorylated, SMERs 10, 18, and 28 had no such effects (Figures 20a and 20b). However, the proportion of total S6K1 that was phosphorylated in the presence of SMER 28 may be slightly less than the control-treated cells (although the effect is nothing like that seen with rapamycin). [00178] The SMERs did not affect the levels of various autophagy regulators, such as

Beclin-1 (Atg6), Atg5, Atg7 and Atgl2 (Figures 29a-29d). Our data suggest that these SMERs are acting in an mTOR-independent fashion with regard to mammalian autophagy. However, it is also conceivable that the SMERs impinge upon a hitherto unknown component of the mTOR autophagy pathway downstream of mTOR. This second possibility may be the more likely option, given the experimental readout of the primary yeast screen, which selected for small-molecule modulators of the effects of rapamycin on cellular growth, as opposed to the effects on rapamycin on a specific cellular pathway. The increased sensitivity afforded by using cellular growth as a screening readout may be one reason why not all SMERs or SMIRs that emerged from the primary screen modulate autophagy, one of many cellular pathways downstream of TOR. Unfortunately, it is very difficult to directly test these hypotheses, as the mTOR substrate(s) and the relevant downstream effectors that mediate mammalian autophagy have not been clarified.

[00179] We also used a stable HeLa cell line expressing Ub^G76V-EGFP degron that is a fluorescent specific proteasome substrate (Dantuma et al. Shortlived green fluorescent proteins for quantifying ubiquitin/proteasomedependent proteolysis in living cells. Nat. Biotechnol. 18, 538-43 (2000); incorporated herein by reference), to assess if these SMERs induce autophagy by impairing the ubiquitin-proteasome pathway, as has been proposed recently (Iwata et al. HDAC6 and microtubules are required for autophagic degradation of aggregated huntingtin. J. Biol. Chem. (2005); incorporated herein by reference). The SMERs did not cause accumulation of the Ub^G76V-EGFP degron, in contrast to the proteasome inhibitor lactacystin. Thus, these SMERs do not induce autophagy by causing major impairments in the ubiquitin-proteasome pathway (Figure 20c).

SMERs and rapamycin have an additive effect on the clearance of mutant aggregate-prone proteins in mammalian cells

[00180] We next studied if these SMERs, which enhance rapamycin' s cytostatic effects in yeast, could also enhance the clearance of mutant proteins by rapamycin in mammalian cells. We assessed the clearance of A53T α-synuclein in stable PC12 cells at an early time-point of 8 hours (instead of 24 hours as shown earlier), where we do not see dramatic reductions of the levels of this autophagy substrate when the cells are treated with either of the compounds alone. SMERs 10, 18, 28 or rapamycin had small but significant effects even at this early time-point (Figures 21a— 21c). Combined treatment of SMERs 10, 18, or 28 with rapamycin dramatically enhanced the effects of rapamycin (or the SMER) alone on A53T α-synuclein clearance (Figures 21a-21c). Here, we have used saturating doses of rapamycin (Sarkar et al. Lithium induces autophagy by inhibiting inositol monophosphatase. J. Cell Biol. 170, 1101-11 (2005); incorporated herein by reference) to demonstrate the combined effects.

[00181] We further confirmed this enhanced protective effect due to dual treatment on mutant huntingtin aggregation in CO S -7 cells. Treatment with SMERs 10, 18, or 28 and rapamycin had an additive effect in reducing EGFP-HDQ74 aggregates and toxicity, compared to the single treatments of rapamycin or the SMERs (Figures 21d-21f).

11 Structural-activity relationship (SAR) analysis of SMERs 10, 18 and 28 as enhancers of autophagic clearance of mutant aggregate-prone proteins

[00182] We next performed limited SAR on SMERs 10, 18 and 28. We tested three commercially available structural analogs of SMERlO, twelve commercially available structural analogs of SMERl 8, and we synthesized twelve structural analogs of SMER28 (see Figure 31 for structures). We screened for clearance of A53T α-synuclein in stable PC12 cells treated with these structural analogs alone. We found that one SMERlO analog (SMERlOa), nine SMER18 analogs (SMER18a, c-h, j, 1), and all twelve SMER28 analogs (SMER28a-l) significantly enhanced the clearance of this autophagy substrate, though in the most instances none do so better than the parent compounds (Figures 22a-22c). [00183] SMERlO is an aminopyrimidone. The pyrimidone functionality of SMERlO is important for its autophagy-inducing activity, because bulky substitution of a phenyl group at the 2 position (SMERlOb), or creating a fused tetrazole (SMERlOc), nearly abolishes activity (Figure 22a). However, removal of the amino group at the 3 position yielding hypoxanthine (SMERlOa) may slightly increase activity compared to the parent compound. [00184] SMERl 8 is a vinylogous amide. The SMERl 8 analog series assesses the tolerance of the two terminal aromatic rings to regiosubstitutions (Figure 22b). For example, changing the hydroxyl group from the meta position either to the para (SMERl 8g) or the ortho (SMERl 8f) positions reduces but does not abolish activity; yet removing the hydroxyl group (SMERl 8i) does abolish activity, suggesting its importance to activity. Similarly, removing the vinyl spacer (SMERl 8d) reduces but does not completely abolish activity. [00185] SMER28 is a bromo-substituted quinazoline. A similar SAR pattern emerges here: the majority of substitutions are well tolerated individually, multiple concurrent substitutions fare worse, and none of the analogs are more potent than the parent compound (Figure 22c). For example, the desbromo version (SMER28b) of SMER28 retains most of the original activity. Likewise the reduced version (SMER28f) of SMER28 also exhibits activity. The greatest loss of potency occurs when the bromo group is replaced with a hydroxyl group (SMER28J). Interestingly, two analogs of SMER28 (SMER28h and i) that have been functionalized for affinity chromatography are also active, the desbromo version less so, an observation that may enable biochemical target identification studies in the future. [00186] We further tested the effects of these structural analogs on mutant huntingtin aggregation in COS-7 cells. Most of them that enhanced the clearance of A53T α- synuclein also reduced EGFP-HDQ74 aggregation, such as SMERlOa, SMERl 8a, c- h, and SMER28a, c-1 (Figures 22d-22f). Thus, we have identified further candidate drugs of potential therapeutic relevance.

[00187] In conclusion, we have identified a new high-throughput screening strategy for identifying pharmacological modulators of mammalian autophagy, as SMERs in yeast appear to act as autophagy inducers on their own in mammalian cells. Autophagy inducers may have value for a range of neurodegenerative diseases and against infective agents. Autophagy inhibitors may also have utility for certain cancers. Thus, the discoveries of new compounds that modulate this process have great clinical potential. The screening strategy we have employed allows the rapid identification of potential autophagy modulators from large libraries of small molecules.

Materials and Methods

Yeast strains and media

[00188] BY4742 (MATα his3Δl leu2Δ0 lys2Δ0 ura3Δ0) and BY4741 (MATa his3Δl leu2Δ0 metl5Δ0 ura3Δ0) were obtained from American Tissue Culture Collection (ATCC). RMl 1-la (MATa leu2Δ ura3Δ) was a generous gift of B. Garvik (Fred Hutchison Cancer Research Center, USA). We thank Dr. Ian Roberts (National Collection of Yeast Cultures, UK) for kindly providing S. paradoxus, S. bayanus, and S. mikatae. Rich media (YPD) is 2% yeast extract, 2% peptone and 2% glucose. Complete synthetic media (CSM) is 6.7g/L yeast nitrogen base (YNB), 0.05% ammonium sulfate (AS), and 2% glucose; 0.05% urea is substituted for AS where appropriate.

