WO2008122038A1 - Autophagie régulatrice - Google Patents

Autophagie régulatrice Download PDF

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Publication number
WO2008122038A1
WO2008122038A1 PCT/US2008/059129 US2008059129W WO2008122038A1 WO 2008122038 A1 WO2008122038 A1 WO 2008122038A1 US 2008059129 W US2008059129 W US 2008059129W WO 2008122038 A1 WO2008122038 A1 WO 2008122038A1
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Prior art keywords
autophagy
substituted
moiety
cell
branched
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PCT/US2008/059129
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English (en)
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James Elliot Bradner
John Paul Shen
Ethan Oren Perlstein
David Rubinsztein
Sovan Sarkar
Stuart L. Schreiber
John Wood
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President And Fellows Of Harvard College
Dana-Farber Cancer Institute, Inc.
Cambridge Enterprise Ltd.
Yale University
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Publication of WO2008122038A1 publication Critical patent/WO2008122038A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings
    • C07D487/14—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin

Definitions

  • 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
  • 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.
  • 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).
  • intracellular metabolites e.g., during starvation or trophic factor withdrawal
  • architectural remodeling e.g., during development
  • damaging cytoplasmic components e.g., during oxidative stress, infection, accumulation of protein aggregates.
  • 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.
  • 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.
  • TOR-independent signalling in the autophagy apparatus 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.
  • IP3 inositol or inositol- 1,4,5-triphosphate
  • 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.
  • 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.
  • 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).
  • cancer e.g., leukemia, multiple myeloma
  • proliferative diseases e.g., 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).
  • infectious diseases e.g., viral
  • the modulators of autophagy are also modulators of acetylation or deacetylation activity in the cell (e.g., HDAC inhibitors, tubulin deacetylase (TDAC) inhibitors).
  • 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 published as US2006/0239909, on October 26, 2006, which is incorporated herein by reference.
  • 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.
  • 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.
  • the identified agents or derivatives thereof may be formulated for administration to a subject (e.g., a human) for the treatment of a disease.
  • a subject e.g., a human
  • Several of the identified autophagy modulators e.g. , fluoxetine, loperamide, doxorubicin, tamoxifen
  • drugs already approved for use in humans are known drugs already approved for use in humans.
  • 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.
  • proliferative diseases such as cancer
  • inflammatory diseases e.g., 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.
  • inhibitors of autophagy may be used to treat proliferative diseases such as cancer.
  • 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.
  • 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.
  • an autophagy modulator is used in combination with a proteasome inhibitor such as bortezomib in the treatment of cancer or other proliferative diseases.
  • an autophagy modulator is used in combination with a protein kinase inhibitor in the treatment of cancer or other proliferative diseases.
  • an autophagy modulator is used in combination with a growth factor pathway inhibitor in the treatment of cancer or other proliferative diseases.
  • 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).
  • 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.
  • 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.
  • an inhibitor of autophagy and an inhibitor of a growth factor pathway e.g., a kinase inhibitor
  • 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.
  • the invention provides a novel approach to the treatment of cancer or other proliferative diseases using an autophagy modulator and a proteasome inhibitor.
  • the autophagy modulator used in combination with a proteasome inhibitor is not an HDAC inhibitor.
  • 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/ ⁇ sto-lactacystin- ⁇ -lactone), NLVS (N
  • promoters of autophagy are used to treat certain diseases.
  • promoters of autophagy may be used to clear aggregate-prone, disease- causing proteins.
  • 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 ⁇ -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).
  • 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.
  • tuberculosis, Group A Streptococcus infections, and viral infections e.g., herpes simples virus type I
  • herpes simples virus type I may be treated with promoters of autophagy.
  • the invention also provides agents that modulate the biological activity (e.g., the cytoxicity) of rapamycin, which is a known inducer of autophagy.
  • agents that modulate the biological activity e.g., the cytoxicity
  • 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.
  • these agents are administered in combination with rapamycin or other autophagy modulator such as those described herein.
  • the invention provides a system for identifying modulators of autophagy.
