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WO2008135639A2 - Novel useful inhibitors - Google Patents

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Publication number
WO2008135639A2
WO2008135639A2 PCT/FI2008/050246 FI2008050246W WO2008135639A2 WO 2008135639 A2 WO2008135639 A2 WO 2008135639A2 FI 2008050246 W FI2008050246 W FI 2008050246W WO 2008135639 A2 WO2008135639 A2 WO 2008135639A2
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Prior art keywords
phd3
cells
induced
aggregation
protein aggregation
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WO2008135639A3 (en
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Panu Jaakkola
Krista Rantanen
Juha Pursiheimo
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    • 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
    • A61K31/01—Hydrocarbons
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/191—Carboxylic acids, e.g. valproic acid having two or more hydroxy groups, e.g. gluconic acid
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis
    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/20—Dermatological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders
    • G01N2800/245—Transplantation related diseases, e.g. graft versus host disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/285—Demyelinating diseases; Multipel sclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2871—Cerebrovascular disorders, e.g. stroke, cerebral infarct, cerebral haemorrhage, transient ischemic event
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/40—Disorders due to exposure to physical agents, e.g. heat disorders, motion sickness, radiation injuries, altitude sickness, decompression illness

Definitions

  • the present invention relates to methods and assays for identifying compounds that inhibit the protein aggregation induced by PHD3. Such compounds are useful in treating, preventing and/or alleviating disorders related to hypoxia and/or PHD3 signalling.
  • Hypoxia forms a key component of multiple diseases, including cardiovascular diseases, stroke, inflammatory diseases, degenerative disorders and progression of solid tumors. Hypoxia and in some cases the following reoxygenation pose considerable stress to the cells. Cells facing these conditions need to survive with reduced amount of energy from oxidative phosphorylation, with increased production of reactive oxygen species (ROS) and low pH. Mammalian cells have evolved a molecular machinery to protect cells in these conditions and in sever hypoxia to drive the cells into apoptosis. The best characterized molecular responses to hypoxia are mediated through hypoxia-inducible factor (HIF) transcription factor complex.
  • HIF hypoxia-inducible factor
  • HIF- ⁇ regulatory ⁇ -subunit
  • Pro402 and 564 proline residues
  • pVHL von Hippel-Lindau tumor suppressor protein
  • pVHL von Hippel-Lindau tumor suppressor protein
  • HPD prolyl hydroxylase domain proteins
  • HPH HIF prolyl hydroxylases
  • Egl-9 homologues Egl-9 homologues
  • the three PHD isoforms (PHD1 -3) have similar requirements for O 2 and co-substrates Fe" and 2-oxoglutarate. However, their function and characteristics differ in several aspects.
  • the isoforms show different preference for HIF-1 ⁇ and -2 ⁇ as well as for the two prolyl hydroxylation sites.
  • PHD2 has been demonstrated to be the most important isoform for the downregulation of HIF in normoxic as well as mild hypoxic conditions.
  • the PHD isoforms show different tissue distribution. PHD2 demonstrates most abundant mRNA expression across tissues while PHD3 (also called EGLN3 or HPH1 ) is expressed mainly in the cardiac and neural tissue.
  • PHD2 and PHD3 are upregulated transiently by hypoxia in a HIF- dependent manner out of which PHD3 shows most robust induction.
  • PHD3 or the murine homolog SM-20 are upregulated e.g. by vascular tissue injury, ageing of cells and NGF removal in neural cells.
  • the murine homolog of PHD3, SM-20 has been reported to bear an apoptotic function in neural cells. The mechanism by which PHD3 activates apoptosis and whether this occurs in other cells than neural cells, are not known.
  • Protein aggregation is induced in response to various cellular stress and when a cell's capacity to degrade misfolded proteins is exceeded as well as during degenerative processes.
  • the aggregates generally contain components of the 26S proteasome, diverse ubiquitylated proteins and chaperones such as HSP70 and TRiC. Both cytotoxic and cytoprotective functions have been reported for these structures. In some situations the accumulation of protein aggregates impairs the function of the ubiquitin- proteasomal system and may lead to caspase-dependent apoptosis. Protein aggregation has been implicated in the pathogenesis of several neurodegenerative diseases, such as Parkinson's, Huntington's and Alzheimer's diseases and amyotrophic lateral sclerosis (ALS) as well as alcoholic liver disease.
  • ALS amyotrophic lateral sclerosis
  • the protein aggregation is a tightly regulated and dynamic process.
  • the number, size, subcellular localization and protein content of the aggregates vary depending on the aggregating protein and cell type. Multiple aggregates varying in size and scattered around the cytoplasm are in many instances referred to as aggresome-like structures.
  • the small cytoplasmic protein aggregates can be transported along the microtubules (MT) towards the perinuclear region where they converge and form large structures at the microtubule organizing center (MTOC) and are termed aggresomes. Aggresomes may disrupt the organization of cytoskeleton leading to formation of vimentin cage around the aggresome.
  • MT microtubules
  • MTOC microtubule organizing center
  • Aggresomes are induced by various proteins such as ⁇ -synuclein and HDAC6 deacetylase in Parkinson's disease and prions in prion-associated disease. Smaller scattered protein aggregates or aggresome-like structures are detected for example with PLIC-1 and p62/SQSTM1. No association between hypoxia and protein aggregation / aggresome formation has been described.
  • PHD3 is known to induce apoptosis in rat neural (PC-12) cells after growth factor removal. PHD3 is undetectable or expressed at low level in normoxic cells. The expression is strongly induced during hypoxia. However, in hypoxia PHD3 remains mainly inactive and the full activity is restored upon reoxygenation.
  • PH D3 may also lead to unexpected disease states in humans depending on the tissue oxygenation status and cell types involved in a given disease state. There is thus an identified need of further elucidating the mechanism by which PHD3 activates cell death and in which cell types this occurs.
  • the present invention relates to a use of a prolyl hydroxylase inhibitor for the manufacture of a medicament for inhibiting PHD3-induced protein aggregation in a disease, and to a method of treating, preventing and/or alleviating a condition associated with PHD3-induced protein aggregation.
  • Said disease or condition may be selected from a group consisting of cytotoxicity or mechanical trauma induced tissue damage, reperfusion associated disorders, ischemic diseases, myocardiopathies, delayed or impaired wound healing, rejection or tissue damage in organ transplantation, alcoholic liver disease, and neurodegenerative diseases.
  • Said inhibitor may be selected from a group consisting of 2-oxoglutarate analogs, oxalo-amino acids, hydroxamic acids, hydroxylated aromatic compounds, pyridine N-oxide derivatives, 3-hydroxyquinolone 2-carboximide derivatives and pyhdylcarbonyl glycines, malonic acid, 3-nitroproprionic acid, and theonyl trifluoroacetate.
  • said neurodegenerative disease is a frontotemporal dementia with motoneuron disease and said inhibitor is a 2- oxoglutarate analog.
  • the present invention further relates to a method for identifying a therapeutic lead compound comprising the steps of a) providing a cell culture comprising cells expressing PHD3, b) treating said cells with a compound suspected to inhibit the aggregation of PHD3 d) analysing said cells for the inhibition of PHD3 aggregation, and e) identifying said compound as a therapeutic lead compound if the determination in step d) is positive.
  • the present invention also relates to a method for the production of a pharmaceutical composition, wherein a therapeutic lead compound is identified by embodiments according to the present invention, and further mixed with a pharmaceutically acceptable excipient.
  • the present invention still further relates to compounds capable of inhibiting PHD3-induced protein aggregation, identified by embodiments according to the present invention.
