[go: up one dir, main page]

EP2300004A2 - Compositions et procédés ayant trait aux composés activant le facteur de transcription de choc thermique et cibles correspondantes - Google Patents

Compositions et procédés ayant trait aux composés activant le facteur de transcription de choc thermique et cibles correspondantes

Info

Publication number
EP2300004A2
EP2300004A2 EP09747702A EP09747702A EP2300004A2 EP 2300004 A2 EP2300004 A2 EP 2300004A2 EP 09747702 A EP09747702 A EP 09747702A EP 09747702 A EP09747702 A EP 09747702A EP 2300004 A2 EP2300004 A2 EP 2300004A2
Authority
EP
European Patent Office
Prior art keywords
group
duke
ord
docket
hsfl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP09747702A
Other languages
German (de)
English (en)
Other versions
EP2300004A4 (fr
Inventor
Dennis J. Thiele
Daniel W. Neef
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Duke University
Original Assignee
Duke University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Duke University filed Critical Duke University
Publication of EP2300004A2 publication Critical patent/EP2300004A2/fr
Publication of EP2300004A4 publication Critical patent/EP2300004A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/4155—1,2-Diazoles non condensed and containing further heterocyclic rings
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/14—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D231/38—Nitrogen atoms
    • C07D231/40—Acylated on said nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/38—Nitrogen atoms
    • C07D277/42—Amino or imino radicals substituted by hydrocarbon or substituted hydrocarbon radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention relates to Heat Shock Transcription Factor (HSF) activating compounds, methods for their discovery, and their research and therapeutic uses.
  • HSF Heat Shock Transcription Factor
  • the present invention provides compounds capable of facilitating HSFl activation, and methods of using such compounds as therapeutic agents to treat a number of conditions associated with diseases and other pathophysiological states caused by or associated with defective protein folding.
  • Protein folding diseases can be divided into two groups: in the first, excessive quantities of incorrectly folded proteins collect in the form of uncontrolled piles of molecular rubbish. This is a group of diseases known as amyloidoses, of which Alzheimer's disease is a well-known example. In the other, a small error in the genetic blueprint leads to incomplete folding of a protein, which affects its function.
  • amyloidoses A common characteristic of all amyloidoses is the collection of aggregates or plaques of insoluble protein in the extracellular tissue, which cannot be broken down by enzymes. Their ordered structure gives them crystal-like properties: they are made up of long filaments (fibrils) that are formed from densely packed ⁇ -pleated sheets of identical proteins. There are at least 20 different proteins that can act as the building blocks of these fibrils, each of which is associated with a different disease. In so-called systemic amyloidoses, the precursors of these plaques are transported through the bloodstream from their point of origin to their point of deposition. Localized amyloidoses are of greater clinical significance, as they affect the central nervous system, which is particularly susceptible to damage, as well as the heart and other organs and tissues.
  • compositions and methods for treating diseases associated with improper protein folding, aggregation and/or the clearance of damaged proteins are needed.
  • the present invention relates to HSFl activating compounds, methods for their discovery, and their research and therapeutic uses.
  • the present invention provides compounds capable of facilitating HSFl activation (e.g., homotrimerization), and methods of using such compounds as therapeutic agents to treat a number of conditions associated with protein misfolding.
  • HSFl when added to cells, and activate expression of heat shock proteins (e.g., HSP70, HSP25).
  • HSP70, HSP25 heat shock proteins
  • the present invention provides compositions capable of HSF activation.
  • the compositions are not limited to a particular type of HSF.
  • the HSF is HSFl, HSF2 or HSF4.
  • the compositions are not limited by the manner in which they result in HSF activation.
  • HSF activation includes, but is not limited to, activation of HSFl homo-trimerization, activation of HSF target protein expression (e.g., Heat Shock Proteins including but not limited to HSP70 and HSP25), activation of protein chaperone activity (e.g., increased protein folding, increased protein solubilization, protein degradation), and/or reducing protein aggregation (e.g., aggregation of poly-glutamine proteins in neuronal cells).
  • HSF target protein expression e.g., Heat Shock Proteins including but not limited to HSP70 and HSP25
  • protein chaperone activity e.g., increased protein folding, increased protein solubilization, protein degradation
  • reducing protein aggregation e.g., aggregation of poly-glutamine proteins in neuronal cells.
  • the composition comprises a compound described by the
  • the compound is not limited to particular definitions of Ri through R 9 groups.
  • the Ri through R 9 groups define a compound capable of HSF activation, identifiable using screening techniques
  • Ri is a compound described herein.
  • Xi is Docket No. DUKE-30386/WO-l/ORD 4/66 , or Xi is absent.
  • X 2 is S or C.
  • X 3 is S or C.
  • R9 is -OCH 3 .
  • R 5 is S or C.
  • R 6 is H or O .
  • R 7 is H or O .
  • Rg is or
  • Rio is chlorine or CH 3 . In some embodiments,
  • Rn is substituted or unsubstituted alkyl such as, for example, or
  • the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-N-phenyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the composition comprises a compound described by the compounds shown in Figure 7. Docket No. DUKE-30386/WO-l/ORD 9/66
  • the composition comprises a functional derivative of a
  • HSFlA a functional derivative of HSFlA
  • the composition comprises a compound described by the
  • Rl through R8 groups define a compound capable of HSF activation, identifiable using screening techniques described herein.
  • Rl is
  • R2 is
  • R 3 R 4 I-S or Docket No. DUKE-30386/WO-l/ORD 10/66 .
  • R5 is S or C.
  • R6 is H or O .
  • R7 is H or O .
  • R8 is or In some embodiments, R9 is chlorine or CH3.
  • RlO is substituted or unsubstituted alkyl such as, for example, In certain embodiments, the compound is
  • the present invention provides methods for treating a condition associated with protein misfolding.
  • the present invention is not limited to a particular method for treating a condition associated with abnormal protein folding.
  • the methods comprise administering to a subject (e.g., human being, cat, dog, mouse, rat, ape, monkey) having misfolded proteins a composition capable of facilitating HSF (e.g., HSFl) activation.
  • a subject e.g., human being, cat, dog, mouse, rat, ape, monkey
  • HSF e.g., HSFl
  • conditions associated with irregular HSF include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington disease, Amyotrophic Lateral Sclerosis, a prion-based disease, cataract, age-related cataract, glaucoma, macular degeneration, age-related macular degeneration, retinitis pigmentosa, cardiovascular disease and stroke, heat stroke, spinocerebellar ataxia, Machado Joseph disease, stress-related neuronal degeneration, aging, cancer, and type 2 diabetes mellitus.
  • the methods are directed towards crystallins in age-related cataracts.
  • the methods are directed towards myocillin in glaucoma.