Plasmid constructs

[00189] HD gene exon 1 fragment with 74 polyQ repeats in pEGFP-Cl (Clontech)

(EGFP-HDQ74) construct was characterized previously (Narain et al. A molecular investigation of true dominance in Huntington's disease. J. Med. Genet. 36, 739-46 (1999)); incorporated herein by reference). EGFP-LC3 construct was obtained as kind gift from T.

Yoshimori.

Primary screen [00190] Culturing of yeast as well as media formulation was done as previously described (28; incorporated herein by reference). We screened the Chembridge Microformat library and a custom collection of bioactive compounds in duplicate. An overnight culture of BY4742 was appropriately diluted in rich media; 25 μL were dispensed into NUNC 384-well, clear-bottom, untreated, sterile plates (VWR, #62409-604) using the Microfill liquid handler (Biotek); compound from library stock plates was robotically pinned (Seiko Instruments) into assay plates; an additional 15 μL of media containing enough rapamycin (acquired by prescription) to yield a 50 nM final concentration in each well were dispensed into assay plates. Inoculated assay plates were grown without agitation on the bench top at ambient temperature conditions for 48-96 hours and visually inspected for primary assay positives. Primary assay positives were ordered either from Chembridge Corporation or from Biomol in 5 mg quantities and resuspended in dimethyl sulfoxide (DMSO).

Dose responses and selectivity profiling

[00191] SMIRs and SMERs were manually arrayed into plastic 384-well plates as twofold dilution series. EC50 values were determined using GraphPad Prism v. 4.01 (GraphPad Software, Inc.). Yeast were dispensed into 384-well plates and compound was pinned into plates as described above, substituting synthetic media for rich media where appropriate. The following SMPs were used in modifier profiling at the listed concentrations: 555nM cycloheximide (GR-310); 18.9 μM anisomycin (Biomol, #ST-102); 595 nM tunicamycin (Biomol, #CC-104); 29 μM and 14.5 μM menadione (Sigma-Aldrich, #M5625); 16.6 μM nocodazole (Biomol, T-101).

Mammalian cell lines

[00192] African green monkey kidney cells (COS-7), human cervical carcinoma cells

(HeLa), stable HeLa cells expressing EGFP-LC3 (Bampton et al. The dynamics of autophagy visualised in live cells: from autophagosome formation to fusion with endo/lysosomes. Autophagy 1, 23-36 (2005)); (kind gift from A.M. Tolkovsky), and wild- type Atg5 (Atg5⁺ ) and Atg5 -deficient (Atg5^{~ ~}) mouse embryonic fibroblasts (Mizushima et al. Dissection of autophagosome formation using Apg5 -deficient mouse embryonic stem cells. J. Cell Biol. 152, 657-68 (2001); incorporated herein by reference); (MEFs) (kind gift from N. Mizushima) were maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin/streptomycin and 2 mM L-glutamine (Sigma) at 37°C, 5% CO₂. HeLa cells stably expressing Ub^G76V-GFP reporter (Dantuma et al. Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells. Nat. Biotechnol. 18, 538-543 (2000); incorporated herein by reference) (kind gift from N. P. Dantuma) were grown in the same media used for COS-7 cells supplemented with 0.5 mg/ml G418. [00193] Inducible PC12 stable cell line expressing HA-tagged A53T α-synuclein mutant, previously characterized (Webb et al. Alpha-Synuclein is degraded by both autophagy and the proteasome. J. Biol. Chem. 278, 25009-13 (2003); incorporated herein by reference), was maintained at 75 μg/ml hygromycin B (Calbiochem) in DMEM with 10% horse serum, 5% FBS, 100 U/ml penicillin/streptomycin, 2 mM L-glutamine and 100 μg/ml G418 (GIBCO) at 37 ⁰C, 10% CO₂.

[00194] Cells were transfected with the constructs for 4 hours using Lipofectamine or

Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol, fixed with 4% paraformaldehyde (Sigma) after 24 h or 48 h (EGFP-HDQ74), or 24h (EGFP-LC3) post- transfection and mounted in citifluor (Citifluor Ltd.) containing 4',6-diamidino-2- phenylindole (DAPI; 3 μg/ml; Sigma-Aldrich).

Quantification of aggregate formation and cell death

[00195] Approximately 200 EGFP-positive cells were counted by fluorescence microscope for the proportion of cells with EGFP-HDQ74 aggregates, as described previously (Sarkar et al. Lithium induces autophagy by inhibiting inositol monophosphatase. J. Cell Biol. 170, 1101-11 (2005); Ravikumar et al. Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy. Hum MoI Genet 11: 1107-17 (2002); Narain et al. A molecular investigation of true dominance in Huntington's disease. J. Med. Genet. 36, 739-46 (1999); each of which is incorporated herein by reference). Nuclei were stained with DAPI and those showing apoptotic morphology were considered abnormal. Experiments were done in triplicate and with the scorer blinded to treatment.

Clearance of mutant huntingtin and a-synucleins

[00196] Stable inducible PC 12 cell line expressing A53T α-synuclein mutant was induced with 1 μg/ml doxycycline (Sigma) for 48 hours and the transgene expression was switched off by removing doxycycline from medium (Webb et al. Alpha-Synuclein is degraded by both autophagy and the proteasome. J. Biol. Chem. 278:25009-13 (2003); incorporated herein by reference). Cells were treated with or without compounds for time- points as indicated in experiments. Clearance of A53T α-synuclein was measured by immunoblotting with antibody against HA respectively and densitometry analysis relative to actin.

Western Blot Analysis

[00197] Cell pellets were lysed on ice in Laemmli buffer (62.5 mM Tris-HCl (pH 6.8),

5% β-mercaptoethanol, 10% glycerol, and 0.01% bromophenol blue) for 30 minutes in the presence of protease inhibitors (Roche Diagnostics). Primary antibodies include anti-EGFP (8362-1, Clontech), anti-HA (12CA5, Covance), anti-mTOR (2972), anti-Phospho-mTOR (Ser2448) (2971), anti-p70 S6 Kinase (9202), anti Phospho-p70 S6 Kinase (Thr389) (9206), anti-4E-BPl (9452), anti-Phospho-4E-BP 1 (Thr37/46) (9459) (all from Cell Signaling Technology), anti-Beclin- 1 (3738, Cell Signaling), anti-Atg (Webb et al. Alpha-Synuclein is degraded by both autophagy and the proteasome. J. Biol. Chem. 278, 25009-13 (2003); incorporated herein by reference); (abl9130, Abeam), anti-Atg7 (600-401-487, Rockland), anti-Atg 12 (36-6400, Zymed Laboratories), anti-actin (A2066, Sigma). Blots were probed with anti-mouse or anti-rabbit IgG-HRP and visualised using ECL detection kit (Amersham).

Microscopy

[00198] Transfected cells were analysed by Nikon Eclipse E600 fluorescence microscope (plan-apo 60x/1.4 oil immersion lens at room temperature) (Nikon, Inc.). Images of EGFP-LC 3 HeLa stable cells were acquired on a Zeiss LSM510 META confocal microscope (63x 1.4NA plan-apochromat oil immersion lens) at room temperature using Zeiss LSM510 v3.2 software (Carl Zeiss, Inc.), and Adobe Photoshop 6.0 (Adobe Systems, Inc.) was used for subsequent image processing.