  • the present invention further relates to the use of a compound capable of inhibiting PHD3-induced protein aggregation, identifiable by a method according to embodiments of the present invention, for the manufacture of a medicament for treating, preventing and/or alleviating a condition or a disease associated with protein aggregation / aggresome formation, such as cytotoxicity or mechanical trauma induced tissue damage, reperfusion associated disorders, ischemic diseases, myocardiopathies, rejection or tissue damage in organ transplantation, delayed or impaired wound healing, and alcoholic liver disease, neurodegenerative disorders.
  • a condition or a disease associated with protein aggregation / aggresome formation such as cytotoxicity or mechanical trauma induced tissue damage, reperfusion associated disorders, ischemic diseases, myocardiopathies, rejection or tissue damage in organ transplantation, delayed or impaired wound healing, and alcoholic liver disease, neurodegenerative disorders.
  • the present invention further relates to a method for treating, preventing and/or alleviating a condition associated with PHD3-induced protein aggregation, and to a method of inhibiting the PHD3 -induced protein aggregation in a mammal, said methods comprising administering to a patient in need thereof a therapeutically effective amount of a compound capable of inhibiting said protein aggregation, identifiable by a method according embodiments of the present invention.
  • the present invention relates to a method of diagnosing a neurodegerative disease comprising the following steps: a) staining a tissue sample obtained from a mammal, said mammal suspected to suffer from a neurodegenerative disorder, using an antibody against PHD3, and b) determining the presence of PHD3-induced protein aggregates in said samples, the presence of PHD3 aggregates being indicative of a neurodegenerative disorder.
  • FIGS 1A - 1 E show that PHD3 induces apoptosis in HeLa cells in a hydroxylase-dependent manner.
  • Fig. 1A HeLa cells expressing enhanced green fluorescent protein (EGFP) (cont), PHD3-EGFP, hydroxylase mutant PHD3 (PHD3R206K-EGFP) or PHD3-EGFP exposed to 8-hour hydroxylase inhibition (DMOG) were stained for activated caspase-3 (white cells). Caspase- 3 activation induced by PHD3 was suppressed by the inhibition of hydroxylase activity.
  • FIG. 1 B Quantification of the PHD3-induced apoptosis by caspase-3 activation with the indicated stimuli.
  • FIG. 1 C Quantification of PHD3-induced apoptosis by nuclear fragmentation.
  • FIG. 1 D Apoptosis measured by FACS analysis in HeLa cells expressing PHD3 for the indicated time.
  • FIG. 1 E Apoptosis measured by FACS analysis in HeLa cells transfected with the indicated amount of PHD3.
  • Figures 2A - 2D show that PHD3 forms subcellular aggregates (Fig.
  • FIG. 2A HeLa cells transfected with PHD3-EGFP or PHD2-EGFP and visualized 24 hours after transfection by confocal microscopy demonstrating the aggregation of PHD3.
  • FIG. 2B Localization of PHD3 24 hours after transfection in inmortalized keratinocytes (HaCaT) and squamous carcinoma cells from oral mucosa (SCC2).
  • FIG. 2C Quantification of PHD3-EGFP expression in cells by confocal microscopy with visible aggregates (lower cell) and cells expressing PHD3 evenly (upper cell).
  • FIG. 2D Quantification of the PHD3 expression level in 15 cells (mean and SD) indicating similar expression levels regardless of the subcellular localization of PHD3.
  • FIGS 3A - 3C show that PHD3 aggregation requires oxygen and hydroxylase activity.
  • FIG. 3A HeLa cells 24 hours after transfection with wild type PHD3-EGFP, PHD3-EGFP expressing cells exposed to either 6 hour hypoxia (1 % O 2 ) or 40 ⁇ M hydroxylase inhibitor dimethyloxaloylglycine (DMOG) or transfected with mutant PHD3R206K-EGFP. Aggregation of PHD3 was attenuated by the inhibition of hydroxylase activity either by DMOG or by the point mutation of PHD3.
  • FIG. 3B Quantification of cells with PHD3 aggregates. 200 - 300 cells were studied and judged to either have or to not have PHD3 aggregates. Means and range for two independent experiments are shown.
  • FIG. 3C Quantification of the cells with aggregated PHD3 under increasing level of the indicated hydroxylase inhibitior DMOG.
  • FIGS 4A - 4G show that PHD3 co-localises with aggresome-like structures.
  • Fig. 4A PHD3 and proteasomal subunit LMP-2 co-transfected into HeLa cells and detected by fluoresence confocal microscopy.
  • Fig. 4B Profiling of the localisation demonstrates co-localisation of LMP-2 with PHD3 within the aggregates, but not outside them. The line indicates the profile location.
  • FIG. 4C PHD3-EGFP transfected into HeLa cells and detected by fluoresence together with the endogenous large subunit (20S) of the proteasome by immunocytochemistry.
  • Fig. 4C PHD3-EGFP transfected into HeLa cells and detected by fluoresence together with the endogenous large subunit (20S) of the proteasome by immunocytochemistry.
  • FIG. 4D Profiling of the localisation of PH D3 and 2OS proteasome demonstrates co-localisation of 2OS proteasome and PHD3.
  • FIG. 4E Localization of PHD3-EGFP and endogenous HSP70 by immunocytochemistry showing near complete co-localization.
  • FIG. 4F Co- localization of transfected PHD3-EGFP and HA-ubiquitin.
  • FIG. 4G Co- localization of the endogenous PHD3 with transfected LMP2-EGFP after 2-day hypoxia and 1 hour reoxygenation. The endogenous PH D3 partially co- localizes with the proteosomal LMP2 marker.
  • Figures 5A - 5C show the dynamics of PHD3 aggregation.
  • FIG. 5A shows the dynamics of PHD3 aggregation.
  • FIG. 5B PHD3-EGFP was used for fluorescence recovery after photobleaching analysis (FRAP). A representative study of a cytoplasmic aggregate is shown. The arrowhead points to the photobleached aggregate. Re-apperance of PHD3-EGFP expression witin the aggregate demonstrated fast movement of PHD3 into the aggregate.
  • FIG. 5C Histogram of the FRAP analysis in Figure 5B.
  • FIGS. 6A-6J show that PHD3 induces aggregation of 26S proteasome.
  • HeLa cells were left untransfected (Figs. 6A-6B) or transfected with PHD3-EGFP construct (Figs. 6C-6H) and stained for endogenous 2OS proteasome.
  • Fig. 6B Proteosomal inhibition by MG132 caused aggregation of 2OS proteasome (arrowheads).
  • FIGs. 6C and 6D The forced expression of PH D3 led to the development of aggregates comparable to that induced by proteasomal inhibition by MG-132.
  • FIGs. 6E and 6F PHD3 expressing cells exposed to DMOG showed breakdown of 2OS aggregation.
  • FIG. 6G and 6H Expression of the mutant PHD3 demonstrated no visible 2OS aggregation.
  • FIG. 6I Localization of PHD3R206K-EGFP with endogenous 2OS. Aggregates formed by PHD3R206K do not co-localize with proteasomes.
  • FIG. 6J Localization of PHD3-EGFP expressing cells exposed to DMOG with endogenous 2OS. Aggregates formed during hydroxylase inhibition do not co- localize with the proteasome.
  • Figures 7A-7C show that the PHD3-induced aggregation coincides with apoptosis.
  • Fig. 7A HeLa cells transfected with PHD3-EGFP and stained for caspase-3 24 hours post-transfection. Only cells with PHD3 aggregation show activated caspase-3.
  • Fig. 7B Quantification of cells demonstrating visible aggregates in cells with (+) or without (-) caspase-3 activation.
  • Fig. 7C Quantification of caspase-3 activation from cells showing either aggregation of PHD3 (aggr) or even expression (even) 48 hours post-transfection.
  • FIG. 8 shows that PHD3 aggregates are found in frontotemporal dementia (FTD) with motoneuron disease. Sections from a spinal cord from a patient were stained for PHD3. Arrows point to the motoneurons showing aggregated PHD3.