  • the composition is co-administered with one or more therapeutic agents (e.g., anticonvulsant agents, antipsychotic agents, rauwolfia alkaloids, antidepressants, dopamine prodrugs, dopamine agonists, catechol-O- methyltransferase (COMT) inhibitors, anticholinergics, MAO-B inhibitors, N-methyl-D- aspartic acid inhibitors, AChE inhibitors, NMDA antagonists, free-radical scavengers, glutamate pathway antagonists, antispastic agents, Congo red and its analogs, anthracyclines, amphotericin B and its analogs, sulfated polyanions, tetrapyrroles, sulfonylurea agents, meglitinides, biguanides, thiazolidinediones, dipeptidyl peptidase IV (DPP-4) inhibitors, incretin mimetics, amylin analogs,
  • the present invention provides methods for identifying HSFl activating agents.
  • the present invention is not limited to a particular method for identifying HSFl activating agents.
  • the method comprises a) providing a yeast yhsf ⁇ strain expressing human HSFl, wherein the yhsf.
  • ⁇ strain comprises a yeast HSF gene coupled with an inducible promoter (e.g., GAL promoter); b) growing the yhsf ⁇ strain on a medium having the inducer (e.g., galactose); c) exposing the yhsf ⁇ strain to a candidate compound; d) switching the yhsf ⁇ strain to a repressive growth medium; e) assessing the growth of the yhsf ⁇ strain; and f) characterizing the candidate compound as a HSFl activating agent if the yhsf ⁇ strain grows on the non-inducer medium.
  • the human HSF is expressed via a pRS424-GPD-hHSFl plasmid.
  • the repressive medium is a glucose medium.
  • FIG. 1 shows a model for the activation of human HSFl.
  • HSFl is shown as an inactive monomer in the cytoplasm in the absence of stress.
  • HSFl is localized to the cytoplasm as an inactive monomer.
  • HSFl homotrimerizes, localizes to the nucleus, binds to DNA Heat Shock Elements (HSEs) becomes hyperphosphorylated, and activates gene transcription.
  • Black oval the HSFl DNA binding domain; hatched rectangles: LZ 1-3 (large), LZ4 (small), which form intermolecular coiled-coils in the homo-trimer; P: phosphorylation.
  • HSFllz4m harbors a point mutation in LZ4 that renders HSFl constitutive Iy homo-trimerized, perhaps by breaking intramolecular coiled-coil interactions. Docket No. DUKE-30386/WO-l/ORD 13/66
  • FIG. 2 shows yeast-based screen for small molecule activators of human HSFl (hHSFl).
  • Yeast cells expressing the essential yeast Heat Shock Transcription Factor (yHSF) under control of the repressible GALl promoter are dependent on galactose for growth.
  • yHSF essential yeast Heat Shock Transcription Factor
  • FIG. 3 shows microtiter plate analysis of human HSFl activation.
  • Yeast hsf ⁇ cells harboring the G ⁇ ZJ -yeast HSF (yHSF) plasmid.
  • yHSF G ⁇ ZJ -yeast HSF
  • yHSF G ⁇ ZJ -yeast HSF
  • glu glucose
  • yhsf ⁇ cells expressing a constitutively trimerized human HSFl protein (HSFllz4m) were viable in the absence of yeast HSF. All wells within a given row of the microtiter plate section contained the same yeast strain to show consistency.
  • Figures 4 show growth of the yhsf ⁇ : human HSFl yeast strain in the presence of library compounds ( Figure 4A) and in the presence of HSFlA derivatives ( Figure 4B).
  • Yeast cells expressing hHSFl were seeded into 96-well plates at a concentration of -1,000 cells/well in glucose and treated with 10 micromolar of various compounds from a chemical library or the DMSO solvent control. Growth was monitored by determining the optical density (O.D.600) for 96 hours.Note that compounds in the 1391 and 1393 series are structurally related but distinct from the 1261 series.
  • Figure 5 shows structures of three independent compounds positive in the yeast screen for human HSFl activator molecules, designated HSFl-A, HSFl-B and HSFl-C. Yeast cell growth in the presence of these molecules at 10 uM is shown in Figure 4.
  • Figure 6 shows synthetic routes for HSFlA, HSFlB and HSFlC. The structural relatedness of the three lead compounds simplified synthesis as a two-step reaction.
  • Figure 7 shows HSFl activating compounds.
  • Figure 7 shows compounds identified as HSFl activating compounds.
  • Figure 8 shows HSFl activating compounds identified thorugh screens conducted with compounds from the LOPAC and Prestwick chemical libraries Docket No. DUKE-30386/WO-l/ORD 14/66
  • FIG. 9 shows that HSFlA activates the expression of the HSP70 and HSP25 heat shock protein (chaperone) genes in Mouse Embryonic Fibroblast (MEF) cells.
  • HSFlA activates the expression of the HSP70 and HSP25 heat shock protein (chaperone) genes in Mouse Embryonic Fibroblast (MEF) cells.
  • Figure 10 shows HSFlA dependent activation of HSP70 is dependent on the presence of the gene encoding HSFl.
  • Figure 11 shows HSFlA acts synergistically with heat shock to activate expression of the HSP70 protein chaperone.
  • Figure 12A and 12B show that HSFlA promotes expression of HSP70 and reduces the aggregation of poly-glutamine (polyQ) proteins in rat neuronal precursor (PC- 12) cells.
  • polyQ poly-glutamine
  • Figure 12 shows that HSFlA promotes expression of HSP70 and reduces aggregation of poly-glutamine (polyQ) proteins in rat neuronal precursor (PC- 12) cells.
  • polyQ poly-glutamine
  • Figure 13 shows that HSFlA functions to ameliorate eye degeneration in a fruit fly model of poly glutamine disease (Example VIII).
  • FIG 14 shows that compositions of some embodiments of the present invention activate HSFl independently of HSP90 binding (Example IX).
  • Neuronal tissues and cells are increasingly sensitive to defects in protein folding, aggregation and clearance and these defects are causally or correlatively associated with diseases that include Huntington's disease, Parkinson's disease, Alzheimer's disease, Amyotropic Lateral Sclerosis, prion diseases and other neurodegenerative disorders (see, e.g., Bonini, N. M. (2002) Proc. Natl. Acad. ScL, USA 99:16407-16411; Muchowski, P.J. (2002) Neuron 35: 9-12; Morimoto, R. I. (2006) Docket No. DUKE-30386/WO-l/ORD 15/66
  • Protein chaperones facilitate the folding, stabilization, solubilization and degradation of cellular proteins and are often included in the group of Heat Shock Proteins (Hsps) because Docket No. DUKE-30386/WO-l/ORD 16/66
  • Hsp70 and Hsp40 can synergize in the suppression of polyQ-mediated neuronal degeneration and that arimoclomal, an inducer of Hsp synthesis, significantly delays disease progression in a mouse model of ALS (see, e.g., Kieran, D., et al., (2004) Nature Medicine 10: 402-405; herein incorporated by reference in its entirety).
  • Hsp synthesis is coordinately induced in response to stress conditions that result in protein unfolding, aggregation and proteolysis by stress-responsive transcription factors.
  • Hsps Heat Shock Elements
  • HSF Heat Shock Transcription Factor
  • yeast HSF directly activates a broad range of genes encoding proteins that function as chaperones, in protein turnover and a variety of additional stress protection roles (see, e.g., Hahn, J.-S., et al., (2004) Molecular and Cellular Biology 24:5249-5256; herein incorporated by reference in its entirety).
  • HSFl activation is a multi-step process that occurs posttranslationally in response to elevated temperatures, the accumulation of unfolded proteins and other stressful conditions (see, e.g., Wu, C. (1995) Ann. Rev. Cell Dev. Biol. 11 : 441-469; Pirkkala, L., et al., (2001) FASEB J. 15: 1118-1131; Baler, R., et al., (1993) MoI. Cell. Biol.
  • HSFl is present largely in the cytoplasm as a monomer, and is thought to be associated with Hsp90, Docket No. DUKE-30386/WO-l/ORD 18/66
  • Hsp70 and other proteins see, e.g., Zuo, J., et al, (1998) Cell 94: 471-480; AIi, A., et al, (1998) MoI. Cell. Biol. 18: 4949-4960; Guo, Y., et al., (2001) J. Biol. Chem. 276: 45791- 45799; each of which are herein incorporated by reference in their entireties).
  • HSFllz4m Leucine Zipper 4
  • HSFl Leucine Zipper 4
  • Rabindran S. K., et al., (1993) Science 259: 230-234; herein incorporated by reference in its entirety.
  • HSFl is converted to a homo-trimer that is thought to be stabilized by inter-molecular coiled coil interactions and accumulates in the nucleus, where it engages in high affinity binding to HSEs within target gene promoters and activates target gene transcription.