Drosophila methods

[00199] Fly culture and crosses were carried out at 25 ⁰C and at 70% humidity, using

Instant Fly Food (Philip Harris, Ashby de Ia Zouch, UK) unless otherwise stated. Flies were raised with a 12 h light: 12 h dark cycle. Aliquots of SMERs in DMSO, or DMSO alone, were added to the water that was used to prepare the instant fly food. [00200] Virgin female flies of genotype y w; gmr-httNterm(l-l 7I)Ql 20 (gmrQ120)

(23; incorporated herein by reference) were mated with isogenised w¹¹¹⁸ males (Ryder et al. The DrosDel collection: a set of P-element insertions for generating custom chromosomal aberrations in Drosophila melanogaster. Genetics 167:797-813 (2004); incorporated herein by reference) in food vials for 48 hours. Flies were then transferred to vials containing instant fly food containing either SMERs in DMSO or DMSO alone. Progeny were collected 0-4 hours after eclosion, kept on food of the same composition as they had been reared on, and scored for photoreceptor degeneration using the pseudopupil technique (Franceschini, N. in Information processing in the visual system of Drosophila (ed. Wehner, R.) 75-82 (Berlin: Springer, 1972); incorporated herein by reference) two days after eclosion.

Statistical analysis

[00201] Pooled estimates for the changes in aggregate formation or cell death, resulting from perturbations assessed in multiple experiments, were calculated as odds ratios with 95% confidence intervals. Odds ratios and/> values were determined by unconditional logistical regression analysis, using the general log-linear analysis option of SPSS 9 software (SPSS, Chicago). Densitometry analysis on the immunoblots was done by Scion Image Beta 4.02 software (Scion Corporation) from three independent experiments («=3). Significance for the clearance of mutant proteins was determined by factorial ANOVA test using STATVIEW software, version 4.53 (Abacus Concepts), where the control condition was set to 100%. The y-axis values are shown in percentage (%) and the error bars denote standard error of mean. ***,p < 0.001; **,p < 0.01; *,p < 0.05; NS, Non-significant.

Statistical Analysis for Counting Aggregation, Cell Death, and EGFP-LCi Vesicles [00202] We have counted approximately 200 EGFP-positive cells per sample for the proportion of EGFP-positive cells with green fluorescent EGFP-HDQ74 aggregates, as described previously (Narain et al. A molecular investigation of true dominance in Huntington's disease. J. Med. Genet. 36, 739-46 (1999); incorporated herein by reference). If an EGFP-positive cell has one or many aggregates, the aggregate score is "one". If an EGFP- positive cell does not have any aggregate, the aggregate score is "zero". For example, the statement "SMERs significantly reduced EGFP-HDQ74 aggregates" means that the SMERs significantly reduced the proportion of EGFP-positive cells with EGFP-HDQ74 aggregates. Nuclei were stained with DAPI, and those showing apoptotic morphology (fragmentation or pyknosis) were considered abnormal. These criteria are specific for cell death, which highly correlate with propidium iodide staining in live cells (Wyttenbach et al. Heat shock protein 27 prevents cellular polyglutamine toxicity and suppresses the increase of reactive oxygen species caused by huntingtin. Hum. MoI. Genet. 11, 1137-51 (2002); incorporated herein by reference). Only EGFP-positive cells were counted so that we count only the transfected cells. Analysis was performed with the observer blinded to the identity of slides. Slides were coded, and the code was broken after completion of experiment. All experiments were done in triplicate at least twice.

[00203] Similar analysis in triplicate was done for counting the proportion of EGFP- positive cells with EGFP-LC3 vesicles (Sarkar et al. Lithium induces autophagy by inhibiting inositol monophosphatase. J. Cell Biol. 170, 1101-11 (2005); incorporated herein by reference). Approximately 200 EGFP-positive cells were counted for the proportions of EGFP-positive cells with >5 LC3-positive vesicles. We considered an EGFP-positive cell as having a score of "zero" if there were 5 or fewer vesicles (as cells have basal levels of autophagy) and cells scored "one" if they had >5 LC3-positive vesicles. [00204] Pooled estimates for the changes in aggregate formation, cell death or

EGFP-LC3 vesicles, resulting from perturbations assessed in multiple experiments, were calculated as odds ratios with 95% confidence intervals [Odds ratio of aggregation = (percentage of cells expressing construct with aggregates in perturbation conditions/percentage of cells expressing construct without aggregates in perturbation conditions )/(percentage of cells expressing construct with aggregates in control conditions/percentage of cells expressing construct without aggregates in control conditions)]. Odds ratios were considered to be the most appropriate summary statistic for reporting multiple independent replicate experiments of this type, because the percentage of cells with aggregates under specified conditions can vary between experiments on different days, whereas the relative change in the proportion of cells with aggregates induced by an experimental perturbation is expected to be more consistent. We have used this method frequently in the past to allow analysis of data from multiple independent experiments (Wyttenbach et al. Heat shock protein 27 prevents cellular polyglutamine toxicity and suppresses the increase of reactive oxygen species caused by huntingtin. Hum. MoI. Genet. 11, 1137-51 (2002); Sarkar et al. Lithium induces autophagy by inhibiting inositol monophosphatase. J. Cell Biol. 170, 1101-11 (2005); Wyttenbach et al. Polyglutamine expansions cause decreased CRE-mediated transcription and early gene expression changes prior to cell death in an inducible cell model of Huntington's disease. Hum. MoI. Genet. 10, 1829-45 (2001); each of which is incorporated herein by reference). Odds ratios and p values were determined by unconditional logistical regression analysis, using the general log-linear analysis option of SPSS 9 software (SPSS, Chicago). When EGFP-LC3 vesicle counts were expressed as a percentage of cells, the error bars denote standard error of mean. ***, p < 0.001; **, p < 0.01; *, p < 0.05; NS, Non-significant.

Statistical Analysis for Densitometry on Western Blots

[00205] Densitometry analysis on the immunoblots was done by Scion Image Beta

4.02 software (Scion Corporation) from three independent experiments (n=3). Significance for the clearance of mutant proteins was determined by factorial ANOVA test using STATVIEW software, version 4.53 (Abacus Concepts). The control condition was set to 100% and the error bars denote standard error of mean. ***, p < 0.001; **, p < 0.01; *, p < 0.05; NS, Non-significant.

SMER 28 structural analog synthesis

[00206] The substituted quinazolinone was generated by reaction of an anthranilic acid with formamide in a microwave assisted Neimentowski reaction (Alexandre et al. Novel series of 8H-quinazolino[4,3-b]quinazolin-8-ones via two Niementowski condensations. Tetrahedron 59, 1413-1419 (2003); incorporated herein by reference). Treatment of the quinazolinone with phosphorus oxychloride gave the chloroquinazoline in high yield. The chloroquinazoline was then treated with a variety of primary amines to give the final aminoquinazolines.