  • FTD frontotemporal dementia
  • the present invention is based on the finding that PHD3 activates protein aggregation. It has surprisingly been found that PHD3 activates protein aggregation in HeLa cells during well -oxygenated conditions through its hydroxylase-dependent activity and that this activates cell death. PHD3 forms dynamic subcellular aggresome-like structures, which contain components of the 26S proteasome, HSP70 chaperone and ubiquitin. Expression of PHD3 is sufficient to induce the aggregation of 26S proteasomes. The ability of PHD3 to induce the aggresome-like structures is lost in hypoxia and requires the enzymatic activity of PHD3.
  • the data presented herein links protein aggregation and aggresome formation, which are implicated in several disease states, to the cellular oxygen-sensing pathway.
  • the present invention thus provides means for developing therapeutics for conditions or diseases related to protein aggregation such as neurodegenerative diseases, liver disorders as well as ischemia - reperfusion related conditions for example during treatment of heart infarction, myocardiopathies and tissue transplantation. More specifically, the present invention provides a method for identifying a therapeutic lead compound, which inhibits aggregation of PHD3.
  • the term "aggregation of PHD3" is intended to include aggresome formation induced by PHD3.
  • the method according to the present invention comprises the steps of: a) providing a cell culture comprising cells expressing PHD3, b) treating said cells with a compound suspected to inhibit the aggregation of PHD3 c) analysing said cells for the inhibition of PHD3 aggregation, d) identifying said compound as a therapeutic lead compound if the analysis in step d) is positive.
  • said cell culture is a mammalian cell culture comprising e.g. carcinoma, muscle, myocardial, endothelial, glioma and/or neuronal cells.
  • said cells of the cell culture have been treated to as to induce overexpression of PHD3. This can be achieved e.g. by transfecting said cells with a DNA encoding PHD3 protein and expressing said protein in said cells. Suitable methods for the transfection include, but are not limited to, calcium precipitation and liposome transfection, as well as electroporation. Suitable reagents, vectors and DNAs for said transfection are readily available in the art.
  • upregulation or overexpression of endogenous PHD3 may be obtained by exposing cell to stress such as hypoxia, reoxygenation, neural growth factor (NGF) withdrawal or others.
  • stress such as hypoxia, reoxygenation, neural growth factor (NGF) withdrawal or others.
  • cells may be exposed to hypoxia by culturing them under reduced pressure of oxygen (e.g. 1 % O2 for 16 hours) or by treating cells with a suitable chemical hypoxia mimetic, such as cobalt chloride.
  • Reoxygenation exposure may be achieved by exposing hypoxic cells to normal pressure of oxygen.
  • NGF withdrawal may be performed by differentiating cells, such as PC-12 cells, to neural cells by culturing them in the presence of NGF followed by withdrawing NGF.
  • Suitable modifications and other methods for upregulating PHD3 expression are apparent to a person skilled in the art.
  • Analysing the cells for the inhibition of PHD3 aggregation in step c) may be performed by any suitable method known in the art, such as microscopy based image analysis, for example by confocal microscopy including but not limited to fluorescence microscopy. Said analysing may comprise comparing the level of PHD3 aggregation in cells treated with a candidate or test compound to the level of PHD3 aggregation in non-treated control cells. A compound capable of inhibiting the aggresome formation induced by PHD3 is identified based on its ability to break down the aggresome bodies induced by PHD3.
  • the present invention also relates to a method for producing a pharmaceutical composition, said method comprising a method for identifying compound(s) that inhibit the aggresome formation induced by PHD3, and mixing the compound identified with a pharmaceutically acceptable excipient.
  • a pharmaceutically acceptable excipient Such excipients are readily available to those skilled in the art.
  • Preferably said identification is performed by the method described above.
  • Compounds or therapeutic lead compounds that may be identified according to the embodiments of the present invention include natural and synthetic chemical compounds, small molecules, natural and synthetic proteins and peptides, as well as vectors encoding any suitable peptide or protein.
  • hydroxylated aromatic compounds such as protocatechuic acid; pyridine N-oxide derivatives; 3- hydroxyquinolone 2-carboximide derivatives; pyridylcarbonyl glycines; malonic acid; 3-nitroproprionic acid, and theonyl thfluoroacetate are useful in preventing, treating and/or alleviating conditions or disease associated with PHD3-induced aggresome formation including cytotoxicity or mechanical trauma induced tissue damage; ischemia-reperfusion injury in the infarction of brain or heart and related ischemic diseases; other ischemic diseases such as complications of arterial blockage; myocardiopathies; alcoholic liver disease; rejection in organ transplantation; delayed or impaired wound healing; several neurodegenerative disorders such as, but not restricted to, Parkinson's disease, Alzheimer's disease, Huntington's disease amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) with motoneuron disease.
  • ALS amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • therapeutic lead compounds identifiable according to the embodiments of the present invention and said prolyl hydroxylase inhibitors may be used for the manufacture of a medicament for treating, preventing and/or alleviating such conditions or diseases.
  • 2-oxoglutarate analog are useful in treating, preventing and/or alleviating frontotemporal dementia (FTD) with motoneuron disease.
  • PHD3 indeed aggregates in cells of patients suffering from neurodegerative diseases, such as frontotemporal dementia (FTD) with motoneuron disease.
  • the present invention thus further relates to a method of diagnosing neurodegenerative disorders such as, but not restricted to, Parkinson's disease, Alzheimer's disease, Huntington's disease amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) with motoneuron disease.
  • neurodegenerative disorders such as, but not restricted to, Parkinson's disease, Alzheimer's disease, Huntington's disease amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) with motoneuron disease.
  • Such a method may comprise the following steps: a) staining a tissue sample obtained from a mammal, said mammal suspected to suffer from a neurodegenerative disorder, using an antibody against PHD3, and b) determining the presence of PHD3 aggregates in said sample, the presence of PHD3 aggregates being indicative of a neurodegenerative disorder.
  • determining the presence of PHD3 aggregates includes determining the level and subcellular localization of PHD3.
  • PHD3 aggregation and PHD3-induced aggresome formation are indicative of PHD3-induced cytotoxicity to the cells. The aggregation impairs the function of the cells and thereby causes or exacerbates the said disorders.
  • PHD3 induces apoptosis in neural cells when accumulated after NGF withdrawal.
  • PHD3 has the apoptosis-inducing capacity in cancer cells and whether the hydroxylase activity of PHD3 is required for apoptosis using HeLa cells as a model.
  • HeLa cells were transfected with PHD3 followed by 8-hour exposure to a well-characterized hydroxylase inhibitor dioxaloylmethylglycine (DMOG) at 40 uM (Cayman Chemical, Ann Arbor, Ml) and detection of an apoptosis marker caspase-3 activation by immunocytochemistry.
  • DMOG dioxaloylmethylglycine
  • Cells were grown in Dulbecco's minimum essential eagle modified medium (Sigma-Aldrich) supplemented with 10% fetal calf serum, 20 U/ml penicillin, 50 ⁇ g/ml streptomycin and 2 mM L-glutamin. Cells were transfected at optimal confluency using either FuGene HD (Roche Applied Science) or Effectene transfection reagent (Qiagen Inc., Chatsworth, CA) according to manufacturers protocols.
  • FuGene HD Roche Applied Science
  • Effectene transfection reagent Qiagen Inc., Chatsworth, CA
  • Activated caspase-3 was detected in immunohistochemistry using activated caspase-3 antibody at 1 :400 dilution (Promega, Madison, Wl). Transfection of PHD3 clearly activated caspase-3, and this was inhibited by DMOG (Fig. 1A).