  • HSFl Heat shock induced Hsp target gene activation by HSFl is transient, and correspondingly, HSFl is ultimately converted back to the low affinity DNA binding monomeric form in the cytosol.
  • HSFl is phosphorylated both under basal conditions where this modification is thought to maintain the protein in an inactive state and in response to stress, with this latter modification having functional consequences that are not well understood (see, e.g., Cotto, J. J., et al., (1996) J. Biol. Chem. 271 : 3355-3358; Guettouche, T., (2005) BMC Bochem. 6:1-14; herein incorporated by reference in its entirety).
  • the present invention provides small molecules (e.g., compounds) capable of activating heat shock factors (e.g., facilitating HSFl homo-trimerization), activating heat shock factor (e.g., HSFl) target gene expression (e.g., Heat Shock genes) and protein Docket No. DUKE-30386/WO-l/ORD 19/66
  • heat shock factors e.g., facilitating HSFl homo-trimerization
  • activating heat shock factor e.g., HSFl
  • target gene expression e.g., Heat Shock genes
  • compositions and methods of the present invention are described in more detail in the following sections: I. HSF Activating Compound Screens; II. HSF Activating Compounds; III. Pharmaceutical Compositions; and IV. Therapeutic Applications.
  • the present invention provides screens for identifying activators of heat shock factor (e.g., activators capable of facilitating HSFl function), and for identifying activators of heat shock factor (e.g., HSFl) target gene expression (e.g., Heat Shock genes) and protein expression (e.g., Heat Shock Proteins).
  • the screen is not limited to identifying activators of a particular heat shock factor.
  • the screens identify HSFl activators, HSF2 activators, and/or HSF4 activators.
  • the present invention is not limited to identifying a particular type of heat shock factor activator. Examples of activators include, but are not limited to, small molecules (see, e.g., the compounds provided in Section II - Exemplary Compounds).
  • the present invention is not limited to a particular type of screen for identifying heat shock factor (e.g., HSFl) activators.
  • the present invention provides a yeast based screen.
  • the present invention is not limited to use of a particular type of yeast. Docket No. DUKE-30386/WO-l/ORD 20/66
  • the screen comprises genetically modified yeast.
  • the screen is not limited to a particular type of genetically modified yeast.
  • the screens provide yeast that are genetically modified such that expression of yeast HSF is regulated.
  • the screens provide yeast that are genetically modified such that the yeast express human HSFl . While human HSFl and yeast HSF have similar structures, bind as homo-trimers to conserved HSEs and activate functionally common Hsp genes, expression of wild type human HSFl cannot suppress the viability defect associated with yeast HSF deletion (yhsf ⁇ ) cells (see, e.g., Liu, X.D., et al, (1997) EMBO J. 16: 6466-6477; herein incorporated by reference in its entirety).
  • Biochemical analysis of human HSFl demonstrated that human HSFl exists in yeast as a monomer and is not able to homo-trimerize under basal or stress conditions. Indeed, expression in yhsf ⁇ cells of the human HSFllz4m mutant, which is constitutively trimerized in culturized human cells, is able to rescue the yhsf ⁇ viability defect, bind to and activate stress-inducible target gene transcription such as from the yeast Hsp70 gene, and exist as a homo-trimer in yeast (see, e.g., Liu, X.D., et al., (1997) EMBO J. 16: 6466-6477; Liu, P.C.C., and Thiele, D.J.
  • the screens of the present invention identify activators (e.g., compounds) capable of facilitating homotrimerization of HSFl.
  • the screens are not limited to a particular manner of genetically modifying yeast HSF expression.
  • genetically modified yeast HSF expression occurs through deleting the HSF gene open reading frame, thereby rendering a yhsf ⁇ strain that is inviable.
  • the yhsf ⁇ strains have a yeast HSF gene coupled with an inducible promoter (e.g., GALl-IO) thereby rendering growth of such yhsf ⁇ strains viable on a selectable medium (e.g., galactose).
  • yhsf ⁇ strains express a yeast HSF gene coupled with an GAL promoter
  • the strain is inviable at any temperature or under any condition tested when cells are shifted to a glucose medium thereby extinguishing yeast HSF espression.
  • the yhsf ⁇ strains expressing a yeast HSF gene coupled with a inducible promoter additionally harbor a plasmid configured for human HSFl Docket No. DUKE-30386/WO-l/ORD 21/66
  • the plasmid configured for expression of hHSF is pRS424-GPD-hHSFl (wherein GPD is the constitutively expressed glucose phosphate dehydrogenase promoter).
  • the yhsf ⁇ strains expressing a yeast HSF gene coupled with a inducible promoter configured for expression hHSFl are used for identifying activators of hHSFl (e.g., activators capable of facilitating HSFl homotrimerization), identifying activators of HSFl target gene expression (e.g., Heat Shock Proteins) and/or activation or inhibition of protein chaperone activity (e.g., increased protein folding, increased protein solubilization, protein degradation).
  • activators of hHSFl e.g., activators capable of facilitating HSFl homotrimerization
  • identifying activators of HSFl target gene expression e.g., Heat Shock Proteins
  • protein chaperone activity e.g., increased protein folding, increased protein solubilization, protein degradation.
  • This screen has several features, including but not limited to, (1) when yeast HSF expression is extinguished, only cells in which human HSFl has been activated are viable, providing a screen with a very low background; (2) this strain allows for positive selection of human HSFl activator molecules; (3) this strain in conjunction with an additional strain lacking hHSFl expression permits identification of molecules that act exclusively in a human HSFl -dependent manner rather than via the prevention of yeast HSF repression by glucose in yeast; and (4) the screen is amenable to automated liquid handling and optical density determination and is therefore high throughput in nature.
  • the screens are not limited to a particular method for identifying activators of hHSFl.
  • a yhsf ⁇ strain expressing hHSFl is exposed to a small molecule under conditions inviable for growth absent homotrimerization or other means of activation of hHSFl . Growth in such conditions indicates that the small molecule is an activator of hHSFl (see, Figure 2). A lack of growth indicates that the small molecule is not an activator of hHSFl (see, Figure 2).
  • the yeast strains used in the screens are genetically modified to maximize small molecule accumulation.
  • the yeast strains are not limited to a particular manner of genetic modification to maximize small molecule accumulation.
  • genetic modification to maximize small molecule accumulation is accomplished through, for example, sequential deletion of the PDR5, SNQ2 and ERG6 genes in the yhsf ⁇ background.
  • PDR5 and SNQ2 encode ATP binding cassette integral plasma membrane transport proteins which mediate multidrug resistance by exporting compounds with a broad range of structures and relatively low specificity (see, e.g., Emter, R., (2002) Docket No. DUKE-30386/WO-l/ORD 2.V66
  • yeast cells lacking Pdr5 and Snq2 accumulate organic molecules to a significantly higher steady state level than wild type strains.
  • the ERG6 gene encoding delta(24)-sterol C- methyltransferase, is a key enzyme in ergosterol biosynthesis. Erg6 mutants exhibit enhanced diffusion rates of lipophilic molecules across the plasma membrane (see, e.g., Emter, R., (2002) FEBS Letters 521 : 57-61; herein incorporated by reference in its entirety).