Example 2-High-Throughput Image-based Screen for Chemical Modulators of

Autophagy

[00207] To screen for autophagy modulators, stable transfectants of the human glioblastoma cell line LN229 expressing GFP-LC3 were created. LC3 is a cytoplasmic autophagy protein which is cleaved and inserted into the membranes of autophagic vesicles (AV). The GFP-LC3 fusion protein allows for the identification of AV in living cells by their GFP fluorescence (Bampton, E. T., C. G. Goemans, et al. (2005). "The dynamics of autophagy visualized in live cells: from autophagosome formation to fusion with endo/lysosomes" Autophagy 1(1): 23-36; incorporated herein by reference). The LN229 cell line was selected for ample cytoplasm and flat profile, both desirable characteristics for the planned high-throughput microscopy screening. Before undertaking a screen of unknown compounds, it was first necessary to establish a robust phenotype to identify autophagy modulators. When grown in media containing 10% serum at low confluency, a diffuse fluorescence pattern with few puncta was observed using direct fluorescence microscopy in LN229/GFP-LC3 cells. Increased cell culture confluency significantly increased the basal number of AV per cell. Addition of the mTOR inhibitor rapamycin, a known autophagy inducer, or addition of the autophagy inhibitor chloroquine (CQ) resulted in increased AV accumulation compared to DMSO controls (Figure 32A) (Ravikumar, Duden, et al. (2002). "Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy" Hum MoI Genet 11(9): 1107-17; Glaumann and Ahlberg (1987). "Comparison of different autophagic vacuoles with regard to ultrastructure, enzymatic composition, and degradation capacity— formation of crinosomes" Exp. MoI. Pathol. 47(3):346-62; each of which is incorporated herein by reference). To quantitate this phenotype, the commercially available software package MetaXpress was used to create an unbiased algorithm to count AV. Prior to imaging, the cells were fixed in paraformaldehyde and the nuclear stain Hoechst was added to identify nuclei and aid in the automated focus of the microscope. Nuclei were identified by their size characteristics and intensity relative to the local background in the DAPI channel. Similarly, AV were identified by size characteristics and intensity over local background in the GFP channel, each AV was then assigned to the nearest nuclei for analysis purposes. Comparison of the analysis overlay to fluorescent images showed excellent correlation {Figure 32B).

[00208] Robust automated identification of nuclei and AV allowed for the subsequent calculation of the number of AV per cell, as well as vesicle size and intensity. Treatment of LN229/GFP-LC3 cells with either the autophagy inducer rapamycin or the autophagy inhibitor chloroquine resulted in a significant increase in the mean number of AV per cell (Figure 32C). Chloroquine treatment also resulted in a significant increase in the mean vesicle area compared to controls, likely due to autophagosome-autophagosome fusions as has been previously reported (Jahreiss, Menzies, et al. (2008) "The Itinerary of Autophagosomes: From Peripheral Formation to Kiss-and-Run Fusion with Lysosomes" Traffic; incorporated herein by reference). Treatment with rapamycin did not significantly change mean vesicle area compared to controls, in agreement with prior reports that rapamycin increases autophagosome formation but does not affect subsequent autophagosomal dynamics. Visual inspection of images revealed that occasionally one or two cells with numerous (50+) AV would exist surrounded by cells with very few (<5) AV. To reduce the influence of such outliers in the screening assay, a cut-off method to identify cells as being "positive" or "negative" for autophagy modulation was employed (Klionsky, Abeliovich, et al. (2008). "Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes" Autophagy 4(2): 151-75; incorporated herein by reference). Cells with > 7 AV were considered positive cells, which accounted for <10% of LN229/GFP- LC3 cells treated with DMSO. The percentage of positive cells increased with increasing concentrations of rapamycin (Figure 40A).

[00209] A library of 3,332 compounds from a collection of known bioactive molecules and FDA-approved drugs was used to screen for autophagy modulators in LN229/GFP-LC3 cells in 384 well plate format. Compounds were screened at concentrations selected based on previously documented biological activity. Included on each plate were between 42 to 64 DMSO control wells, for a total of 782 such control wells in the entire screen. Using >7 AV per cell as the cut-off for positive cells, we calculated the percentage of positive cells for each compound and compared this to the percentage of positive cells in DMSO controls. Compounds with Z-score greater than 2 were considered hits. In order to identify potential autophagy inhibitors that prevented the completion of autophagy, compounds with Z-score greater than 2 for mean vesicle area (similar to the autophagy inhibitor chloroquine) were also included as candidate compounds. Both the DMSO control population and the experimental compound population yielded a Gaussian distribution, with 96.5% of experimental compounds within two standard deviations of DMSO average for percentage of cells > 7 AV and 93.8% of compounds within two standard deviations for mean vesicle area (Figure 40B). 103 compounds (3.1%) met threshold criteria for increase in the percentage of positive cells, and 180 compounds (5.4%) met threshold criteria for increased mean vesicle area. Of these 283 hits 47 compounds met both criteria, yielding a total set of 236 unique compounds (Figure 39). Among the putative autophagy modulators identified in this screen were several compounds previously reported as autophagy enhancers including pimozide, trifluoperazine, and loperamide (Figure 32D) (Zhang, Yu, et al. (2007). "Small molecule regulators of autophagy identified by an image-based high-throughput screen" Proc. Natl. Acad. Sci. USA 104(48): 19023-8; incorporated herein by reference). Several classes of known autophagy inhibitors were also noted including the microtubule destabilizers nocodazole (Bampton, Goemans, et al. (2005). "The dynamics of autophagy visualized in live cells: from autophagosome formation to fusion with endo/lysosomes" Autophagy l(l):23-36; incorporated herein by refernce), vinblastine (Punnonen and Reunanen (1990). "Effects of vinblastine, leucine, and histidine, and 3-methyladenine on autophagy in Ehrlich ascites cells" Exp. MoI. Pathol. 52(l):87-97; Munafo and Colombo (2002). "Induction of autophagy causes dramatic changes in the subcellular distribution of GFP-Rab24" Traffic 3(7):472-82; Munafo and Colombo (2001). "A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation" J. Cell Sci. 114(Pt 20): 3619-29; each of which is incorporated herein by reference), and colchicine (Nicolescu, Frangopol, et al. (1984). "Increased autophagocytosis induced by colchicine in rat sympathetic neurons" Morphol. Embryol. (Bucur) 30(4): 251-4; incorporated herein by reference) and the actin filament assembly inhibitor wiskostatin (Guerriero and Weisz (2007). "N-WASP inhibitor wiskostatin nonselectively perturbs membrane transport by decreasing cellular ATP levels" Am. J. Physiol. Cell. Physiol. 292(4): C1562-6; Zhang, L., J. Yu, et al. (2007). "Small molecule regulators of autophagy identified by an image-based high-throughput screen." Proc Natl Acad Sci USA 104(48): 19023-8; each of which is incorporated herein by reference). The anti-malarial quinacrine, structurally similar to chloroquine (Barth, H., K. Meiling-Wesse, et al. (2001). "Autophagy and the cytoplasm to vacuole targeting pathway both require AutlOp." FEBS Lett 508(1): 23-8; incorporated herein by reference), as well as the known autophagy inhibitor monensin significantly increased both AV number and area (Mollenhauer, H. H., D. J. Morre, et al. (1990). "Alteration of intracellular traffic by monensin; mechanism, specificity and relationship to toxicity" Biochim. Biophys. Acta 1031(2): 225-46; incorporated herein by reference).