  • DMOG Fig. 1A
  • PHD3 2-oxoglutarate coordinating arginine is mutated into lysine (PHD3R206K) and has been reported to inactivate the hydroxylase activity.
  • PHD3R206-EGFP was made by QuikChange site-directed mutagenesis kit (Stratagene) using 5'- CCCTCTTACGCAACCAAATATGCT ATGACTGTCTGG-3' oligonucleotide (the mutated residue is underlined).
  • Fig. 1A Quantification of either caspase-3 activation (Fig. 1 B) or nuclear fragmentation (Fig. 1 C) demonstrated near complete loss of the apoptotic activity of PHD3 by the inhibition of hydroxylase activity.
  • FACS studies further demonstrated that the apoptotic activity is both time- (Fig. 1 D) and PHD3 concentration- dependent (Fig. 1 E).
  • PHD3 forms subcellular aggregates in an oxygen and hydroxylase-dependent manner.
  • PHD3-EGFP expressing cells contained subcellular bodies or aggregates, which were not present in e.g. PHD2-EGFP transfected cells (Fig. 2A).
  • the aggregates were mainly located in the cytoplasmic or perinuclear departments and occasionally in the nucleus.
  • the aggregation of PHD3 did not depend on the tag used, as other PHD3 fusion proteins with different both N- and C- terminal tags showed similar aggregates.
  • the aggregation was not cell-type dependent and was also seen in HaCaT keratinocytes and squamous carcinoma cells (SCC) (Fig. 2B).
  • SCC HaCaT keratinocytes and squamous carcinoma cells
  • HeLa cells expressing ectopical PHD3-EGFP were exposed to hypoxia (1 % O2) or normoxia for 8 hours. Hypoxic treatments were performed in Invivo2 400 incubator (Ruskinn technologies, UK) in 1 % O2, 5 % CO2, 90 % moisture. Oxygen was replaced with 99,5% pure N 2 (AGA, Finland). The aggregation seen in normoxia was strongly reduced in hypoxic conditions (Fig. 3A).
  • PHD3 co-localizes with aggresomes.
  • PHD3 aggregates strongly resembled aggresome-like structures. These structures contain components of the 26S proteasome.
  • LMP2 an essential structural component of the proteasome
  • PHD3-dsRed A red variant of EGFP fused to PHD3 (PHD3-dsRed) was used to detect PHD3 with LMP2-EGFP fusion protein.
  • PHD3-dsRed complete PHD3 ORF was aquired by PCR and cloned into pDsRed-Monomer- N1 vector (Clontech) using Hindlll and Smal sites.
  • PCR primer oligonucleotides used were ⁇ '-GAGCAAGCTTTTATGCCCCTGGGACAC-S' and ⁇ '-CCTCCCGGGTCAGTCTTCAGTGAGG-S'.
  • Cells expressing both constructs showed nearly complete co-localization one day after transfection (Fig. 4A and B).
  • 2OS proteasome subunit expression was detected by anti-20S proteasome core subunits (Biomol) at 1 :2000 dilution and Cy5-conjugated secondary antibody (Jackson Immunochemicals, West Grove, PA) at 1 :1000 dilution.
  • the endogenous 2OS demonstrated co-localization with PHD3 (Fig. 4C and D). Furthermore, we detected co-localization of endogenous HSP70 chaperone (Fig. 4E) and transfected HA-ubiquitin (Fig. 4F) with PHD3-EGFP. Endogenous HSP70 was detected by a monoclonal antibody (SPA-810) at 1 :1000 dilution (Stressgen) and HA-Ubiquitin was detected by immunocytochemistry using antibody against haemagglutin (clone 3F10) at 1 :1000 dilution (Roche Diagnostics). We next asked whether the endogenous PHD3 co-localises with proteosomal structures.
  • Protein aggregates may be transported along the microtubules and in the case of aggresome formation they are transported towards the perinuclear region. Inhibition of microtubule polymerization by nocodazole abrogates the transportation. Supporting the hypothesis of PHD3 transport along the microtubuli, 5 ⁇ M nocodazole (Sigma-Aldhch) nearly completely inhibited the aggregation of PHD3 (Fig. 5A). Moreover, suggesting the requirement of proteasomal function for the localization of the PHD3 bodies, the inhibition of proteosomal function by MG132 (Sigma-Aldrich) caused PHD3 to cluster along the microtubuli (Fig. 5B). Aggresomes are highly dynamic structures.
  • a region of interest was selected and imaged (prebleach). Then ROI was photobleached with argon laser 488nm at maximum power for 100 iterations. The recovery of the bleached region was then imaged with 2% laser power with 2s intervals. The mobile fraction and the half-life of the recovery was calculated usin FRApCaIc program (Dr. Rolf Sara, Turku Centre for Biotechnology).
  • FRAP was studied from several cytoplasmic PHD3 aggregates and from larger perinuclear aggregates. The cytoplasmic aggregates showed rapid recovery of PHD3 within the aggregates indicating fast movement of PHD3 into these structures.
  • PHD3 expression activates aggresome formation in an oxygen- and hydroxylase-dependent manner.

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Abstract

The invention relates to a method for identifying therapeutic lead compounds useful in treating, preventing and/or alleviating disorders related to PHD3-induced aggresome formation. The invention further relates to pharmaceutical compositionsand methods for detecting PHD3- induced disorders.

Description

NOVEL USEFUL INHIBITORS
FIELD OF THE INVENTION
The present invention relates to methods and assays for identifying compounds that inhibit the protein aggregation induced by PHD3. Such compounds are useful in treating, preventing and/or alleviating disorders related to hypoxia and/or PHD3 signalling.
BACKGROUND OF THE INVENTION
Hypoxia forms a key component of multiple diseases, including cardiovascular diseases, stroke, inflammatory diseases, degenerative disorders and progression of solid tumors. Hypoxia and in some cases the following reoxygenation pose considerable stress to the cells. Cells facing these conditions need to survive with reduced amount of energy from oxidative phosphorylation, with increased production of reactive oxygen species (ROS) and low pH. Mammalian cells have evolved a molecular machinery to protect cells in these conditions and in sever hypoxia to drive the cells into apoptosis. The best characterized molecular responses to hypoxia are mediated through hypoxia-inducible factor (HIF) transcription factor complex. Under normoxic conditions the regulatory α-subunit (HIF-α) is post-translationally hydroxylated at two proline residues (Pro402 and 564). These are recognized by the von Hippel-Lindau tumor suppressor protein (pVHL) that subsequently leads to ubiquitination and proteosomal destruction of HIF-α. Three human prolyl hydroxylases that use O2 as a co-substrate, have been characterized and termed prolyl hydroxylase domain proteins (PHD), HIF prolyl hydroxylases (HPH) or Egl-9 homologues (EGLN). Under restricted O2 availability the PHD activity decreases and the degradation of HIF-α is blocked leading to the transcription of a wide range of genes. Such genes have key functions in cell survival and apoptosis regulation.