  • HSF activating compounds e.g., compounds capable of facilitating HSFl homotrimerization, compounds capable of activating HSFl target gene expression (e.g., Heat Shock Elements), compounds capable of activating HSFl protein function (e.g., Heat Shock Proteins), compounds capable of activating or inhibiting protein chaperone activity (e.g., increased protein folding, increased protein solubilization, protein degradation)).
  • HSF activating compounds e.g., compounds capable of facilitating HSFl homotrimerization, compounds capable of activating HSFl target gene expression (e.g., Heat Shock Elements), compounds capable of activating HSFl protein function (e.g., Heat Shock Proteins), compounds capable of activating or inhibiting protein chaperone activity (e.g., increased protein folding, increased protein solubilization, protein degradation)).
  • the composition comprises a compound described by the
  • Ri is , H, or halogen (e.g., chlorine ).
  • Xi is , or Xi is absent.
  • X 2 is S or C.
  • X 3 is S or C.
  • R 9 is -OCH 3 .
  • R 5 is S or C.
  • R 6 is H or O .
  • R 7 is H or O .
  • Rg is or
  • Rio is chlorine or CH 3 . In some embodiments,
  • Rn is substituted or unsubstituted alkyl such as, for example, or
  • the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-N-phenyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the present invention provides HSF activating compounds described by any of the the compounds shown in Figure 7.
  • the present invention provides HSF activating compounds described by the following formulas: Docket No. DUKE-30386/WO-l/ORD 28/66
  • Rl is , H, or halogen (e.g., Chlorine);
  • R3 and R4 are C or N, but R3 and R4 are not the same); wherein R5 is S or C; wherein R6 is H or O ; wherein R7 is H or O ; Docket No. DUKE-30386/WO-l/ORD 29/66
  • R9 is C hlorine or CH3; and wherein RlO is • , or
  • the present invention provides the following HSFl activating compounds:
  • a functional derivative of a compound capable of HSF activation e.g., a compound capable of HSF activation
  • the present invention provides any of the compounds described herein with further functionalization.
  • the present invention provides any of the compounds functionalized with a biotin moiety (e.g., HSFlA-biotin)
  • Such functionalized compounds can be used for identification of agents (e.g., protein(s)) with which they interact. Docket No. DUKE-30386/WO-l/ORD 31/66
  • the compounds are also useful for preparing medicaments for treating other disorders wherein the effectiveness of the compounds are known or predicted.
  • disorders include, but are not limited to, neurological disorders.
  • the methods and techniques for preparing medicaments of a compound of the present invention are well-known in the art. Exemplary pharmaceutical formulations and routes of delivery are described below.
  • any one or more of the compounds described herein, including the many specific embodiments, are prepared by applying standard pharmaceutical manufacturing procedures. Such medicaments can be delivered to the subject by using delivery methods that are well-known in the pharmaceutical arts.
  • compositions are administered alone, while in some other embodiments, the compositions are preferably present in a pharmaceutical formulation comprising at least one active ingredient/agent, as defined above, together with a solid support or alternatively, together with one or more pharmaceutically acceptable carriers and optionally other therapeutic agents.
  • Each carrier must be "acceptable” in the sense that it is compatible with the other ingredients of the formulation and not injurious to the subject. Docket No. DUKE-30386/WO-l/ORD 3-V66
  • Contemplated formulations include those suitable oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary administration.
  • formulations are conveniently presented in unit dosage form and are prepared by any method known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association (e.g. , mixing) the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, wherein each preferably contains a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient is presented as a bolus, electuary, or paste, etc.
  • tablets comprise at least one active ingredient and optionally one or more accessory agents/carriers are made by compressing or molding the respective agents.
  • compressed tablets are prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g., povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. , sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose)surface-active or dispersing agent.
  • a binder e.g., povidone, gelatin, hydroxypropylmethyl cellulose
  • lubricant e.g., inert diluent
  • preservative e.g. , sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose
  • Molded tablets are made by molding in a suitable machine a mixture of the powdered compound (e.g., active ingredient) moistened with an inert liquid diluent. Tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or Docket No. DUKE-30386/WO-l/ORD 33/66
  • sucrose and acacia sucrose and acacia
  • mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • compositions for topical administration are optionally formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.
  • topical formulations comprise patches or dressings such as a bandage or adhesive plasters impregnated with active ingredient(s), and optionally one or more excipients or diluents.
  • the topical formulations include a compound(s) that enhances absorption or penetration of the active agent(s) through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide (DMSO) and related analogues.
  • DMSO dimethylsulfoxide
  • the aqueous phase of a cream base includes, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane- 1, 3 -diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof.
  • a polyhydric alcohol i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane- 1, 3 -diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof.
  • oily phase emulsions of this invention are constituted from known ingredients in a known manner.
  • This phase typically comprises a lone emulsifier (otherwise known as an emulgent), it is also desirable in some embodiments for this phase to further comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil.
  • a hydrophilic emulsifier is included together with a lipophilic emulsifier so as to act as a stabilizer. It some embodiments it is also preferable to include both an oil and a fat.
  • Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.
  • oils or fats for the formulation is based on achieving the desired properties (e.g., cosmetic properties), since the solubility of the active compound/agent in most oils likely to be used in pharmaceutical emulsion formulations is very low.
  • creams should preferably be a non-greasy, non-staining and washable Docket No. DUKE-30386/WO-l/ORD 34/66
  • Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used. Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the agent.
  • a suitable carrier especially an aqueous solvent for the agent.
  • Formulations for rectal administration may be presented as a suppository with suitable base comprising, for example, cocoa butter or a salicylate.
  • Formulations suitable for vaginal administration may be presented as pessaries, creams, gels, pastes, foams or spray formulations containing in addition to the agent, such carriers as are known in the art to be appropriate.
  • Formulations suitable for nasal administration include coarse powders having a particle size, for example, in the range of about 20 to about 500 microns which are administered in the manner in which snuff is taken, i.e., by rapid inhalation (e.g., forced) through the nasal passage from a container of the powder held close up to the nose.
  • suitable formulations wherein the carrier is a liquid for administration include, but are not limited to, nasal sprays, drops, or aerosols by nebulizer, an include aqueous or oily solutions of the agents.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs.