Phenotype Validation & Dose-Response

[00210] Autophagy is a multi-component intracellular process that is regulated by a number of oncogenes and tumor suppressor genes. To enrich for molecules that reproducibly modulate autophagy across cancer phenotypes, the screening hits from the LN229/GFP-LC3 assay were tested in a second unrelated human cancer cell line whose growth was presumably driven by a distinct combination of oncogenes and tumor suppressor genes. Several human cancer cell lines expressing GFP-LC3 were generated to find a cell line with a low basal number of AV per cell, ideal for identifying autophagy inhibitors that block the completion of autophagy. The human non-small cell lung cancer (NSCLC) cell line H1299 was selected for its low basal levels of AV relative to the closely related PC-9 human NSCLC cell line or HeIa human cervical cancer cells (Figure 33A,B). In contrast to LN229/GFP-LC3 cells, treatment of H1299/GFP-LC3 cells with the autophagy enhancer rapamycin did not result in a significant increase in mean AV count per cell compared to DMSO control. Hydroxychloroquine (HCQ) treatment of H1299/GFP-LC3 cells resulted in a significant increase in the mean vesicle count per cell compared to controls (Figure 33C). These results indicated that compounds which induced AV accumulation in H1299/GFP-LC3 cells line would be enriched for compounds that inhibit the completion of autophagy as opposed to compounds that increase autophagosomal formation. In contrast to the effects on mean vesicle area observed in chloroquine treated LN299/GFP-LC3 cells, treatment of H1299/GFP-LC3 cells with chloroquine derivatives did not result in a significant increase in mean AV area. (Figure 41).

[00211] Based on these results, the 236 compounds identified as autophagy modulators in the LN229/GFP-LC3 screen were used to screen for autophagy inhibitors in H1299/GFP- LC3 cells. This approach identified 35 candidate autophagy inhibitors (Figure 33D). Included in this group of 35 compounds were many drugs identified as autophagy inhibitors by other investigators including nocodazole (Bampton, Goemans, et al. (2005). "The dynamics of autophagy visualized in live cells: from autophagosome formation to fusion with endo/lysosomes" Autophagy l(l):23-36; incorporated herein by reference), bafilomycin Al (Yamamoto, Tagawa, et al. (1998). "Bafilomycin Al prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells" Cell Struct Fund 23(1): 33-42; incorporated herein by reference), wiskostatin (Guerriero and Weisz (2007). "N-WASP inhibitor wiskostatin nonselectively perturbs membrane transport by decreasing cellular ATP levels" Am. J. Physiol. Cell. Physiol. 292(4):C1562-6; incorporated herein by reference), and monensin (Mollenhauer, Morre, et al. (1990). "Alteration of intracellular traffic by monensin; mechanism, specificity and relationship to toxicity." Biochim Biophys Acta 1031(2): 225-46; incorporated herein by reference), but notably absent were the known autophagy enhancers trifluoperazine, pimozide and loperamide (Zhang, L., J. Yu, et al. (2007). "Small molecule regulators of autophagy identified by an image-based high-throughput screen." Proc Natl Acad Sci USA 104(48): 19023-8; incorporated herein by reference) (Figure 33E; Figure 34). Structure-activity relationship analysis highlighted the conserved bisindolyl maleimide (BIM) structural feature in the compounds K252A, Go6976, and GF-109203X, all known to be kinase inhibitors with differing selectivities (Figure 33F). Treatment of H1299/GFP-LC3 cells with each of these three compounds resulted in a dose-dependent increase in percentage of cells with >7 AV (Figure 33G). Doses of K252 greater than 10 μM were toxic to the H1299/GFP-LC3 cells preventing accurate measurement of AV.

K252A, Go6076, and GF-109230X are inhibitors of autophagy

[00212] In order to confirm that the bis-indolyl maleimide kinase inhibitors discovered in the tandem GFP-LC3 fluorescence screen were in fact inhibitors of autophagy, a series of secondary validation assays were performed. LC3 is a cytoplasmic protein which is cleaved and conjugated to a phosphatidylethanolamine (PE) during the formation of AV. The cleaved, PE conjugated product LC3-II is inserted in the membranes of AV. Therefore the ratio of LC3-II to LC3-I can be used to monitor autophagy (Klionsky, Abeliovich, et al. (2008). "Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes" Autophagy 4(2): 151-75; incorporated herein by reference). Treatment of H1299/GFP-LC3 cells with K252A, Go6079, and GF-109230X led to a dose-dependent increase in the LC3-II/LC3I ratio compared to DMSO controls (Figure 35A). The effect of these compounds on intracellular protein turnover, a reflection of autophagic flux, was investigated by radiolabled pulse-chase measurement of the degradation of proteins which had incorporated ³H-tyrosine. K252A, Go6076, and GF-109230X significantly decreased the rate of protein turnover relative to DMSO control in H1299/GFP-LC3 cells (Figure 35B). Electron microscopy (EM) of H1299/GFP-LC3 cells treated with K252A characterized the morphological evidence of AV accumulation in treated versus control cells (Figure 35C). Quantification of AV demonstrated that K252a treatment of H1299/GFP-LC3 cells increased the number of AV observed by EM relative to DMSO control (Figure 35D).

Structure-Activity Relationship of K252A analogs

[00213] A series of structural analogs of K252A (shown below) were generated by

Professor John Wood of Colorado State University. These analogs were screened to determine characteristics of the molecule necessary for autophagy inhibition and areas amenable to functionalization. Treatment of H1299/GFP-LC3 cells with twenty -two structural analogues of K252A identified several compounds which resulted in a higher percentage of cells with >7 AV per cell compared to the parent compound (Figure 36A). Substitution at the C3 position in the five-membered ring linking the two indole groups increased activity, while substitution at the C2 position rendered the molecule inactive (Figure 36B). Dose-ranging studies demonstrated that in addition to greater efficacy, the C3 substituted K252-11 was also more potent than K252A (Figure 36C).

Kinase Inhibitors in Clinical Development are Also Autophagy Inhibitors

[00214] Given the structural similarity of K252A to several kinase inhibitors currently in clinical development, a broad range of clinically relevant kinase inhibitors were assayed in the GFP-LC3 screen. Amongst a panel of 18 structurally diverse kinase inhibitors treatment of H1299/GFP-LC3 cells with sunitinib, UCNOl, ruboxistaurin, and PKC412 resulted in greater than 20% of cells with >7 AV per cell (Figure 37A). These compounds were identified as putative inhibitors of autophagy. Structurally, UCNOl, ruboxistaurin, and PKC412 are BIMs, while sunitinib contains an indole linked to a substituted pyrrole (Figure 37B). Treatment of H1299/GFP-LC3 cells with each of these compounds resulted in a dose- dependent increase in the percentage of cells with >7 AV per cell. Sunitinib and UCNOl were more potent than ruboxistaurin and PKC412 in their ability to cause accumulation of AV (Figure 37C). In addition to the accumulation of AV, K252A and UCNOl also altered the morphology of the H1299/GFP-LC3 cells creating long, spindle-like cytoplasmic projections. This effect was not seen with sunitinib, ruboxistaurin, or PKC412 (Figure 42). Treatment of H1299/GFP-LC3 cells with sunitinib, UCNOl, ruboxistaurin, and PKC412 also resulted in increased accumulation of LC3-II compared to DMSO control on LC3 immunoblot. The kinase inhibitors did not increase LC3-II accumulation relative to balfilomycin Al alone when the LC3 immunoblot was repeated in the presence of a saturating dose of the autophagy inhibitor bafilomycin Al (Figure 37D). Additionally, treatment of H1299/GFP-LC3 cells with UCNOl led to an increased number of AV visualized by electron microscopy (Figure 37E,F).