  • 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.
  • 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.
  • 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.
  • Figure 2 shows an exemplary high throughput, high content screen for small molecule modulators of autophagy based on EGFP-LC3 positive puncta.
  • 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.
  • Figure 4 shows a negative control (DMSO) and a positive control (rapamycin,
  • 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.
  • 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.
  • Figure 7 shows the well distibutions for DMSO (negative control) and rapamycin (positive control) versus percent of cells with greater than seven puncta.
  • 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.
  • Figure 9 shows a plot for vesicle average area versus concentration of chloroquine, rapamycin, and DMSO.
  • Figure 10 are photographs of cells treated with bafilomycin A, carnitine, trimethobenzamide, and monensin.
  • Figure 11 is the single agent toxicity of IRESSA in lung cancer.
  • Figure 12 shows the increased toxicity of IRESSA in combination with chloroquine.
  • Figure 13 demonstrates the synergistic effect of combining chloroquine (a modulator of autophagy) and IRESSA (a kinase inhibitor).
  • Figure 14 shows the synergistic effect of combining IRESSA with a small molecule inhibitor of rapamycin (SMIR 20).
  • Figure 15 shows the synergistic effect of combining GLEEVEC with an autophagy inhibitor such as chloroquine, and combining GLEEVEC with SMIR 20.
  • 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.
  • 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 OD 6 oo 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 ⁇ M anisomycin or 16.6 ⁇ M nocodazole or 595 nM tunicamycin or 29 ⁇ M (used in A) and 14.5 ⁇ M (used in B) menadione.
  • Black indicates no interaction between small-molecule modifiers and assay compounds; intense green corresponds to low half-maxi
  • FIG. 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 ⁇ -synuclein was induced with doxycycline for 48h, and expression of the trans gene was switched off for 24 hours, with DMSO (control), 47 ⁇ M SMERlO, 43 ⁇ M SMER18 or 47 ⁇ M SMER28 added in the switch- off period.
  • ⁇ -syn levels of A53T ⁇ -synuclein ( ⁇ -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). /K ⁇ .0001 (all SMERs).
  • 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.
  • 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 ⁇ M SMERlO, 43 ⁇ M SMERl 8 or 4 7 ⁇ M SMER28 for 24 hours.
  • 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 ⁇ M SMERlO, 43 ⁇ M SMER18 or 47 ⁇ M 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).
  • Figure 19 demonstrates that SMERs 10, 18 and 28 protect against neurodegeneration in Drosophila model of Huntingdon's disease.
  • FIG. 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 ⁇ M SMERlO, 43 ⁇ M SMER18, 47 ⁇ M SMER28, or 0.2 ⁇ M 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 C.
  • Figure 22 shows a screen of the chemical analogs of the autophagy- inducing
  • A-C Clearance of A53T ⁇ -synuclein ( ⁇ -syn) in stable PC12 cells as in Figure 18b, treated for 24 hours with either DMSO (control), or with 47 ⁇ M SMERlO and its analogs (SMERl 0a-c) (a), 43 ⁇ M SMERl 8 and its analogs (SMERl 8a-l) (b), or 47 ⁇ M SMER28 and its analogs (SMER28a-l) (c), was analysed by immunoblotting with anti-HA antibody (i) and densitometry analysis relative to actin (ii).
  • Figure 23 shows the chemical structures of SMIRs. Twenty-one structurally non-redundant SMIRs that were identified from the primary assay positives.
  • Figure 24 shows the chemical structures of SMERs. Twelve structurally non- redundant SMERs that were identified from the primary assay positives.
  • 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).
  • 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 ⁇ -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 ( ⁇ -syn) was analysed by immunoblotting with antibody against HA (A) and densitometry analysis relative to actin (B).
  • 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 ⁇ -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 ⁇ -synuclein ( ⁇ -syn) was analysed by immunoblotting with antibody against HA (A) and densitometry analysis relative to actin (B).
  • 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.