The three PHD isoforms (PHD1 -3) have similar requirements for O2 and co-substrates Fe" and 2-oxoglutarate. However, their function and characteristics differ in several aspects. The isoforms show different preference for HIF-1 α and -2α as well as for the two prolyl hydroxylation sites. PHD2 has been demonstrated to be the most important isoform for the downregulation of HIF in normoxic as well as mild hypoxic conditions. Moreover, The PHD isoforms show different tissue distribution. PHD2 demonstrates most abundant mRNA expression across tissues while PHD3 (also called EGLN3 or HPH1 ) is expressed mainly in the cardiac and neural tissue. Both PHD2 and PHD3 are upregulated transiently by hypoxia in a HIF- dependent manner out of which PHD3 shows most robust induction. Besides hypoxia, PHD3 or the murine homolog SM-20, are upregulated e.g. by vascular tissue injury, ageing of cells and NGF removal in neural cells. Moreover, unlike the two other PHD isoforms, the murine homolog of PHD3, SM-20, has been reported to bear an apoptotic function in neural cells. The mechanism by which PHD3 activates apoptosis and whether this occurs in other cells than neural cells, are not known. Protein aggregation is induced in response to various cellular stress and when a cell's capacity to degrade misfolded proteins is exceeded as well as during degenerative processes. The aggregates generally contain components of the 26S proteasome, diverse ubiquitylated proteins and chaperones such as HSP70 and TRiC. Both cytotoxic and cytoprotective functions have been reported for these structures. In some situations the accumulation of protein aggregates impairs the function of the ubiquitin- proteasomal system and may lead to caspase-dependent apoptosis. Protein aggregation has been implicated in the pathogenesis of several neurodegenerative diseases, such as Parkinson's, Huntington's and Alzheimer's diseases and amyotrophic lateral sclerosis (ALS) as well as alcoholic liver disease.
The protein aggregation is a tightly regulated and dynamic process. The number, size, subcellular localization and protein content of the aggregates vary depending on the aggregating protein and cell type. Multiple aggregates varying in size and scattered around the cytoplasm are in many instances referred to as aggresome-like structures. The small cytoplasmic protein aggregates can be transported along the microtubules (MT) towards the perinuclear region where they converge and form large structures at the microtubule organizing center (MTOC) and are termed aggresomes. Aggresomes may disrupt the organization of cytoskeleton leading to formation of vimentin cage around the aggresome. Aggresomes are induced by various proteins such as α-synuclein and HDAC6 deacetylase in Parkinson's disease and prions in prion-associated disease. Smaller scattered protein aggregates or aggresome-like structures are detected for example with PLIC-1 and p62/SQSTM1. No association between hypoxia and protein aggregation / aggresome formation has been described. PHD3 is known to induce apoptosis in rat neural (PC-12) cells after growth factor removal. PHD3 is undetectable or expressed at low level in normoxic cells. The expression is strongly induced during hypoxia. However, in hypoxia PHD3 remains mainly inactive and the full activity is restored upon reoxygenation. Therefore, the expression of PH D3 may also lead to unexpected disease states in humans depending on the tissue oxygenation status and cell types involved in a given disease state. There is thus an identified need of further elucidating the mechanism by which PHD3 activates cell death and in which cell types this occurs.
BRIEF DESCRIPTION OF THE INVENTION
The present invention relates to a use of a prolyl hydroxylase inhibitor for the manufacture of a medicament for inhibiting PHD3-induced protein aggregation in a disease, and to a method of treating, preventing and/or alleviating a condition associated with PHD3-induced protein aggregation. Said disease or condition may be selected from a group consisting of cytotoxicity or mechanical trauma induced tissue damage, reperfusion associated disorders, ischemic diseases, myocardiopathies, delayed or impaired wound healing, rejection or tissue damage in organ transplantation, alcoholic liver disease, and neurodegenerative diseases. Said inhibitor may be selected from a group consisting of 2-oxoglutarate analogs, oxalo-amino acids, hydroxamic acids, hydroxylated aromatic compounds, pyridine N-oxide derivatives, 3-hydroxyquinolone 2-carboximide derivatives and pyhdylcarbonyl glycines, malonic acid, 3-nitroproprionic acid, and theonyl trifluoroacetate. In one specific embodiment, said neurodegenerative disease is a frontotemporal dementia with motoneuron disease and said inhibitor is a 2- oxoglutarate analog.
The present invention further relates to a method for identifying a therapeutic lead compound comprising the steps of a) providing a cell culture comprising cells expressing PHD3, b) treating said cells with a compound suspected to inhibit the aggregation of PHD3 d) analysing said cells for the inhibition of PHD3 aggregation, and e) identifying said compound as a therapeutic lead compound if the determination in step d) is positive.
The present invention also relates to a method for the production of a pharmaceutical composition, wherein a therapeutic lead compound is identified by embodiments according to the present invention, and further mixed with a pharmaceutically acceptable excipient.
The present invention still further relates to compounds capable of inhibiting PHD3-induced protein aggregation, identified by embodiments according to the present invention.
Furthermore, the present invention further relates to the use of a compound capable of inhibiting PHD3-induced protein aggregation, identifiable by a method according to embodiments of the present invention, for the manufacture of a medicament for treating, preventing and/or alleviating a condition or a disease associated with protein aggregation / aggresome formation, such as cytotoxicity or mechanical trauma induced tissue damage, reperfusion associated disorders, ischemic diseases, myocardiopathies, rejection or tissue damage in organ transplantation, delayed or impaired wound healing, and alcoholic liver disease, neurodegenerative disorders. The present invention further relates to a method for treating, preventing and/or alleviating a condition associated with PHD3-induced protein aggregation, and to a method of inhibiting the PHD3 -induced protein aggregation in a mammal, said methods comprising administering to a patient in need thereof a therapeutically effective amount of a compound capable of inhibiting said protein aggregation, identifiable by a method according embodiments of the present invention.
Moreover, the present invention relates to a method of diagnosing a neurodegerative disease comprising the following steps: a) staining a tissue sample obtained from a mammal, said mammal suspected to suffer from a neurodegenerative disorder, using an antibody against PHD3, and b) determining the presence of PHD3-induced protein aggregates in said samples, the presence of PHD3 aggregates being indicative of a neurodegenerative disorder.
BRIEF DESCRIPTION OF THE DRAWINGS In the following the invention will be described in greater detail by means of some embodiments with reference to the attached drawings, in which:
Figures 1A - 1 E show that PHD3 induces apoptosis in HeLa cells in a hydroxylase-dependent manner. (Fig. 1A) HeLa cells expressing enhanced green fluorescent protein (EGFP) (cont), PHD3-EGFP, hydroxylase mutant PHD3 (PHD3R206K-EGFP) or PHD3-EGFP exposed to 8-hour hydroxylase inhibition (DMOG) were stained for activated caspase-3 (white cells). Caspase- 3 activation induced by PHD3 was suppressed by the inhibition of hydroxylase activity. (Fig. 1 B) Quantification of the PHD3-induced apoptosis by caspase-3 activation with the indicated stimuli. (Fig. 1 C) Quantification of PHD3-induced apoptosis by nuclear fragmentation. (Fig. 1 D) Apoptosis measured by FACS analysis in HeLa cells expressing PHD3 for the indicated time. (Fig. 1 E) Apoptosis measured by FACS analysis in HeLa cells transfected with the indicated amount of PHD3. Figures 2A - 2D show that PHD3 forms subcellular aggregates (Fig.
2A) HeLa cells transfected with PHD3-EGFP or PHD2-EGFP and visualized 24 hours after transfection by confocal microscopy demonstrating the aggregation of PHD3. (Fig. 2B) Localization of PHD3 24 hours after transfection in inmortalized keratinocytes (HaCaT) and squamous carcinoma cells from oral mucosa (SCC2). (Fig. 2C) Quantification of PHD3-EGFP expression in cells by confocal microscopy with visible aggregates (lower cell) and cells expressing PHD3 evenly (upper cell). (Fig. 2D) Quantification of the PHD3 expression level in 15 cells (mean and SD) indicating similar expression levels regardless of the subcellular localization of PHD3. Figures 3A - 3C show that PHD3 aggregation requires oxygen and hydroxylase activity. (Fig. 3A) HeLa cells 24 hours after transfection with wild type PHD3-EGFP, PHD3-EGFP expressing cells exposed to either 6 hour hypoxia (1 % O2) or 40 μM hydroxylase inhibitor dimethyloxaloylglycine (DMOG) or transfected with mutant PHD3R206K-EGFP. Aggregation of PHD3 was attenuated by the inhibition of hydroxylase activity either by DMOG or by the point mutation of PHD3. (Fig. 3B) Quantification of cells with PHD3 aggregates. 200 - 300 cells were studied and judged to either have or to not have PHD3 aggregates. Means and range for two independent experiments are shown. (Fig. 3C) Quantification of the cells with aggregated PHD3 under increasing level of the indicated hydroxylase inhibitior DMOG.