Autophagy Inhibitors are Toxic to Multiple Myeloma Cell Lines

[00215] To confirm that the putative autophagy inhibitors discovered from screening solid tumor cell lines were also active in multiple myeloma (MM) cells, LC3 immunoblot and EM were repeated in the RPMI-8826 cell line. Treatment of RPMI-8826 cells with sunitinib, UCNOl, ruboxistaurin, and PKC412 resulted in increased accumulation of LC3-II compared to DMSO control (Figure 38A). Electron micrographs of RPMI-8826 cells treated with BIM kinase inhibitors K252A and UCNOl provided morphological conformation of autophagy inhibition (Figure 38B). Quantitiation of these images confirmed that treatment with both K252A and UCNOl resulted in increased AV accumulation relative to DMSO control (Figure 38C). Despite their activity in H 1299 cells, sunitinib and the bis-indolyl maleimide (BIM) autophagy inhibitors were selectively cytotoxic to myeloma cell lines (Figure 38D).

Other Embodiments

[00216] The foregoing has been a description of certain non-limiting preferred embodiments of the invention. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

Claims

ClaimsWhat is claimed is:

1. A method of inhibiting autophagy in a cell, the method comprising: contacting a cell with an amount of a compound selected from the group consisting of cefamandole, monensin, astemizole, spiramycin, (lS,9R)-beta-hydrastine, carnitine, tomatine, K252A, atranorin, tetrandrine, amlopidine, benzyl isothiocyanate, pristimerin, homochlorocyclizine, fluoxetine, bafilomycin Al, wiskostatin, quinacrine, nocodazole, colchicine, puromycin, bepridil, spiramycin, migericin, 2-methylcinngel, amiprilose, carnitine, tyrphostin 9, salinomycin, PPl, lavendustin A, ZL3VS, astemizole, GO6976, RWJ- 60475 -(AM)3, D609, mefenamic acid, cytochalasin D, E6 berbamine, beta-peltatin, aesculin, GF-109203D, benzyl isothiocyanate, podophyllotoxin, thimerosal, maprotiline hydrochloride, vinblastine, norethindrone, gramacidin, sunitinib, UCNOl, PKC412, and ruboxistaurin, effective to inhibit autophagy in the cell.

2. A method of inhibiting autophagy in a cell, the method comprising: contacting a cell with an amount of a compound of formula:

wherein each V is independently -CH₂-, -(C=O)-, or -CH(OH)-; each occurrence of a dashed line independently represents a single bond, double bond, or the absence of a bond;

Ri is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -C(O)RA; -CO₂RA; -SOR_A; -SO₂RA; -N(RA)₂; -NHC(0)R_A; or - C(R_A)₃; wherein each occurrence of R_A is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₂ is is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -C(O)R₈; -CO₂R_B; -SOR_B; -SO₂R_B; -N(RB)₂; -NHC(O)R_B; or - C(R_B)₃; wherein each occurrence of R_B is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; and pharmaceutically acceptable forms thereof, effective to inhibit autophagy in the cell.

3. A method of modulating autophagy in a cell, the method comprising: contacting a cell with an amount of a compound selected from the group consisting of LY-83583, pimozide, gramicidin, manoalide, doxorubicin, daunorubicin, rhodomyrtoxin B, isogedunin, solanine alpha (solanidine), elliticine, amiprilose, gentian violet, wiskostatin, loperamide, manumycin A, tetrandrine, trimethobenzamide, tamoxifen, RWJ-60475-(AM)3, amphotericin B, hexetidine, maprotiline, D609, GO6976, nigericin, methyl benzethonium chloride, nocodazole, GF-109203X, FK-506, PPl, strophanthidinic acid lactone, mitoxantrone, tyrothricin, puromycin, chukrasin, tyrphostin 9, norethindrone, colchicine, vinblastine, metixene, clemastine, thioridazine, creatinine, phorbol 12-myristate 13 -acetate, ZL3VS, and triflupromazine, effective to modulate autophagy in the cell.

4. A method of stimulating autophagy in a cell, the method comprising: contacting a cell with an amount of a compound selected from the group consisting of SMERlO, effective to stimulate autophagy in the cell.

5. The method of claim 1, 2, 3, or 4, wherein the cell is a eukaryotic cell.

6. The method of claim 1, 2, 3, or 4, wherein the cell is a mammalian cell.

7. The method of claim 1, 2, 3, or 4, wherein the cell is a human cell.

8. The method of claim 1, 2, 3, or 4, wherein the concentration of the compound ranges from 0.01 μM to 100 μM.

9. The method of claim 8, wherein the concentration of the compound ranges from 0.1 μM to 10 μM

10. A method of treating a subject with a proliferative disease, the method comprising steps of: administering a therapeutically effective amount of a compound selected from the group consisting of cefamandole, monensin, astemizole, spiramycin, (lS,9R)-beta-hydrastine, carnitine, tomatine, K252A, atranorin, tetrandrine, amlopidine, benzyl isothiocyanate, pristimerin, homochlorocyclizine, and fluoxetine, to a subject.

11. A method of treating a subject with a proliferative disease, the method comprising steps of: administering a therapeutically effective amount of a compound selected from the group consisting of bafilomycin Al, wiskostatin, monensin, quinacrine, nocodazole, vinblastine, colchicine, puromycin, bepridil, spiramycin, nigericin, 2-methylcinngel, amiprilose, carnitine, tyrphostin 9, salinomycin, PPl, lavendustin A, ZL3VS, astemizole, GO6976, RWJ-60475-(AM)3, D609, mefenamic acid, cytochalasin D, E6 berbamine, beta- peltatin, aesculin, GF-109203D, benzyl isothiocyanate, monensin, podophyllotoxin, thimerosal, maprotiline hydrochloride, vinblastine, norethindrone, and gramacidin, to a subject.

12. A method of treating a subject with a proliferative disease, the method comprising steps of: administering a therapeutically effective amount of a compound selected from the group consisting of sunitinib, UCNOl, PKC412, and ruboxistaurin, to a subject.

13. A method of treating a subject with a proliferative disease, the method comprising steps of: administering to a subject a therapeutically effective amount of a compound of formula:

wherein each V is independently -CH₂-, -(C=O)-, or -CH(OH)-; each occurrence of a dashed line independently represents a single bond, double bond, or the absence of a bond;

Ri is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -C(=O)R_A; -CO₂R_A; -SOR_A; -SO₂R_A; -N(RA)₂; -NHC(0)R_A; or - C(R_A)₃; wherein each occurrence of R_A is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety;

R₂ is is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -C(=O)R_B; -CO₂R_B; -SOR_B; -SO₂R_B; -N(R_B)₂; -NHC(O)R₈; or - C(R_B)₃; wherein each occurrence of R_B is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; and pharmaceutically acceptable forms thereof.

14. A method of treating a subject with a proliferative disease, the method comprising steps of: administering a therapeutically effective amount of a compound of selected from the group consisting of LY-83583, pimozide, gramicidin, manoalide, doxorubicin, daunorubicin, rhodomyrtoxin B, isogedunin, solanine alpha (solanidine), elliticine, amiprilose, gentian violet, wiskostatin, manumycin A, tetrandrine, trimethobenzamide, tamoxifen, RWJ-60475- (AM)3, amphotericin B, hexetidine, maprotiline, D609, GO6976, nigericin, methyl benzethonium chloride, nocodazole, GF-109203X, FK-506, PPl, strophanthidinic acid lactone, mitoxantrone, tyrothricin, puromycin, chukrasin, tyrphostin 9, norethindrone, colchicine, vinblastine, metixene, clemastine, thioridazine, creatinine, phorbol 12-myristate 13-acetate, ZL3VS, and triflupromazine, to a subject.