  • Figure 29 shows the effect of SMERs 10, 18, and 28 on Beclin-1, Atg5, Atg7 and Atgl2.
  • Figure 30 shows saturating concentrations of SMERs 10, 18, and 28 for enhancing the clearance of A53T ⁇ -synuclein. A-C.
  • FIG. 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).
  • Figure 32 shows a Forward Chemical Genetic Study of Autophagy.
  • 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.
  • FIG. 34 is a table of autophagy modulators identified from H1299 GFP-LC3 screening.
  • Figure 35 focues on the autophagy inhibitors: K252A, Go6976, and GF-
  • Figure 36 shows the structure-activity relationship of K252A analogs.
  • FIG. 37 (A) Rank-ordered activity of a panel of clinically relevant kinase inhibitors (all 6.3 ⁇ M) 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).
  • FIG. 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 ⁇ M). (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).
  • Figure 39 is table listing all LN229 EGFP-LC3 assay positives with > 7 (Z- score > 2).
  • Figure 40 (A) Quantitation of number of AV in LN229 GFP-LC3 cells. (B)
  • Figure 41 Mean vesicle area in H 1299 GFP-LC3 cells treated with either
  • FIG. 42 Images of H1299 GFP-LC3 cells treated with K252A, sunitinib,
  • Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis- and ⁇ r ⁇ ws-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.
  • 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.
  • a particular enantiomer of a compound of the present invention 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.
  • 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.
  • protecting group 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.
  • 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.
  • oxygen, sulfur, nitrogen, and carbon protecting groups may be utilized.
  • Hydroxyl protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), ⁇ -butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p- methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), ⁇ -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-methoxyt
  • 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 -methoxyethylid
  • 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-
  • 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.
  • substituted refers to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent.
  • 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.
  • the substituent may be either the same or different at every position.
  • substituted is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • 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.
  • 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.
  • 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.
  • aliphatic 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.
  • aliphatic is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties.
  • alkyl includes straight, branched and cyclic alkyl groups.
  • alkyl alkenyl
  • alkynyl alkynyl
  • lower alkyl is used to indicate those alkyl groups (cyclic, acyclic, substituted, unsubstituted, branched or unbranched) having 1-6 carbon atoms.
  • 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.
  • 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, -CH 2 -cyclopropyl, vinyl, allyl, n-butyl, sec- butyl, isobutyl, tert-butyl, cyclobutyl, -CH 2 -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.
  • alkoxy refers to an alkyl group, as previously defined, attached to the parent molecule through an oxygen atom or through a sulfur atom.
  • the alkyl, alkenyl, and alkynyl groups contain 1-20 alipahtic carbon atoms.
  • the alkyl, alkenyl, and alkynyl groups contain 1-10 aliphatic carbon atoms.
  • the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms.
  • 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.
  • 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.
  • alkylamino refers to a group having the structure -NHR', wherein
  • R is aliphatic, as defined herein.
  • the aliphatic group contains 1-20 aliphatic carbon atoms.
  • the aliphatic group contains 1-10 aliphatic carbon atoms.
  • the aliphatic group employed in the invention contain 1-8 aliphatic carbon atoms.
  • the aliphatic group contains 1-6 aliphatic carbon atoms.
  • the aliphatic group contains 1-4 aliphatic carbon atoms.
  • 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.
  • 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.
  • the aliphatic groups contains 1- 20 aliphatic carbon atoms.
  • the aliphatic groups contains 1-10 aliphatic carbon atoms.
  • the aliphatic groups employed in the invention contain 1-8 aliphatic carbon atoms.
  • the aliphatic groups contains 1-6 aliphatic carbon atoms.
  • the aliphatic groups contains 1-4 aliphatic carbon atoms.
  • 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.
  • R and R' are linked to form a cyclic structure.
  • cyclic structure may be aromatic or non-aromatic.
  • cyclic diaminoalkyl groups include, but are not limted to, aziridinyl, pyrrolidinyl, piperidinyl, morpholinyl, pyrrolyl, imidazolyl, 1,3,4-trianolyl, and tetrazolyl.