  • the formulations are presented/formulated in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, Docket No. DUKE-30386/WO-l/ORD 35/66
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Preferred unit dosage formulations are those containing a daily dose or unit, daily subdose, as herein above -recited, or an appropriate fraction thereof, of an agent.
  • the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavoring agents. It also is intended that the agents, compositions and methods of this invention be combined with other suitable compositions and therapies.
  • Still other formulations optionally include food additives (suitable sweeteners, flavorings, colorings, etc.), phytonutrients ⁇ e.g., flax seed oil), minerals ⁇ e.g., Ca, Fe, K, etc.), vitamins, and other acceptable compositions ⁇ e.g., conjugated linoelic acid), extenders, and stabilizers, etc.
  • food additives suitable sweeteners, flavorings, colorings, etc.
  • phytonutrients ⁇ e.g., flax seed oil
  • minerals e.g., Ca, Fe, K, etc.
  • vitamins and other acceptable compositions ⁇ e.g., conjugated linoelic acid
  • extenders e.g., and stabilizers, etc.
  • the compounds of the present invention are provided in unsolvated form or are in non-aqueous solutions (e.g., ethanol).
  • the compounds may be generated to allow such formulations through the production of specific crystalline polymorphs compatible with the formulations.
  • the compounds of the present invention are provided in conjunction with thermal therapy such as a thermal bath.
  • the present invention provides instructions for administering said compound to a subject.
  • the present invention provides instructions for using the compositions contained in a kit for the treatment of conditions characterized by the dysregulation of apoptotic processes in a cell or tissue ⁇ e.g., providing dosing, route of administration, decision trees for treating physicians for correlating patient- specific characteristics with therapeutic courses of action).
  • the present invention provides instructions for using the compositions contained in the kit to treat a variety of medical conditions associated with misfolded proteins or irregular HSFl activitiy (e.g., medical conditions involving misfolded proteins or irregular HSFl activity) (e.g., medical conditions involving irregular chaperone activity) (e.g., Alzheimer's disease, Parkinson's disease, Huntington disease, Amyotrophic Lateral Sclerosis, a prion-based disease, cataract, age-related cataract, glaucoma, macular degeneration, age-related macular degeneration, retinitis pigmentosa, cardiovascular disease and stroke, heat stroke, Docket No. DUKE-30386/WO-l/ORD 36/66
  • the methods are directed towards crystallins in age-related cataracts. In some embodiments, the methods are directed towards myocillin in glaucoma.
  • Various delivery systems are known and can be used to administer therapeutic agents (e.g., exemplary compounds as described in Section III above) of the present invention, e.g., encapsulation in liposomes, microparticles, microcapsules, receptor-mediated endocytosis, and the like.
  • Methods of delivery include, but are not limited to, intra-arterial, intramuscular, intravenous, intranasal, and oral routes.
  • the agents identified can be administered to subjects or individuals susceptible to or at risk of developing pathological growth of target cells and correlated conditions.
  • the agent When the agent is administered to a subject such as a mouse, a rat or a human patient, the agent can be added to a pharmaceutically acceptable carrier and systemically or topically administered to the subject.
  • a tissue sample is removed from the patient and the cells are assayed for sensitivity to the agent.
  • Therapeutic amounts are empirically determined and vary with the pathology being treated, the subject being treated and the efficacy and toxicity of the agent. When delivered to an animal, the method is useful to further confirm efficacy of the agent.
  • in vivo administration is effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations are carried out with the dose level and pattern being selected by the treating physician. Docket No. DUKE-30386/WO-l/ORD 37/66
  • Suitable dosage formulations and methods of administering the agents are readily determined by those of skill in the art.
  • the compounds are administered at about 0.01 mg/kg to about 200 mg/kg, more preferably at about 0.1 mg/kg to about 100 mg/kg, even more preferably at about 0.5 mg/kg to about 50 mg/kg.
  • the effective amount may be less than when the agent is used alone.
  • the pharmaceutical compositions can be administered orally, intranasally, parenterally or by inhalation therapy, and may take the form of tablets, lozenges, granules, capsules, pills, ampoules, suppositories or aerosol form. They may also take the form of suspensions, solutions and emulsions of the active ingredient in aqueous or nonaqueous diluents, syrups, granulates or powders. In addition to an agent of the present invention, the pharmaceutical compositions can also contain other pharmaceutically active compounds or a plurality of compounds of the invention.
  • an agent of the present invention also referred to herein as the active ingredient, may be administered for therapy by any suitable route including, but not limited to, oral, rectal, nasal, topical (including, but not limited to, transdermal, aerosol, buccal and sublingual), vaginal, parental (including, but not limited to, subcutaneous, intramuscular, intravenous and intradermal) and pulmonary. It is also appreciated that the preferred route varies with the condition and age of the recipient, and the disease being treated.
  • the agent should be administered to achieve peak concentrations of the active compound at sites of disease. This may be achieved, for example, by the intravenous injection of the agent, optionally in saline, or orally administered, for example, as a tablet, capsule or syrup containing the active ingredient. Desirable blood levels of the agent may be maintained by a continuous infusion to provide a therapeutic amount of the active ingredient within disease tissue.
  • the use of operative combinations is contemplated to provide therapeutic combinations requiring a lower total dosage of each component antiviral agent than may be required when each individual therapeutic compound or drug is used alone, thereby reducing adverse effects. Docket No. DUKE-30386/WO-l/ORD 38/66
  • the present invention also includes methods involving co-administration of the compounds described herein with one or more additional active agents. Indeed, it is a further aspect of this invention to provide methods for enhancing prior art therapies and/or pharmaceutical compositions by co-administering a compound of this invention.
  • the agents may be administered concurrently or sequentially.
  • the compounds described herein are administered prior to the other active agent(s).
  • the pharmaceutical formulations and modes of administration may be any of those described above.
  • the two or more co -administered chemical agents, biological agents or radiation may each be administered using different modes or different formulations.
  • the agent or agents to be co-administered depends on the type of condition being treated.
  • the condition being treated is a neurological disorder (e.g., Huntington Disease)
  • the additional agent can be an anticonvulsant medication.
  • the additional agents to be co-administered can be any of the well-known agents in the art for a particular disorder, including, but not limited to, those that are currently in clinical use and/or experimental use.
  • the present invention provides methods (e.g., therapeutic applications) for treating conditions associated with protein misfolding.
  • the present invention is not limited to a particular type of method.