15. The method of claim 10, 11, 12, 13, or 14, wherein the proliferative disease is cancer.

16. The method of claim 10, 11, 12, 13, or 14, wherein the proliferative disease is an inflammatory disease.

17. The method of claim 10, 11, 12, 13, or 14, wherein the proliferative disease is a benign neoplasm.

18. The method of claim 10, 11, 12, 13, or 14, wherein the proliferative disease is an autoimmune disease.

19. The method of claim 10, 11, 12, 13, or 14 further comprising step of: administering a therapeutically effective amount of an agent that inhibits a growth factor pathway.

20. The method of claim 19, wherein the agent is a kinase inhibitor.

21. The method of claim 20, wherein the kinase inhibitor is a receptor tyrosine kinase.

22. The method of claim 20, wherein the kinase inhibitor is selected from the group consisting of erlotinib (TARCEVA^®), gefitinib (IRESSA^®), cetuximab, sorafenib (NEXAVAR), dasatinib, ZD6474 (ZACTIMA), lapatinib (TYKERB), STI571, imatinib (GLEEVEC), lestaurtinib (CEP-701), sunitinib maleate (SUTENT), panitumumab, EMD 72000, TheraCIM hR3, EKB-569, 2C4, AMG706, MP -412, XL647, XL 999, MLN518, PKC412, AMN107, AEE708, OSI-930, OSI-817, and AG-013736.

23. The method of claim 10, 11, 12, 13, or 14 further comprising step of: administering a therapeutically effective amount of a proteasome inhibitor.

24. The method of claim 23, wherein the proteasome inhibitor is selected from the group consisting of bortezomib (VELCADE^®), peptide boronates, salinosporamide A (NPI-0052), lactacystin, epoxomicin (Ac(Me)-Ile-Ile-Thr-Leu-EX), MG- 132 (Z-Leu-Leu-Leu-al), PR- 171, PS-519, eponemycin, aclacinomycin A, CEP-1612, CVT-63417, PS-341 (pyrazylcarbonyl-Phe-Leu-boronate), PSI (Z-Ile-Glu(OtBu)-Ala-Leu-al), MG-262 (Z-Leu- Leu-Leu-bor), PS-273 (MNLB), omuralide (c/αsto-lactacystin-β-lactone), NLVS (Nip-Leu- Leu-Leu-vinyl sulfone), YLVS (Tyr-Leu-Leu-Leu-vs), dihydroeponemycin, DFLB (dansyl- Phe-Leu-boronate), ALLN (Ac-Leu-Leu-Nle-al), 3,4-dichloroisocoumarin, 4-(2-aminoethyl)- benzenesulfonyl fluoride, TMC-95A, gliotoxin, EGCG ((-)-epigallocatechin-3-gallate), and YUlOl (Ac-hFLFL-ex).

25. The method of claim 23, wherein the proteasome inhibitor is bortezomib

(VELCADE ι®"\).

26. A method of treating a subject with a neurodegenerative disease, the method comprising: administering a therapeutically effective amount of a compound selected from the group consisting of LY-83583, pimozide, gramicidin, manoalide, doxorubicin, daunorubicin, rhodomyrtoxin B, isogedunin, solanine alpha (solanidine), elliticine, amiprilose, gentian violet, wiskostatin, loperamide, manumycin A, tetrandrine, trimethobenzamide, tamoxifen, RWJ-60475-(AM)3, amphotericin B, hexetidine, maprotiline, D609, GO6976, nigericin, methyl benzethonium chloride, nocodazole, GF-109203X, FK-506, PPl, strophanthidinic acid lactone, mitoxantrone, tyrothricin, puromycin, chukrasin, tyrphostin 9, norethindrone, colchicine, vinblastine, metixene, clemastine, thioridazine, creatinine, phorbol 12-myristate 13-acetate, ZL3VS, triflupromazine, and SMERlO to a subject.

27. A method of treating a subject with reperfusion injury or at risk for reperfusion injury, the method comprising: administering a therapeutically effective amount of a compound selected from the group consisting of LY-83583, pimozide, gramicidin, manoalide, doxorubicin, daunorubicin, rhodomyrtoxin B, isogedunin, solanine alpha (solanidine), elliticine, amiprilose, gentian violet, wiskostatin, loperamide, manumycin A, tetrandrine, trimethobenzamide, tamoxifen, RWJ-60475-(AM)3, amphotericin B, hexetidine, maprotiline, D609, GO6976, nigericin, methyl benzethonium chloride, nocodazole, GF-109203X, FK-506, PPl, strophanthidinic acid lactone, mitoxantrone, tyrothricin, puromycin, chukrasin, tyrphostin 9, norethindrone, colchicine, vinblastine, metixene, clemastine, thioridazine, creatinine, phorbol 12-myristate 13-acetate, ZL3VS, triflupromazine, and SMERlO to a subject.

28. A method of treating a subject with ischemic cardiac disease, the method comprising: administering a therapeutically effective amount of a compound selected from the group consisting of LY-83583, pimozide, gramicidin, manoalide, doxorubicin, daunorubicin, rhodomyrtoxin B, isogedunin, solanine alpha (solanidine), elliticine, amiprilose, gentian violet, wiskostatin, loperamide, manumycin A, tetrandrine, trimethobenzamide, tamoxifen, RWJ-60475-(AM)3, amphotericin B, hexetidine, maprotiline, D609, GO6976, nigericin, methyl benzethonium chloride, nocodazole, GF-109203X, FK-506, PPl, strophanthidinic acid lactone, mitoxantrone, tyrothricin, puromycin, chukrasin, tyrphostin 9, norethindrone, colchicine, vinblastine, metixene, clemastine, thioridazine, creatinine, phorbol 12-myristate 13-acetate, ZL3VS, triflupromazine, and SMERlO to a subject.

29. A method of treating a subject with an infectious disease, the method comprising: administering a therapeutically effective amount of a compound selected from the group consisting of LY-83583, pimozide, gramicidin, manoalide, doxorubicin, daunorubicin, rhodomyrtoxin B, isogedunin, solanine alpha (solanidine), elliticine, amiprilose, gentian violet, wiskostatin, loperamide, manumycin A, tetrandrine, trimethobenzamide, tamoxifen, RWJ-60475-(AM)3, amphotericin B, hexetidine, maprotiline, D609, GO6976, nigericin, methyl benzethonium chloride, nocodazole, GF-109203X, FK-506, PPl, strophanthidinic acid lactone, mitoxantrone, tyrothricin, puromycin, chukrasin, tyrphostin 9, norethindrone, colchicine, vinblastine, metixene, clemastine, thioridazine, creatinine, phorbol 12-myristate 13-acetate, ZL3VS, triflupromazine, and SMERlO to a subject.

30. A pharmaceutical composition comprising a therapeutically effective amount of a compound selected from the group consisting of cefamandole, monensin, astemizole, spiramycin, (lS,9R)-beta-hydrastine, carnitine, tomatine, K252A, atranorin, tetrandrine, amlopidine, benzyl isothiocyanate, pristimerin, homochlorocyclizine, fluoxetine, LY-83583, pimozide, gramicidin, manoalide, doxorubicin, daunorubicin, rhodomyrtoxin B, isogedunin, solanine alpha (solanidine), elliticine, amiprilose, gentian violet, wiskostatin, manumycin A, tetrandrine, trimethobenzamide, tamoxifen, RWJ-60475-(AM)3, amphotericin B, hexetidine, maprotiline, D609, GO6976, nigericin, methyl benzethonium chloride, nocodazole, GF- 109203X, FK-506, PPl, strophanthidinic acid lactone, mitoxantrone, tyrothricin, puromycin, chukrasin, tyrphostin 9, norethindrone, colchicine, vinblastine, metixene, clemastine, thioridazine, creatinine, phorbol 12-myristate 13-acetate, ZL3VS, and triflupromazine.