  • 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; -NO 2 ; - CN; -CF 3 ; -CH 2 CF 3 ; -CHCl 2 ; -CH 2 OH; -CH 2 CH 2 OH; -CH 2 NH 2 ; -CH 2 SO 2 CH 3 ; -C(O)R x ; - CO 2 (R x ); -CON(R X ) 2 ; -OC(O)R x ; -OCO 2 R x ; -OCON(
  • aryl and heteroaryl 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.
  • 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.
  • heteroaryl 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.
  • 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; -NO 2 ; -CN; -CF 3 ; -CH 2 CF 3 ; -CHCl 2 ; - CH 2 OH; -CH 2 CH 2 OH; -CH 2 NH 2 ; -CH 2 SO 2 CH 3 ; -C(O)R x ; -CO 2 (R x
  • heteroaliphatic 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.
  • 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; -NO 2 ; -CN; -CF 3 ; -CH 2 CF 3 ; -CHCl 2 ; -CH 2 OH; -CH 2 CH 2 OH; -CH 2 NH 2 ; - CH 2 SO 2 CH 3 ; -C(O)R x ; -CO 2 (R x ); -CON(R X ) 2 ; -OC(O)R x ; -OC
  • heterocyclic 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.
  • heterocycles include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.
  • a "substituted heterocycloalkyl or heterocycle” group 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; -NO 2 ; -CN; -CF 3 ; -CH 2 CF 3 ; -CHCl 2 ; - CH 2 OH; -CH 2 CH 2 OH; -CH 2 NH 2 ; -CH 2 SO 2 CH 3 ; -C(O)R x ;
  • 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.
  • 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.
  • compound or "chemical compound” as used herein can include organometallic compounds, organic compounds, metals, transitional metal complexes, and small molecules.
  • polynucleotides are excluded from the definition of compounds.
  • polynucleotides and peptides are excluded from the definition of compounds.
  • 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.
  • phrases, "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.
  • 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.
  • small molecule refers to a non-peptidic, non- oligomeric organic compound either synthesized in the laboratory or found in nature.
  • Small molecules 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.
  • administration includes routes of introducing the compound of the invention(s) to a subject to perform their intended function.
  • routes of administration 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.
  • 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.
  • 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.
  • 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.
  • biological activities refers to phenotypic changes.
  • biological activity refers to cytotoxicity, inhibition of autophagy, stimulation of autophagy, inhibition of deacetylation or acetylation activity, or stimulation of deacetylation or acetylation activity.
  • 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.
  • a therapeutically effective amount of compound of the invention 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.
  • 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.
  • 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.
  • 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.
  • the effective dosage of a compound of the invention used for treatment may increase or decrease over the course of a particular treatment.
  • Therapeutic agent 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.).
  • a disease e.g., a proliferative disease, a neurodegenerative disease, infectious disease, cardiovascular disease, a protein misfolding state, protein mishandling state, etc.
  • 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.
  • this phrase is intended to include cellular element degradation by autophagy.
  • 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.
  • prodrug includes compounds with moieties that can be metabolized in vivo.
  • 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.
  • 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
  • prodrugs include derivatives of compounds of any of the formulae disclosed herein that comprise -NO, -NO 2 , -ONO, or -ONO 2 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.
  • 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.
  • 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.
  • mammals e.g., rodents, e.g., mice
  • 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.
  • 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..
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • Ri and R 2 are taken together to form an optionally substituted heteroyclic moiety. In certain embodiments, Ri and R 2 are taken together to form a substituted heteroyclic moiety. In certain embodiments, Ri and R 2 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 R 2 are taken together to form an optionally substituted 7- membered heteroyclic moiety. In certain embodiments, Ri and R 2 are taken together to form an optionally substituted 8-membered heteroyclic moiety. [00100] In certain embodiments, the compound is of the formula:
  • the compound is of the formula:
  • the compound is of the formula:
  • R 1 , R 2 , and V are defined herein.
  • the compound is of the formula:
  • R 1 , R 2 , and V are defined herein.