  • the methods for treating conditions associated with protein misfolding comprise a) providing: i. target cells having misfolded proteins; and ii. a composition (e.g., a composition comprising exemplary HSFl activating compounds as described in Section III above); and b) exposing the target cells to the composition under conditions such that the exposure results in enhanced HSFl activity.
  • the methods are not limited to treating a particular condition associated with protein misfolding.
  • the condition associated with protein misfolding is a medical condition involving deficient chaperone activity.
  • the condition associated with protein misfolding is enhanced aging, Alzheimer's disease,
  • Parkinson's disease Huntington disease, Amyotrophic Lateral Sclerosis, and prion-based Docket No. DUKE-30386/WO-l/ORD 39/66
  • the condition associated with protein misfolding is type 2 diabetes mellitus (see, e.g., Chung, J., et al., (2008) PNAS 105(5) 1739-1744; herein incorporated by refernece in its entirety).
  • the methods are not limited to a particular type of target cells.
  • the target cells are neurological cells.
  • the target cells are within a living mammal (e.g., human, horse, dog, cat, pig, rat, mouse, ape, monkey).
  • any one or more of these compounds can be used in combination with at least one other therapeutic agent (e.g., potassium channel openers, calcium channel blockers, sodium hydrogen exchanger inhibitors, anticonvulsant agents, antiarrhythmic agents, antiatherosclerotic agents, anticoagulants, antithrombotic agents, prothrombolytic agents, fibrinogen antagonists, diuretics, antihypertensive agents, ATPase inhibitors, mineralocorticoid receptor antagonists, phospodiesterase inhibitors, antidiabetic agents, antiinflammatory agents, antioxidants, angiogenesis modulators, antiosteoporosis agents, hormone replacement therapies, hormone receptor modulators, oral contraceptives, antiobesity agents, antidepressants, antianxiety agents, antipsychotic agents, antiproliferative agents, antitumor agents, antiulcer and gastroesophageal reflux disease agents, growth hormone agents and/or growth hormone secretagogues, thyroid mimetics, anti-infective agents, anti-spastic agents, anti-
  • Additional therapeutic agents for Huntington disease include, but are not limited to, anticonvulsant agents (e.g., valproic acid, clonazepam), antipsychotic agents (e.g., risperidone, haloperidol), rauwolfia alkaloids (e.g., reserpine), antidepressants (e.g., proxetine).
  • Additional therapeutic agents for Parkinson's disease include, but are not limited to, dopamine prodrugs (e.g., levodopa/carbidopa), dopamine agonists (e.g., Docket No. DUKE-30386/WO-l/ORD 40/66
  • catechol-O- methyltransferase (COMT) inhibitors e.g., tolcapone, entacapone, levodopa, carbidopa, entacapone
  • anticholinergics e.g., trihexyphenidyl, benztropine mesylate
  • MAO-B inhibitors e.g., selegiline, rasagiline
  • N-methyl-D-aspartic acid inhibitors e.g., amantadine.
  • Additional therapeutic agents for Alzheimer's disease include, but are not limited to, centrally acting AChE inhibitors (e.g., rivastigmine), NMDA antagonists (e.g., memantine), and free-radical scavengers (e.g., tocopherol).
  • Additional therapeutic agents for Amyotrophic Lateral Sclerosis include, but are not limited to, glutamate pathway antagonists (e.g., riluzole), antispastic agents (e.g., baclofen).
  • Additional therapeutic agents for prion diseases include, but are not limited to, Congo red and its analogs, anthracyclines, amphotericin B and its analogs, sulfated polyanions, and tetrapyrroles.
  • Additional therapeutic agents for type 2 diabetes mellitus include, but are not limited to, sulfonylurea agents (e.g., glipizide, glyburide, glimepiride), meglitinides (e.g., repaglinide, nateglinide), biguanides (e.g., metformin), thiazolidinediones (e.g., pioglitazone, rosiglitazone), dipeptidyl peptidase IV (DPP-4) inhibitors (e.g., sitagliptin), incretin mimetics (e.g., exenatide), amylin analogs (e.g., pramlintide acetate), and alpha-glucosidase inhibitors (e.g., acarbose, miglitol).
  • sulfonylurea agents e.g., glipizide, glyburide, glimepir
  • Additional agents for glaucoma, cataract, retinitis pigmentosa, and/or macular degeneration include, but are not limited to, Levobunolol (Betagan), Timolol maleate/hemihydrate (Timoptic Timoptic XE, Betimol, Istalol), Carteolol (Cartrol, Ocupress), Betaxolol (Betoptic- S), Metipranolol hydrochloride (OptiPranolol), Levobetaxolol (Betaxon), Brimonidine
  • Isosorbide Ismotic
  • Mannitol Osmitrol, Resectisol
  • Glycerin Ophthalgan, Osmoglyn
  • Brimonidine/timolol Combigan
  • Phenylephrine HCl Neo-Synephrine
  • Prednisolone acetate AK-Pred, Pred Forte
  • Dexamethasone Ocu-Dex
  • Ciprofloxacin Ciloxan
  • Erythromycin E-Mycin
  • Nepafenac ophthalmic Nepafenac ophthalmic (Nevanac), Verteporfm (Visudyne), Pegaptanib (Macugen), Ranibizumab (Lucentis), Vitamin A (Aquasol A, DeI-Vi-A), Vitamin E (Aquasol E, Vitec), Ascorbic acid (Cebid, Ascorbicap, Cevalin, Cecon), Lutein or Docket No.
  • This example describes the optimization of growth conditions for the DTY512 strain.
  • the growth conditions for ayhsfA strain harboring 1) a yeast HSF gene coupled with a GAL promoter and 2) a pRS424-GPD-hHSFl plasmid for hHSFl expression were optimized from petri dishes to 96 well microtiter dish format.
  • Cells were grown in Synthetic Complete medium lacking uracil and tryptophan to select for plasmid maintenance, in the presence of the non-inducing/non-repressing carbon source raff ⁇ nose (2%).
  • Galactose concentrations (0.01%) were empirically identified that induce sufficient levels of yeast HSF for robust yeast cell viability, while rendering cells sensitive to strong glucose repression of yeast HSF expression after the addition of 4% glucose.
  • the screen cells are grown to midlog phase in selective synthetic complete medium with 2% raffinose and 0.01% galactose. The culture was then diluted to -5,000 cells/ml in the same growth medium in which 4% glucose was substituted for the galactose, to initiate glucose repression of yeast HSF expression.
  • microtiter well slopes calculated over the course of 96 hours. As shown in Figure 4, the growth of cells was quantitatively followed in each microtiter well, the background growth of cells expressing wild type human HSFl with either no addition or DMSO alone is very low and allowed for facile qualitative detection of positive candidates in the screen.
  • This example describes validation of candidate HSFl activating compounds.
  • appropriate growth conditions galactose induction and glucose repression parameters for application of the yeast-based human HSFl activator screen to a high throughput 96-well format was identified.
  • This screen evaluated a combinatorial chemistry library of -10,500 diverse compounds built from approximately 75 unique scaffold structures in the PPD library (PPD Discovery Research, Research Triangle Park, NC). While this was a modest-sized screen, ⁇ 50 distinct library components were identified that allowed modest to robust yeast cell growth within 48-96 hours post-seeding at a concentration of 10 micromolar.
  • Figure 4A shows a small sample of five compounds that stimulated yeast cell growth with different efficacy, the DMSO solvent control and one compound that was negative from this screen.
  • HSFl-A HSFl-B
  • HSFl-C HSFl-C
  • HSFlADl is a functional derivative Docket No. DUKE-30386/WO-l/ORD 44/66
  • HSFlA e.g., HSFlADl is capable of stimulating yeast cell growth similar to HSFlA.
  • HSFlA As shown in Figure 5 the three chemically distinct compounds HSFlA, HSFlB, and HSFlC exhibited common structural features including the conservation of aryl moieties typical of pyrazol benzamides, as well as polarized bonds to oxygen near the center of each molecule.
  • Figure 6 shows synthetic routes for HSFlA, HSFlB, and HSFlC.