Figures 4A - 4G show that PHD3 co-localises with aggresome-like structures. (Fig. 4A) PHD3 and proteasomal subunit LMP-2 co-transfected into HeLa cells and detected by fluoresence confocal microscopy. (Fig. 4B) Profiling of the localisation demonstrates co-localisation of LMP-2 with PHD3 within the aggregates, but not outside them. The line indicates the profile location. (Fig. 4C) PHD3-EGFP transfected into HeLa cells and detected by fluoresence together with the endogenous large subunit (20S) of the proteasome by immunocytochemistry. (Fig. 4D) Profiling of the localisation of PH D3 and 2OS proteasome demonstrates co-localisation of 2OS proteasome and PHD3. (Fig. 4E) Localization of PHD3-EGFP and endogenous HSP70 by immunocytochemistry showing near complete co-localization. (Fig. 4F) Co- localization of transfected PHD3-EGFP and HA-ubiquitin. (Fig. 4G) Co- localization of the endogenous PHD3 with transfected LMP2-EGFP after 2-day hypoxia and 1 hour reoxygenation. The endogenous PH D3 partially co- localizes with the proteosomal LMP2 marker. Figures 5A - 5C show the dynamics of PHD3 aggregation. (Fig. 5A)
PHD3-EGFP expression in cells exposed to microtubulus inhibitor nocodazole (100 uM) or proteasomal inhibitor MG132 (100 uM). (Fig. 5B) PHD3-EGFP was used for fluorescence recovery after photobleaching analysis (FRAP). A representative study of a cytoplasmic aggregate is shown. The arrowhead points to the photobleached aggregate. Re-apperance of PHD3-EGFP expression witin the aggregate demonstrated fast movement of PHD3 into the aggregate. (Fig. 5C) Histogram of the FRAP analysis in Figure 5B.
Figures 6A-6J show that PHD3 induces aggregation of 26S proteasome. HeLa cells were left untransfected (Figs. 6A-6B) or transfected with PHD3-EGFP construct (Figs. 6C-6H) and stained for endogenous 2OS proteasome. (Fig. 6B) Proteosomal inhibition by MG132 caused aggregation of 2OS proteasome (arrowheads). (Figs. 6C and 6D) The forced expression of PH D3 led to the development of aggregates comparable to that induced by proteasomal inhibition by MG-132. (Figs. 6E and 6F) PHD3 expressing cells exposed to DMOG showed breakdown of 2OS aggregation. (Figs. 6G and 6H) Expression of the mutant PHD3 demonstrated no visible 2OS aggregation. (Fig. 6I) Localization of PHD3R206K-EGFP with endogenous 2OS. Aggregates formed by PHD3R206K do not co-localize with proteasomes. (Fig. 6J) Localization of PHD3-EGFP expressing cells exposed to DMOG with endogenous 2OS. Aggregates formed during hydroxylase inhibition do not co- localize with the proteasome.
Figures 7A-7C show that the PHD3-induced aggregation coincides with apoptosis. (Fig. 7A) HeLa cells transfected with PHD3-EGFP and stained for caspase-3 24 hours post-transfection. Only cells with PHD3 aggregation show activated caspase-3. (Fig. 7B) Quantification of cells demonstrating visible aggregates in cells with (+) or without (-) caspase-3 activation. (Fig. 7C) Quantification of caspase-3 activation from cells showing either aggregation of PHD3 (aggr) or even expression (even) 48 hours post-transfection.
Figure 8 shows that PHD3 aggregates are found in frontotemporal dementia (FTD) with motoneuron disease. Sections from a spinal cord from a patient were stained for PHD3. Arrows point to the motoneurons showing aggregated PHD3.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based on the finding that PHD3 activates protein aggregation. It has surprisingly been found that PHD3 activates protein aggregation in HeLa cells during well -oxygenated conditions through its hydroxylase-dependent activity and that this activates cell death. PHD3 forms dynamic subcellular aggresome-like structures, which contain components of the 26S proteasome, HSP70 chaperone and ubiquitin. Expression of PHD3 is sufficient to induce the aggregation of 26S proteasomes. The ability of PHD3 to induce the aggresome-like structures is lost in hypoxia and requires the enzymatic activity of PHD3. The data presented herein links protein aggregation and aggresome formation, which are implicated in several disease states, to the cellular oxygen-sensing pathway. The present invention thus provides means for developing therapeutics for conditions or diseases related to protein aggregation such as neurodegenerative diseases, liver disorders as well as ischemia - reperfusion related conditions for example during treatment of heart infarction, myocardiopathies and tissue transplantation. More specifically, the present invention provides a method for identifying a therapeutic lead compound, which inhibits aggregation of PHD3. As used herein, the term "aggregation of PHD3" is intended to include aggresome formation induced by PHD3.
The method according to the present invention comprises the steps of: a) providing a cell culture comprising cells expressing PHD3, b) treating said cells with a compound suspected to inhibit the aggregation of PHD3 c) analysing said cells for the inhibition of PHD3 aggregation, d) identifying said compound as a therapeutic lead compound if the analysis in step d) is positive.
In one embodiment according to the present invention, said cell culture is a mammalian cell culture comprising e.g. carcinoma, muscle, myocardial, endothelial, glioma and/or neuronal cells. In some embodiments, said cells of the cell culture have been treated to as to induce overexpression of PHD3. This can be achieved e.g. by transfecting said cells with a DNA encoding PHD3 protein and expressing said protein in said cells. Suitable methods for the transfection include, but are not limited to, calcium precipitation and liposome transfection, as well as electroporation. Suitable reagents, vectors and DNAs for said transfection are readily available in the art.
Alternatively or in addition, upregulation or overexpression of endogenous PHD3 may be obtained by exposing cell to stress such as hypoxia, reoxygenation, neural growth factor (NGF) withdrawal or others. As an example, cells may be exposed to hypoxia by culturing them under reduced pressure of oxygen (e.g. 1 % O2 for 16 hours) or by treating cells with a suitable chemical hypoxia mimetic, such as cobalt chloride. Reoxygenation exposure may be achieved by exposing hypoxic cells to normal pressure of oxygen. As an example, NGF withdrawal may be performed by differentiating cells, such as PC-12 cells, to neural cells by culturing them in the presence of NGF followed by withdrawing NGF. Suitable modifications and other methods for upregulating PHD3 expression are apparent to a person skilled in the art.
Analysing the cells for the inhibition of PHD3 aggregation in step c) may be performed by any suitable method known in the art, such as microscopy based image analysis, for example by confocal microscopy including but not limited to fluorescence microscopy. Said analysing may comprise comparing the level of PHD3 aggregation in cells treated with a candidate or test compound to the level of PHD3 aggregation in non-treated control cells. A compound capable of inhibiting the aggresome formation induced by PHD3 is identified based on its ability to break down the aggresome bodies induced by PHD3.
The present invention also relates to a method for producing a pharmaceutical composition, said method comprising a method for identifying compound(s) that inhibit the aggresome formation induced by PHD3, and mixing the compound identified with a pharmaceutically acceptable excipient. Such excipients are readily available to those skilled in the art. Preferably said identification is performed by the method described above.
Compounds or therapeutic lead compounds that may be identified according to the embodiments of the present invention include natural and synthetic chemical compounds, small molecules, natural and synthetic proteins and peptides, as well as vectors encoding any suitable peptide or protein.