  • the compound is of the formula:
  • R 1 , R 2 , and V are defined herein.
  • the compound is of the formula:
  • 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.
  • modulators i.e., both promoters and inhibitors
  • Other modulators 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 benzethon
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • D609 is a potassium xanthate derived compound and a potential glutathione mimetic
  • 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.
  • 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.
  • the present invention provides analogs of SMERlO.
  • Certain SMERlO analogs have the ability to modify rapamycin's biological activity.
  • 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. In other embodiments, Ri is C 1 -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, R 2 is hydrogen. In certain embodiments, R 2 is C 1 -Ce alkyl. In certain embodiments, R 2 is acyl. In certain embodiments, R2 is -OR B . In certain embodiments, R2 is -N(R B ) 2 . In certain embodiments,
  • R 2 is -NHR ⁇ . In certain embodiments, R 2 is -NH 2 . In certain embodiments, — *' is a
  • — '' is a substituted or unsubstituted, five- or six-membered heteroaryl moiety.
  • ---'' is a substituted or unsubstituted pyrrole. In certain embodiments, is a substituted or unsubstituted imidazole. In certain embodiments, -—'' is a
  • the SMERlO analog is of the formula:
  • R 2 is hydrogen or Ci -Ce alkyl; and Ri is a defined above. In certain particular embodiments, R 2 is hydrogen.
  • 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.
  • the present invention provides analogs of SMERl 8.
  • Certain SMERl 8 analogs have the ability to modify rapamycin's biological activity.
  • 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:
  • Ri is hydrogen. In certain embodiments, Ri is Ci-C ⁇ alkyl. In certain particular embodiments, Ri is methyl. In certain particular embodiments, Ri is ethyl. In certain embodiments, Ri is propyl. In certain embodiments, R 2 is -OR B . In certain embodiments, R 2 is -OH. In certain embodiments, R 2 is halogen. In certain embodiments, R 2 is fluoro. In certain embodiments, R 2 is chloro. In certain embodiments, R 2 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.
  • 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:
  • the SMERl 8 analog is of the formula: wherein R 1 , R 2 , and R3 are defined as above. In certain embodiments, the SMERl 8 analog is of the formula:
  • the SMERl 8 analog is of the formula:
  • the SMERl 8 analog is of the formula:
  • the SMERl 8 analog is of the formula:
  • the SMERl 8 analog is of the formula:
  • the SMERl 8 analog is of the formula:
  • the SMERl 8 analog is of the formula:
  • the SMERl 8 analog is of the formula: wherein Ri and R 2 are defined as above. In certain embodiments, the SMERl 8 analog is of the formula:
  • SMER28 is a substituted quinazoline. Twelve structural analogs (see Figure
  • the present invention provides analogs of SMER28.
  • Certain SMER28 analogs have the ability to modify rapamycin's biological activity.
  • 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 -OR A . In certain embodiments, Ri is -SR A . In certain embodiments, Ri is -NHR A . In certain embodiments, R A is C 1 -Ce aliphatic. In certain embodiments, R A is C 2 -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, R 2 is halogen. In certain embodiments, R 2 is fluoro.
  • R 2 is chloro. In certain embodiments, R 2 is bromo. In certain embodiments, R 2 is -0R B . In certain embodiments, R 2 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:
  • 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.
  • the animal is a vertebrate.
  • the animal is a mammal.
  • the animal is a human.
  • the animal is a domesticated animal such as a dog, cat, horse, etc.
  • autophagy modulators are used to treat proliferative diseases.
  • 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.
  • 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
  • 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.
  • proliferative diseases may also be treated with modulators of autophagy or promoters of autophagy.
  • proliferative diseases are treated with the inhibitors of autophagy described herein.
  • 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).
  • an analog of one of these identified inhibitors of autophagy is used in the treatment of a proliferative disease.
  • 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., maprot
  • an analog of one of these identified modulators is used in the treatment of proliferative disease.
  • proliferative diseases are treated with derivatives of the modulators of autophagy described herein.