  • Figure 7 shows additional compounds, including HSFl-A, HSFl-B, and HSF-IC, identified as HSFl activating compounds identified from the PPD library. It should be understood that derivates of these compounds may also be used in the compositions and methods described herein (see, e.g., HSFlADl).
  • FIG. 8 shows HSFl activating compounds identified thorugh screens conducted with compounds from the LOPAC and Prestwick chemical libraries. It should be understood that derivates of these compounds may also be used in the compositions and methods described herein.
  • Equal amounts of total protein was separated on 10%-20% gradient gel, transferred to a nitrocellulose membrane and analyzed for expression of HSP70, mHSP25, HSFl, Q74-GFP and SODl using anti-HSP70 (SC-24, Santa Cruz), anti-niHSP25 (SPA-801, Stressgen), anti-HSFl (Bethyl), anti-GFP (SC-8334) and anti-SODl (SOD-100, Stressgen) antibodies respectively.
  • Secondary antibodies were either anti-mouse or anti-rabbit- HRP conjugated antibodies from GE Healthcare Life Sciences. Proteins were visualized using the Pico chemio luminescence kit (Pierce).
  • Example IV This example demonstrates that HSFlA activates the expression of the HSP70 and
  • HSP25 heat shock protein (chaperone) genes in Mouse Embryonic Fibroblast (MEF) cells were treated with the indicated concentrations of HSFlA (in micromolar) or the DMSO solvent control in OptiMEM media without serum for 15 hours or heat shocked (HS) in OptiMEM media without serum for 2 hours at 42 0 C and allowed to recover for 15 hr at 37 0 C.
  • Total cellular protein extracts were prepared, fractionated by SDS- polyacrylamide gel electrophoresis, transferred to a solid membrane and the Hsp70, Hsp25 and SODl proteins detected by probing with their respective specific antibodies followed by standard immunoblotting techniques to identify the proteins. Docket No. DUKE-30386/WO-l/ORD 46/66
  • HSFlA dependent activation of HSP70 is dependent on the presence of the gene encoding HSFl.
  • wild-type and hsfl -/- MEF cells were treated with the indicated concentrations of HSFlA or the DMSO solvent as control in OptiMEM media without serum for 15 hr or heat shocked (HS) for 2 hr in
  • the Hsp70, HSFl and SODl proteins were detected by immunoblotting as described in Figure 9.
  • Example VI This example demonstrates that HSFlA can act synergistically with heat shock to activate expression of the HSP70 protein chaperone.
  • MEF cells were treated with the indicated concentrations of HSFlA or the solvent DMSO as control in OptiMEM media without serum for 15 hr at 37 0 C, or with 30 micromolar HSFlA for lhr at 37 0 C, followed by a 1 hr heat shock at a sub-optimal heat shock temperature of 4O 0 C followed by a 15 hr recovery at 37 0 C.
  • Independently, cells were treated at the optimal heat shock temperature of 42 0 C for two hours followed by a 15 hr recovery.
  • the HSP70 and SODl proteins were detected by immunoblotting as described in Figure 9. Note that 30 micromolar HSFlA treatment synergizes with a sub-optimal heat shock temperature to result in higher levels of expression of the HSP70 protein chaperone.
  • HSFlA promotes expression of HSP70 and reduces the aggregation of poly-glutamine (polyQ) proteins in rat neuronal precursor (PC-12) cells.
  • PC-12 cells were treated with the indicated concentrations of HSFlA or the DMSO solvent as control in OptiMEM media without serum for 15 hr or heat shocked in OptiMEM media without serum for 2 hr at 42 0 C and allowed to recover for 15 hr at 37 0 C (HS).
  • HSP70 and SODl proteins were detected by immunoblotting as described in Figure 9.
  • Figure 12B shows PC-12 cells, stably expressing the Huntington Q74-GFP fusion protein were treated with 20 micromolar HSFlA or the solvent DMSO (0.5%) as control in OptiMEM media without serum for 15 hr to stimulate HSP70 expression. After 15 hr incubation, expression of the Q74-GFP protein was induced via the addition of 1 microgram Docket No. DUKE-30386/WO-l/ORD 47/66
  • doxycyclin per milliliter doxycyclin.
  • the cells were incubated in the presence of doxycyclin and HSFlA for an additional 48 hrs after which the cells were harvested and soluble proteins (S) were isolated using cell lysis buffer.
  • Insoluble proteins (P) were pelleted by centrifugation. The pellet was washed once in 1 milliliter cell lysis buffer and solubilized via a mixture urea buffer (cell lysis buffer + 5M Urea) and Laemmli buffer (1 :2 ratio).
  • the polyglutamine-GFP protein (Q74-GFP) was detected by immunoblotting with anti-GFP antibody in a manner similar to that described in Figure 9.
  • Example VIII An example of a neurodegenerative disease that results from protein misfolding is called Huntington's disease, a progressive neurodegenerative disease that leads to neuronal cell death. This disease is due to the aggregation of a protein called Huntingtin, where those patients with this disease gene express a Huntingtin protein that harbors a genetically inherited expansion of the amino acid glutamine (abbreviated Q). The polyQ Huntingtin protein mis-folds and aggregates, thereby causing neuron death and the disease symptoms.
  • a fly model of polyQ protein-mediated neuronal degeneration has been used to test if HSFlA- mediated elevation in protein chaperone production can ameliorate eye cell degeneration in a fly in which polyQ protein is strongly expressed in the eye.
  • Figure 13 shows that HSFlA functions to ameliorate eye degeneration in a fruit fly model of poly glutamine disease.
  • UAS-M JDtrQ78 flies were crossed to gmr-GAL4 flies in the chronic presence of food supplemented with DMSO, 200 ⁇ M HSFlA or 5 ⁇ M 17-AAG (positive control).
  • Control flies were UAS-MJDtrQ78 flies lacking the Gal4 transcription factor.
  • HSFlA did not bind to the Hsp90 protein nor did it compete for binding with a known HSFl activating chemical, geldanamycin, that Docket No. DUKE-30386/WO-l/ORD 48/66
  • HSFlA activates by binding and inhibiting Hsp90.
  • Hsp90 As shown in Figure 14 A, purified Hsp90 (AssayDesigns) was incubated with either 17- AAG, a known Hsp90 binding chemical, or HSFlA at the indicated concentration for 30 min at 4 0 C. 1 ⁇ M Geldanamycin, a known Hsp90 binding chemical, was coupled to the affinity matrix biotin (Geldanamycin-biotin) and was added for 1 hr at 4 0 C to displace 17- AAG and HSFlA from Hsp90. Geldanmycin-biotin bound Hsp90 was captured by the addition of neutravidin-agarose beads (Pierce) at 4 0 C for 30 min. Hsp90 was eluted from beads by heating to 95 0 C for 5 min and analyzed by immunob lotting utilizing and anti-Hsp90 antibody. As shown in Figure 14B, purified Hsp90 was incubated with either 10 ⁇ M
  • GD-B GD-B
  • 100 ⁇ M HSFlA-biotin for 60 min at 4 0 C and bound Hsp90 ⁇ was captured by the addition of neutravidin-agarose beads at 4 0 C for 30 min.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biomedical Technology (AREA)
  • Neurosurgery (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Neurology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Psychology (AREA)
  • Biophysics (AREA)
  • Hospice & Palliative Care (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Psychiatry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des composés activant le HSF, des procédés pour leur découverte, et leur recherche et leurs utilisations thérapeutiques. En particulier, la présente invention concerne des composés capables de faciliter l’activation du HSFl, et des procédés destinés à utiliser de tels composés en tant qu’agents thérapeutiques afin de traiter plusieurs états pathologiques associés au mauvais repliement des protéines.
EP09747702A 2008-05-15 2009-05-15 Compositions et procédés ayant trait aux composés activant le facteur de transcription de choc thermique et cibles correspondantes Withdrawn EP2300004A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US5351308P 2008-05-15 2008-05-15
PCT/US2009/044186 WO2009140621A2 (fr) 2008-05-15 2009-05-15 Compositions et procédés ayant trait aux composés activant le facteur de transcription de choc thermique et cibles correspondantes