The therapeutic lead compounds identifiable according to the embodiments of the present invention, as well as known small molecule prolyl hydroxylase inhibitors including 2-oxoglutarate analogs; oxalo-amino acis, such as N-oxalyl glycine, N-oxalyl alanine, dimethyloxalylglycine (disclosed in Cunliffe et al (1992) J. med. Chem. 35; 2652-2658 and Baeder et al (1994) Biochem J. 300; 525-530); hydroxamic acids, such as benzohydroxyaminen acid (disclosed in Walter et al (1999) Bioorg. Chem 27(1 ); 35-40); hydroxylated aromatic compounds, such as protocatechuic acid; pyridine N-oxide derivatives; 3- hydroxyquinolone 2-carboximide derivatives; pyridylcarbonyl glycines; malonic acid; 3-nitroproprionic acid, and theonyl thfluoroacetate are useful in preventing, treating and/or alleviating conditions or disease associated with PHD3-induced aggresome formation including cytotoxicity or mechanical trauma induced tissue damage; ischemia-reperfusion injury in the infarction of brain or heart and related ischemic diseases; other ischemic diseases such as complications of arterial blockage; myocardiopathies; alcoholic liver disease; rejection in organ transplantation; delayed or impaired wound healing; several neurodegenerative disorders such as, but not restricted to, Parkinson's disease, Alzheimer's disease, Huntington's disease amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) with motoneuron disease. Thus, therapeutic lead compounds identifiable according to the embodiments of the present invention and said prolyl hydroxylase inhibitors may be used for the manufacture of a medicament for treating, preventing and/or alleviating such conditions or diseases. In one specific embodiment, 2-oxoglutarate analog are useful in treating, preventing and/or alleviating frontotemporal dementia (FTD) with motoneuron disease.
As demonstrated in Figure 8, PHD3 indeed aggregates in cells of patients suffering from neurodegerative diseases, such as frontotemporal dementia (FTD) with motoneuron disease. The present invention thus further relates to a method of diagnosing neurodegenerative disorders such as, but not restricted to, Parkinson's disease, Alzheimer's disease, Huntington's disease amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) with motoneuron disease. Such a method may comprise the following steps: a) staining a tissue sample obtained from a mammal, said mammal suspected to suffer from a neurodegenerative disorder, using an antibody against PHD3, and b) determining the presence of PHD3 aggregates in said sample, the presence of PHD3 aggregates being indicative of a neurodegenerative disorder. In one embodiment according to the present invention, determining the presence of PHD3 aggregates includes determining the level and subcellular localization of PHD3. PHD3 aggregation and PHD3-induced aggresome formation are indicative of PHD3-induced cytotoxicity to the cells. The aggregation impairs the function of the cells and thereby causes or exacerbates the said disorders.
It will be obvious to a person skilled in the art that, as the technology advances, the inventive concept can be implemented in various ways. The invention and its embodiments are not limited to the examples described above but may vary within the scope of the claims.
EXAMPLE 1
Forced expression of PHD3 during reoxygenation induces apoptosis in HeLa cells in an oxygen- and hydroxylase-dependent manner.
PHD3 induces apoptosis in neural cells when accumulated after NGF withdrawal. We asked whether PHD3 has the apoptosis-inducing capacity in cancer cells and whether the hydroxylase activity of PHD3 is required for apoptosis using HeLa cells as a model. HeLa cells were transfected with PHD3 followed by 8-hour exposure to a well-characterized hydroxylase inhibitor dioxaloylmethylglycine (DMOG) at 40 uM (Cayman Chemical, Ann Arbor, Ml) and detection of an apoptosis marker caspase-3 activation by immunocytochemistry. Cells were grown in Dulbecco's minimum essential eagle modified medium (Sigma-Aldrich) supplemented with 10% fetal calf serum, 20 U/ml penicillin, 50 μg/ml streptomycin and 2 mM L-glutamin. Cells were transfected at optimal confluency using either FuGene HD (Roche Applied Science) or Effectene transfection reagent (Qiagen Inc., Chatsworth, CA) according to manufacturers protocols.
Activated caspase-3 was detected in immunohistochemistry using activated caspase-3 antibody at 1 :400 dilution (Promega, Madison, Wl). Transfection of PHD3 clearly activated caspase-3, and this was inhibited by DMOG (Fig. 1A). In order to study whether the apoptosis-activating capacity requires PHD3's own enzymatic activity in cancer cells, we introduced an enzyme activity inhibiting point mutation to PHD3. Here, the 2-oxoglutarate coordinating arginine is mutated into lysine (PHD3R206K) and has been reported to inactivate the hydroxylase activity. PHD3R206-EGFP was made by QuikChange site-directed mutagenesis kit (Stratagene) using 5'- CCCTCTTACGCAACCAAATATGCT ATGACTGTCTGG-3' oligonucleotide (the mutated residue is underlined). Similarly to DMOG, the inactivation of PHD3 by a point mutation led to the loss of the apoptotic activity (Fig. 1A). Quantification of either caspase-3 activation (Fig. 1 B) or nuclear fragmentation (Fig. 1 C) demonstrated near complete loss of the apoptotic activity of PHD3 by the inhibition of hydroxylase activity. FACS studies further demonstrated that the apoptotic activity is both time- (Fig. 1 D) and PHD3 concentration- dependent (Fig. 1 E).
EXAMPLE 2
PHD3 forms subcellular aggregates in an oxygen and hydroxylase- dependent manner.
We detected that in addition to cells with dispersed PHD3, a subset of PHD3-EGFP expressing cells contained subcellular bodies or aggregates, which were not present in e.g. PHD2-EGFP transfected cells (Fig. 2A). The aggregates were mainly located in the cytoplasmic or perinuclear departments and occasionally in the nucleus. The aggregation of PHD3 did not depend on the tag used, as other PHD3 fusion proteins with different both N- and C- terminal tags showed similar aggregates. Likewise, the aggregation was not cell-type dependent and was also seen in HaCaT keratinocytes and squamous carcinoma cells (SCC) (Fig. 2B). Importantly, quantification, using confocal microscopy, of the PHD3-EGFP expression level from aggregate-forming and non-forming cells showed that the aggregation does not depend on the amount of the expressed PHD3 (Fig. 2C and 2D).
Next we asked whether the PHD3 aggregate formation depends on oxygen availability. HeLa cells expressing ectopical PHD3-EGFP were exposed to hypoxia (1 % O2) or normoxia for 8 hours. Hypoxic treatments were performed in Invivo2 400 incubator (Ruskinn technologies, UK) in 1 % O2, 5 % CO2, 90 % moisture. Oxygen was replaced with 99,5% pure N2 (AGA, Finland). The aggregation seen in normoxia was strongly reduced in hypoxic conditions (Fig. 3A). Similarly to hypoxia, two chemical hypoxia-mimetics, 100 uM desferrioxamine (DFO, Sigma-Aldrich) and 100 uM cobalt chloride (C0CI2, Sigma-Aldrich), attenuated the aggregate formation. We further investigated whether the aggregation requires the hydroxylase activity of PHD3. First, PHD3 expressing HeLa cells were subjected to 40 uM DMOG for 8 hours. Similarly to the hypoxia, the inhibition of hydroxylase activity nearly abolished visible PHD3 aggregates (Fig. 3A). Furthermore, PHD3 bearing hydroxylase activity inhibiting point mutation (PHD3R206K) showed strongly diminished aggregation. These indicated that besides sufficient oxygen level, the PHD3 aggregation depends on its enzymatic activity.
EXAMPLE 3
PHD3 co-localizes with aggresomes.