  • proliferative diseases are treated with a SMER or SMIR.
  • the SMER or SMIR may be used in conjunction with rapamycin.
  • proliferative diseases are treated with inhibitors of autophagy identified using the screen described herein.
  • proliferative diseases are treated with compounds that are similar to, are analogs of, or are derived from the compounds described herein.
  • the inhibitors described herein may be used as a lead compound to develop other modulators of autophagy.
  • 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.
  • cytotoxic agent such as a cytotoxic agent, kinase inhibitor, proteasome inhibitor, inhibitor of a growth factor pathway, or other anti-neoplastic agent.
  • SMIRs kinase inhibitor
  • 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 ( Saintmeyer-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.
  • the neurodegenerative disease is spinocerebellar ataxia.
  • the neurodegenerative disease is a dementia (e.g., fronto-temporal dementia).
  • the neurodegenerative disease is Alzheimer's disease.
  • the neurodegenerative disease is Parkinson's disease.
  • the neurodegenerative disease is Huntington's disease.
  • the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).
  • a compound described herein is administered to a subject in order to prevent a neurodegenerative disease.
  • Infectious diseases may also be treated with autophagy modulators, SMIRs,
  • 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.
  • the disease is a bacterial infection.
  • the disease is a viral infection.
  • the disease is tuberculosis, which is cause by Mycobacterium tuberculosis.
  • the infectious disease is cause by a Group A Streptococcus.
  • the disease is viral disease.
  • the viral infection is caused by a herpes virus (e.g., herpes simplex virus type
  • 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.
  • such a disease is prevented using the administration of a compound described herein.
  • SMERs, and analogs thereof may also be used to treat cardiac diseases.
  • the cardiac disease is ischemic cardiac diseases.
  • the cardiac disease is cardiac disease due to reperfusion injury.
  • a compound described herein of analog thereof is administered to a subject in order to prevent reperfusion injury.
  • a subject suffering from ischemic heart disease may be administered an autophagy enhancer in order to prevent reperfusion injury once the ischemia is relieved.
  • Neurodegenerative diseases, infectious diseases, cardiac diseases, and diseases characterized by protein misfolding and/or mishandling are typically treated with agents that promote autophagy.
  • these diseases are treated with the inducers of autophagy described herein.
  • 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
  • these diseases are treated with derivatives of the modulators of autophagy described herein.
  • the diseases are treated with inducers of autophagy identified using the screen described herein.
  • 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.
  • the inhibitors described herein may be used as a lead compound to identify or prepare other inducers of autophagy.
  • an inducer of autophagy is used in conjunction with another agent typically used to treat the disease.
  • 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.
  • 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.
  • 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.
  • 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.
  • the compound is administered orally.
  • the compound is administered parenterally.
  • the compound is administered intravenously.
  • 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.
  • 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.
  • 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.
  • the compounds are cytotoxic (e.g., the compound inhibit autophagy) and are useful in the treatment of proliferative diseases.
  • the compounds stimulate autophagy and are useful in treating diseases such as neurodegenerative diseases or diseases associated with protein misfolding or mishandling.
  • the compounds show cytostatic or cytotoxic activity against neoplastic cells such as cancer cells.
  • the compounds inhibit the growth of or kill rapidly dividing cells such as stimulated inflammatory cells.
  • 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.
  • pharmaceutical compositions are provided, wherein these compositions comprise any one of the compounds as described herein, and optionally comprise a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises an activator of autophagy.
  • Exemplary activators of autophagy include SMERlO.
  • 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,
  • 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]
  • these compositions optionally further comprise one or more additional therapeutic agents, e.g., another another anti-proliferative agent.
  • 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.
  • the inhibitor of the growth factor pathway is a small molecule.
  • the inhibitor of the growth factor pathway is a protein.
  • the inhibitor of the growth factor pathway is an antibody or a fragment thereof.
  • the inhibitor of the growth factor pathway is a humanized antibody or antibody fragment.