Publications (2)

Publication Number Publication Date
EP2300004A2 true EP2300004A2 (fr) 2011-03-30
EP2300004A4 EP2300004A4 (fr) 2012-05-30

Family

ID=41319361

Family Applications (1)

Application Number Title Priority Date Filing Date
EP09747702A Withdrawn EP2300004A4 (fr) 2008-05-15 2009-05-15 Compositions et procédés ayant trait aux composés activant le facteur de transcription de choc thermique et cibles correspondantes

Country Status (4)

Country Link
EP (1) EP2300004A4 (fr)
CN (1) CN102088973A (fr)
CA (1) CA2724413C (fr)
WO (1) WO2009140621A2 (fr)

Families Citing this family (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9315449B2 (en) * 2008-05-15 2016-04-19 Duke University Substituted pyrazoles as heat shock transcription factor activators
SG183853A1 (en) 2010-03-17 2012-10-30 Taivex Therapeutics Inc Modulators of hec1 activity and methods therefor
RU2639876C2 (ru) 2010-03-30 2017-12-25 Версеон Корпорейшн Мультизамещенные ароматические соединения в качестве ингибиторов тромбина
ES2736999T3 (es) 2011-11-21 2020-01-09 Taivex Therapeutics Corp Biomarcadores para cánceres que responden a moduladores de la actividad de Hec1
US11071736B2 (en) 2011-11-21 2021-07-27 Taivex Therapeutics Corporation Modulators of HEC1 activity and methods therefor
MX373964B (es) 2013-03-15 2020-07-13 Verseon Corp Compuestos aromaticos multisustituidos como inhibidores de serina proteasa.
US9951025B2 (en) 2013-03-15 2018-04-24 Verseon Corporation Halogenopyrazoles as inhibitors of thrombin
WO2015143653A1 (fr) * 2014-03-26 2015-10-01 Merck Sharp & Dohme Corp. Inhibiteurs de la kinase trka, compositions et méthodes associées
US10189810B2 (en) 2014-09-17 2019-01-29 Verseon Corporation Pyrazolyl-substituted pyridone compounds as serine protease inhibitors
SI3261639T1 (sl) 2015-02-27 2023-01-31 Verseon International Corporation Substituirane pirazolne spojine kot zaviralci serinskih proteaz
US11028061B2 (en) * 2015-07-27 2021-06-08 Sanford Burnham Prebys Medical Discovery Institute Modulators of myocyte lipid accumulation and insulin resistance and methods of use thereof
CN105524056A (zh) * 2016-01-05 2016-04-27 中山大学肿瘤防治中心 一种氨基噻唑化合物及其制备方法和应用
CN107519239A (zh) * 2017-04-25 2017-12-29 兰州大学 黄芩提取物在制备激活hsp70药物中的应用
WO2019241376A1 (fr) 2018-06-14 2019-12-19 The Trustees Of Columbia University In The City Of New York Traitement de troubles cognitifs à l'aide de nitazoxanide (ntz), d'analogues de nitazoxanide (ntz) et de leurs métabolites
JP7189368B2 (ja) 2018-10-30 2022-12-13 ギリアード サイエンシーズ， インコーポレイテッド アルファ４ベータ７インテグリンの阻害のための化合物
KR102630416B1 (ko) 2018-10-30 2024-02-01 길리애드 사이언시즈, 인코포레이티드 알파4베타7 인테그린 억제제로서의 퀴놀린 유도체
US11179383B2 (en) 2018-10-30 2021-11-23 Gilead Sciences, Inc. Compounds for inhibition of α4β7 integrin
WO2020092394A1 (fr) 2018-10-30 2020-05-07 Gilead Sciences, Inc. Dérivés d'imidazopyridine utilisés en tant qu'inhibiteurs de l'intégrine alpha4bêta7
EP3917913A2 (fr) 2019-01-31 2021-12-08 Pfizer Inc. Composés 3-carbonylamino-5-cyclopentyl-1h-pyrazole ayant une activité inhibitrice de cdk2
CN114222730B (zh) 2019-08-14 2024-09-10 吉利德科学公司 用于抑制α4β7整合素的化合物
CN113087783B (zh) * 2021-04-20 2021-10-01 南京医科大学 Hd治疗药物中的小分子多肽及其应用
CN120754089A (zh) * 2025-08-14 2025-10-10 复旦大学附属肿瘤医院 一种hsf1a在制备抗肺癌转移药物中的应用

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MXPA05012281A (es) * 2003-05-14 2006-05-19 Torreypines Therapeutics Inc Compuestos y uso de los mismos en la modulacion beta amiloide.
GB0315111D0 (en) * 2003-06-27 2003-07-30 Cancer Rec Tech Ltd Substituted 5-membered ring compounds and their use
CN1898221A (zh) * 2003-09-06 2007-01-17 沃泰克斯药物股份有限公司 Atp－结合弹夹转运蛋白的调控剂
WO2005044194A2 (fr) * 2003-10-28 2005-05-19 Pharmacia Corporation Traitement ou prevention de la neoplasie a l'aide d'un inhibiteur de la proteine hsp90
EP1682143A2 (fr) * 2003-10-28 2006-07-26 Pharmacia Corporation Association de l'inhibiteur de hsp90 et de l'inhibiteur de la phophodiesterase destinee a traiter ou prevenir la neoplasie
BRPI0514099A (pt) * 2004-08-05 2008-05-27 Hoffmann La Roche composições farmacêuticas, compostos, processo para a sua manufatura, método para o tratamento terapêutico e/ou profilático de enfermidades e utilização dos compostos
US7919603B2 (en) * 2005-12-19 2011-04-05 New York University Heat shock RNA

Also Published As

Publication number Publication date
CA2724413C (fr) 2016-10-18
WO2009140621A3 (fr) 2010-02-25
CN102088973A (zh) 2011-06-08
WO2009140621A2 (fr) 2009-11-19
CA2724413A1 (fr) 2009-11-19
EP2300004A4 (fr) 2012-05-30
WO2009140621A8 (fr) 2011-05-12

Similar Documents

Publication Publication Date Title
CA2724413C (fr) Compositions et procedes ayant trait aux composes activant le facteur de transcription de choc thermique et cibles correspondantes
US9718784B2 (en) Substituted pyrazoles as heat shock transcription factor activators
CN103124724B (zh) Lsd1的基于芳基环丙胺的脱甲基酶抑制剂及其医疗用途
JP6276289B2 (ja) ベンジリデングアニジン誘導体、及びタンパク質ミスフォールディング疾患を治療するための治療的使用
JP5431485B2 (ja) β−アミロイド疾患およびシヌクレイノパチーを処置するための化合物、組成物、および方法
CN104755484B (zh) Bace抑制剂
KR20220078644A (ko) 미토콘드리아 질환 치료를 위한 바티퀴논의 퀴논-, 히드로퀴논- 및 나프토퀴논-유사체
EP2830608B1 (fr) Dérivés de phénylurée et de phénylcarbamate comme inhibiteurs d'agrégation de protéine
EP2888252B1 (fr) Composés de benzofurazane anti-amyloïdes et procédés s'y rapportant
US20040024215A1 (en) Benzamide derivatives and their use as apob-100 and mtp inhibitors
US20170227553A1 (en) Compositions and methods for identifying and treating conditions involving hsf1 activity
JP2009521470A (ja) シヌクレイン障害の治療
DE60110802T2 (de) Heterozyklische-hydroximino-fluorene und ihre verwendung zur inhibierung von proteinkinasen
CN106068269B (zh) 氨基噻嗪化合物
AU2011326130B9 (en) Compositions and methods relating to heat shock transcription factor activating compounds and targets thereof
EP1036069A1 (fr) Composes benzenesulfamides
JP2010502680A (ja) ミトコンドリアナトリウム−カルシウムエクスチェンジャーとして有用な環状スルホン
WO2025160592A1 (fr) Composés dérivés de benzothiazole et d'indole, compositions et leurs utilisations pour la double inhibition de tau 2n4r
CN117794538A (zh) 用于治疗α1-抗胰蛋白酶缺乏症的组合物及其用途

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20101214

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL BA RS

RIC1 Information provided on ipc code assigned before grant

Ipc: C07D 277/60 20060101ALI20120418BHEP

Ipc: A61K 31/4155 20060101ALI20120418BHEP

Ipc: A61K 31/415 20060101AFI20120418BHEP

Ipc: A61K 31/426 20060101ALI20120418BHEP

Ipc: A61K 45/06 20060101ALI20120418BHEP

Ipc: A61P 25/28 20060101ALI20120418BHEP

Ipc: C07D 277/38 20060101ALI20120418BHEP

Ipc: A61P 25/16 20060101ALI20120418BHEP

Ipc: A61P 35/00 20060101ALI20120418BHEP

A4 Supplementary search report drawn up and despatched

Effective date: 20120502

17Q First examination report despatched

Effective date: 20131205

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20180213