31. The pharmaceutical composition of claim 30 comprising a therapeutically effective amount of the compound sufficient to treat a proliferative disease.

32. A pharmaceutical composition comprising a therapeutically effective amount of a compound selected from the group consisting of LY-83583, pimozide, gramicidin, manoalide, doxorubicin, daunorubicin, rhodomyrtoxin B, isogedunin, solanine alpha (solanidine), elliticine, amiprilose, gentian violet, wiskostatin, loperamide, manumycin A, tetrandrine, trimethobenzamide, tamoxifen, RWJ-60475-(AM)3, amphotericin B, hexetidine, maprotiline, D609, GO6976, nigericin, methyl benzethonium chloride, nocodazole, GF- 109203X, FK-506, PPl, strophanthidinic acid lactone, mitoxantrone, tyrothricin, puromycin, chukrasin, tyrphostin 9, norethindrone, colchicine, vinblastine, metixene, clemastine, thioridazine, creatinine, phorbol 12-myristate 13-acetate, ZL3VS, triflupromazine, and SMERlO.

33. The pharmaceutical composition of claim 32 comprising a therapeutically effective amount of the compound sufficient to treat a neurodegenerative disease.

34. The pharmaceutical composition of claim 32 comprising a therapeutically effective amount sufficient to treat a cardiac disease.

35. The pharmaceutical composition of claim 32 comprising a therapeutically effective amount sufficient to treat an infectious disease.

36. The pharmaceutical composition of claim 32 comprising a therapeutically effective amount sufficient to treat a disease associated with protein misfolding or mishandling.

37. The pharmaceutical composition of claim 30 or 32 comprising approximately 0.1 mg to 500 mg of the compound.

38. A pharmaceutial composition comprising a therapeutically effective amount of a autophagy inhibitor and an agent that inhibits a growth factor pathway.

39. The pharmaceutical composition of claim 38, wherein the agent is a kinase inhibitor.

40. The pharmaceutical composition of claim 39, wherein the kinase inhibitor is selected from the group consisting of erlotinib (T ARCEV A^®), gefitinib (IRESSA^®), cetuximab, sorafenib (NEXAVAR), dasatinib, ZD6474 (ZACTIMA), lapatinib (TYKERB), STI571, imatinib (GLEEVEC), lestaurtinib (CEP-701), sunitinib maleate (SUTENT), panitumumab, EMD 72000, TheraCIM hR3, EKB-569, 2C4, AMG706, MP-412, XL647, XL 999, MLN518, PKC412, AMN107, AEE708, OSI-930, OSI-817, and AG-013736.

41. The pharmaceutical composition of claim 38, wherein the autophagy inhibitor is selected from the group consisting of cefamandole, monensin, astemizole, spiramycin, (lS,9R)-beta-hydrastine, carnitine, tomatine, K252A, atranorin, tetrandrine, amlopidine, benzyl isothiocyanate, pristimerin, homochlorocyclizine, fluoxetine, LY-83583, pimozide, gramicidin, manoalide, doxorubicin, daunorubicin, rhodomyrtoxin B, isogedunin, solanine alpha (solanidine), elliticine, amiprilose, gentian violet, wiskostatin, manumycin A, tetrandrine, trimethobenzamide, tamoxifen, RWJ-60475-(AM)3, amphotericin B, hexetidine, maprotiline, D609, GO6976, nigericin, methyl benzethonium chloride, nocodazole, GF- 109203X, FK-506, PPl, strophanthidinic acid lactone, mitoxantrone, tyrothricin, puromycin, chukrasin, tyrphostin 9, norethindrone, colchicine, vinblastine, metixene, clemastine, thioridazine, creatinine, phorbol 12-myristate 13-acetate, ZL3VS, and triflupromazine.

42. A compound of formula:

wherein each V is independently -CH₂-, -(C=O)-, or -CH(OH)-; each occurrence of a dashed line independently represents a single bond, double bond, or the absence of a bond;

Ri is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -C(O)RA; -CO₂RA; -SOR_A; -SO₂R_A; -N(RA)₂; -NHC(0)R_A; or - C(R_A)₃; wherein each occurrence of R_A is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; R₂ is is hydrogen; halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstitued, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; -CC=O)R₈; -CO₂R_B; -SOR_B; -SO₂R_B; -N(RB)₂; -NHC(0)R_B; or - C(R_B)₃; wherein each occurrence of R_B is independently a hydrogen, a protecting group, an aliphatic moiety, a heteroaliphatic moiety, an acyl moiety; an aryl moiety; a heteroaryl moiety; alkoxy; aryloxy; alkylthio; arylthio; amino, alkylamino, dialkylamino, heteroaryloxy; or heteroarylthio moiety; and pharmaceutically acceptable forms thereof.

43. A pharmaceutical composition comprising a compound of claim 42 and a pharmaceutically acceptable excipient.

44. The pharmaceutical composition of claim 43 comprising a therapeutically effective amount of the compound sufficient to treat a proliferative disease.

45. A method of modifying the biological activity of an autophagy modulator, the method comprising steps of: contacting a cell with an autophagy modulator and a compound selected from the group consisting of D609, LY-83583, SMIR7, SMIR8b, SMIRI l, SMIR12, SMIR15, SMIR16, SMIR17, SMIR18, SMIR19a, SMIR19b, SMIR20, SMIR23, SMIR27, SMIR28, SMIR29a, SMIR29b, SMIR30, SMIR32, and SMIR33.

46. A method of modifying the biological activity of an autophagy modulator, the method comprising steps of: contacting a cell with an autophagy modulator and a compound selected from the group consisting of SMER3, SMER6, SMERlO, SMER14, SMER17, SMER18, SMER20, SMER22, SMER23, SMER24, SMER26, and SMER28.

47. The method of claim 45 or 46, wherein the autophagy modulator is rapamycin.

48. A method of identifying modulators of autophagy, the method comprising steps of: contacting a cell with a test compound under suitable conditions to affect autophagy; visualizing the cell for changes in one or more phenotypic characteristics of autophagy; and identifying changes in one or more phenotypic characteristics of autophagy to determine if the test compound is a modulator of autophagy.

49. The method of claim 48 further comprising a step of staining the cells.

50. The method of claim 49, wherein the step of staining comprises staining the cells with EGFP-LC3.

51. The method of claim 49, wherein the step of staining comprises staining the cells with EGFP-LC3.

52. The method of claim 48, wherein the cell is a mammalian cell.

53. The method of claim 48, wherein the cell is a human cell.

54. The method of claim 48, wherein the cell is a LN229 glioblastoma cell.

55. The method of claim 48, wherein the phenotypic characteristic is selected from the group consisting of vesicle size, vesicle shape, number of vesicles, number of EGFP-LC3 positive puncta, and intensity of EGFP-LC3 staining.

56. The method of claim 48, wherein the phenotypic characteristic is number of EGFP- LC3 positive puncta.