  • the other agent is a kinase inhibitor
  • the kinase inhibitor is a receptor tyrosine kinase inhibitor.
  • 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.
  • T ARCEV A ® gefitinib
  • NEXAVAR sorafenib
  • dasatinib ZD6474
  • lapatinib STI
  • 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).
  • the invention provides a combination of an autophagy inhibitor and a kinase inhibitor for the treatment of a proliferative disease.
  • 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, tetran
  • the inventive compositions further comprise a proteasome inhibitor.
  • the agents may be packaged separaterly or together in the same composition.
  • 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),
  • the proteasome inhibitor is bortezomib (VELCAD E ® ).
  • the composition includes an autophagy inhibitor and a proteasome inhibitor.
  • 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, manu
  • 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.
  • 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.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid
  • 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.
  • 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
  • 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.
  • 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.
  • esters include formates, acetates, propionates, butyrates, acrylates, and ethylsuccinates.
  • the esters are cleaved by enzymes such as esterases.
  • pharmaceutically acceptable prodrugs 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.
  • 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.
  • 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.
  • a pharmaceutically acceptable carrier 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.
  • 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.
  • 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
  • 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.
  • 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).
  • proliferative diseases e.g., cancer, benign neoplasms
  • inflammatory disease e.g., autoimmune diseases
  • neurodegenerative disorders e.g., Parkinson's disease
  • 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.
  • the compound or pharmaceutical composition is administered orally.
  • the compound or pharmaceutical composition is
  • a method for the treatment of a proliferative disease 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.
  • an autophagy modulator e.g., an autophagy inhibitor
  • 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.
  • the processing of misfolded or aggregated proteins in cells is increased by contacting the cells with a compound as described herein.
  • the degradation of the proteins of infectious organisms is increased by contacting the cells with a compound as described herein.
  • a method for the treatment of a neurodegenerative disease or a disease associated with protein misfolding and/or mishandling 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • 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.
  • inert diluents commonly used in the art such as, for example, water or
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • the compounds of the invention are mixed with solubilizing agents such an Cremophor, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and combinations thereof.
  • solubilizing agents such an Cremophor, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and combinations thereof.
  • 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.
  • a nontoxic parenterally acceptable diluent or solvent for example, as a solution in 1,3-butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid are used in the preparation of injectables.
  • 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.
  • the rate of drug release can be controlled.
  • 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.
  • 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.
  • 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.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • 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
  • the dosage form may also comprise buffering agents.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • buffering agents include polymeric substances and waxes.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • an additional approved therapeutic agent for use as a combination therapy 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.
  • the present invention also provides a system for screening chemical compounds to identify modulators of 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.
  • the screening system is performed in a high-throughput format allowing for the screening of tens, hundreds, or thousands of compounds at once.
  • 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.
  • the cells are stained before visualization.
  • the cells are stained with EGFP-LC3, which localized to autophagic membranes, to identify EGFP-LC3 positive autophagosomes.
  • 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.
  • 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.
  • one or more characteristics of autophagy is affected befor a test compound is identified as a modulator of autophagy.
  • two or more characteristics of autophagy are altered.
  • 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.
  • a similar screening system may also be used to identify compounds that modulate the activity of an autophagy modulator.
  • 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.
  • 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.
  • 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.
  • the components of the kit are conveniently packaged for use by a researcher.
  • 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.
  • 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.
  • HD Huntington's disease
  • polyQ polyglutamine
  • 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).
  • 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).
  • 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.
  • 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.
  • the 21 SMIRs comprise 18 distinct structural classes; the 12 SMERs comprise 11 structural classes.
  • D609 is a potassium xanthate derivative and a potential glutathione mimetic (Sultana et al. Protective effect of the xanthate, D609, on Alzheimer's amyloid ⁇ -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).
  • the EC50 of suppression spans two orders of magnitude, from >50 ⁇ M to as low as 0.37 ⁇ M.
  • 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 ⁇ M to 1.4 ⁇ M, 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.
  • SMIRs 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.
  • SMERs 10, 18, and 28 reduce mutant huntingtin aggregation and toxicity by autophagy [00171]
  • 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).
  • 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 ⁇ -synuclein in PC12 cells. No overt toxicity was observed with SMERs 10, 18, and 28.
  • SMERs 10, 18 and 28 induce autophagy in mammalian cells
  • 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).
  • LC3-I 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.
  • 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)).
  • EHNA erythro-9-[3-(2- hydroxynonyl)] adenine
  • 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
  • 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).
  • 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.
  • 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).
  • S6K1 ribosomal S6 protein kinase
  • 4E- BPl eukaryotic initiation factor 4E-binding protein 1
  • 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).
  • 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.
  • SMERl 8 is a vinylogous amide. The SMERl 8 analog series assesses the tolerance of the two terminal aromatic rings to regiosubstitutions ( Figure 22b).
  • 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.
  • BY4742 (MAT ⁇ his3 ⁇ l leu2 ⁇ 0 lys2 ⁇ 0 ura3 ⁇ 0) and BY4741 (MATa his3 ⁇ l leu2 ⁇ 0 metl5 ⁇ 0 ura3 ⁇ 0) were obtained from American Tissue Culture Collection (ATCC).
  • RMl 1-la (MATa leu2 ⁇ ura3 ⁇ ) was a generous gift of B. Garvik (Fred Hutchison Cancer Research Center, USA).
  • 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.
  • 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.
  • 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).
  • 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.
  • SMPs were used in modifier profiling at the listed concentrations: 555nM cycloheximide (GR-310); 18.9 ⁇ M anisomycin (Biomol, #ST-102); 595 nM tunicamycin (Biomol, #CC-104); 29 ⁇ M and 14.5 ⁇ M menadione (Sigma-Aldrich, #M5625); 16.6 ⁇ M nocodazole (Biomol, T-101).
  • HeLa HeLa
  • HeLa stable HeLa cells expressing EGFP-LC3
  • Mizushima were maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin/streptomycin and 2 mM L-glutamine (Sigma) at 37°C, 5% CO 2 .
  • 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.
  • 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 ⁇ g/ml; Sigma-Aldrich).
  • DAPI 4',6-diamidino-2- phenylindole
  • Stable inducible PC 12 cell line expressing A53T ⁇ -synuclein mutant was induced with 1 ⁇ g/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 ⁇ -synuclein was measured by immunoblotting with antibody against HA respectively and densitometry analysis relative to actin.
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Autophagy is a multi-component intracellular process that is regulated by a number of oncogenes and tumor suppressor genes.
  • 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).
  • NSCLC human non-small cell lung cancer
  • Figure 33C 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.
  • HCQ Hydroxychloroquine
  • 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).
  • K252A, Go6076, and GF-109230X are inhibitors of autophagy
  • LC3 is a cytoplasmic protein which is cleaved and conjugated to a phosphatidylethanolamine (PE) during the formation of AV.
  • PE phosphatidylethanolamine
  • 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).
  • 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).
  • 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).
  • K252A and UCNOl also altered the morphology of the H1299/GFP-LC3 cells creating long, spindle-like cytoplasmic projections.

Abstract

L'invention concerne l'autophagie, qui est un processus cellulaire par lequel des cellules cannibalisent des éléments cellulaires non essentiels tels que des organelles pour générer des métabolites, ou dans certains cas, pour provoquer une mort cellulaire. La présente invention propose des modulateurs de l'autophagie, qui ont été identifiés en utilisant un criblage phénotypique de capacité élevée sur plus de 3 500 composés. Ces modulateurs sont utiles pour traiter des maladies allant des maladies prolifératives aux maladies neurodégénératives jusqu'aux maladies infectieuses à des états de mauvais repliement de protéine. De plus, l'invention propose un traitement d'une maladie proliférative telle que le cancer avec une combinaison d'inhibiteurs d'autophagie et d'inhibiteurs de protéine kinase.
PCT/US2008/059129 2007-04-02 2008-04-02 Autophagie régulatrice WO2008122038A1 (fr)

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