The PHD3 aggregates strongly resembled aggresome-like structures. These structures contain components of the 26S proteasome. To study whether PHD3 localizes with the proteosomal components, cells were co-transfected with PHD3 and an essential structural component of the proteasome, LMP2. A red variant of EGFP fused to PHD3 (PHD3-dsRed) was used to detect PHD3 with LMP2-EGFP fusion protein. For PHD3-dsRed, complete PHD3 ORF was aquired by PCR and cloned into pDsRed-Monomer- N1 vector (Clontech) using Hindlll and Smal sites. PCR primer oligonucleotides used were δ'-GAGCAAGCTTTTATGCCCCTGGGACAC-S' and δ'-CCTCCCGGGTCAGTCTTCAGTGAGG-S'. Cells expressing both constructs showed nearly complete co-localization one day after transfection (Fig. 4A and B). We further studied the localization of the endogenous proteasomal components using antibody against 2OS together with ectopic PHD3-EGFP. 2OS proteasome subunit expression was detected by anti-20S proteasome core subunits (Biomol) at 1 :2000 dilution and Cy5-conjugated secondary antibody (Jackson Immunochemicals, West Grove, PA) at 1 :1000 dilution. Similarly to LMP2, the endogenous 2OS demonstrated co-localization with PHD3 (Fig. 4C and D). Furthermore, we detected co-localization of endogenous HSP70 chaperone (Fig. 4E) and transfected HA-ubiquitin (Fig. 4F) with PHD3-EGFP. Endogenous HSP70 was detected by a monoclonal antibody (SPA-810) at 1 :1000 dilution (Stressgen) and HA-Ubiquitin was detected by immunocytochemistry using antibody against haemagglutin (clone 3F10) at 1 :1000 dilution (Roche Diagnostics). We next asked whether the endogenous PHD3 co-localises with proteosomal structures. SCC cells were transfected with LMP2-EGFP, followed by 2 day hypoxia and reoxygenation. We detected prominent structures with co-localizing LMP2 and endogenous PHD3, albeit the endogenous PHD3 expression was clearly lower compared to PHD3-EGFP (Fig. 4G) supporting the hypothesis that PHD3 aggregates represent aggresomal structures.
Protein aggregates may be transported along the microtubules and in the case of aggresome formation they are transported towards the perinuclear region. Inhibition of microtubule polymerization by nocodazole abrogates the transportation. Supporting the hypothesis of PHD3 transport along the microtubuli, 5 μM nocodazole (Sigma-Aldhch) nearly completely inhibited the aggregation of PHD3 (Fig. 5A). Moreover, suggesting the requirement of proteasomal function for the localization of the PHD3 bodies, the inhibition of proteosomal function by MG132 (Sigma-Aldrich) caused PHD3 to cluster along the microtubuli (Fig. 5B). Aggresomes are highly dynamic structures. Proteins are trafficked to the cytoplasmic aggresomes, which are transported towards the nucleus and converged into larger structures. We studied the dynamics of the PHD3 aggregates using the fluorescence recovery after photobleaching (FRAP) technique. For live cell microscopy, cells were grown on MatTek glass bottomed dishes (MatTek Corporation, Ashland, MA) and visualized with Zeiss LSM 510 META confocal laser scanning microscope with Physiology software (Carl Zeiss Corporation, Jena, Germany) in a humified cell culture chamber with 5% CO2 in 370C. The experimental conditions for FRAP have been described previously. Briefly, from cells transiently expressing PHD3-EGFP a region of interest (ROI) was selected and imaged (prebleach). Then ROI was photobleached with argon laser 488nm at maximum power for 100 iterations. The recovery of the bleached region was then imaged with 2% laser power with 2s intervals. The mobile fraction and the half-life of the recovery was calculated usin FRApCaIc program (Dr. Rolf Sara, Turku Centre for Biotechnology). FRAP was studied from several cytoplasmic PHD3 aggregates and from larger perinuclear aggregates. The cytoplasmic aggregates showed rapid recovery of PHD3 within the aggregates indicating fast movement of PHD3 into these structures. The half-time of recovery varied from 7 to 50 seconds and the mobile fraction from 20 to 65% (Fig. 5A and B). The live-cell imaging studies also demonstrated movement and convergence of the aggregates. The dynamics within the perinuclear aggregates, demonstrated slower recovery and dynamics of PHD3. Similarly, during inhibition of microtubules by nocodazole, FRAP showed clearly reduced PHD3 movement within the remaining aggregates. Taken together our data implicated that PHD3 forms aggresome-like structures. EXAMPLE 4
PHD3 expression activates aggresome formation in an oxygen- and hydroxylase-dependent manner.
The experiments with forced PH D3 expression suggested that PHD3 could activate the aggregation of proteasomes. To study this more closely, we compared the aggregation induced by proteosomal inhibition to that induced by PHD3 expression. Forced PHD3 expression activated aggregation of the endogenous 2OS component of the proteasome as visible 2OS aggregates were detected only within cells with aggregating PHD3. 2OS expression was studied as indicated in example 3 (Fig. 6C and D). The aggregation was comparable to that induced by MG-132 (Fig. 6B). Inhibition of the hydroxylase activity of PH D3 either by DMOG (Fig. 6E and F) or by the hydroxylase activity inhibiting mutation (Fig. 6G and H) completely inhibited the aggregation of 2OS by PHD3. A small amount of PHD3 aggregation was retained by the inhibition of the PHD3 hydroxylase activity either by hydroxylase inhibitor or by the inactivating mutation (Figs. 3 and 6). However, neither of these were able to induce the aggregation of the 2OS proteasome. We therefore asked whether the small amount of aggregates formed by the mutant PHD3 (Fig. 6I) or the wild type PHD3 with DMOG (Fig. 6J) co-localized with 2OS. Interestingly, neither of these showed co-localization with the proteosomal components, indicating that the aggregates seen with hydroxylase inhibition represent non- aggresomal structures. Taken together this indicated that PHD3 induces the formation of aggresome-like structures and that this requires the enzymatic activity of PHD3.
EXAMPLE 5
Aggregation of PHD3 co-incides with apoptosis.
Both the emergence of PHD3-induced aggresome-like structures (Fig. 3) and the PHD3-induced apoptosis (Fig. 1 ) were strictly dependent on oxygen availability and hydroxylase activity. Furthermore, protein aggregation has been reported to lead to caspase-dependent apoptosis. We therefore asked whether the aggregation of PHD3 co-insides with PHD3-induced caspase-3 activation. HeLa cells were transfected with PHD3-EGFP and caspase-3 activation was detected by immunostaining as described. 48 hours after transfection a clear correlation between caspase activation and PH D3 aggregation was seen. Cells with visible PHD3 aggregates showed both activated caspase-3 and nuclear fragmentation, in contrast to the cells with non-aggregating or evenly PHD3 expressing cells (Fig. 7A). The correlation of caspase activation and PHD3 aggregation was time-dependent. 24 hours post- transfection approximately half of the cells with aggregated PHD3 showed activated caspase-3 and 48 hours post-transfection this was increased to over 90% (Fig. 7B). In a separate experiment we compared the caspase-3 activation in cells with aggregated and non-aggregated PHD3 2 days after transfection. The two cell populations showed a mirror image; over 90% of the cells with PHD3 aggregates also had activated caspase-3. Vice versa, less than 5% of cells with non-aggregating PHD3 demonstrated activated caspase- 3 (Fig. 7C).
We further performed live-cell imaging of HeLa cells with forced PHD3-EGFP expression to detect the correlation between PHD3 aggregation and cell death. Mitochondrial respiration (Mitotracker) and blebbing of the cells, which is typical for apoptotic cells, were used as a marker for cell viability. A population of 9 cells was followed from 14 to 24 hours post-transfection. Out of the 9 cells 7 showed PHD3 expression and 5 of these aggregating PHD3. All aggregate forming cells (5/5) showed signs of cell death during the follow-up time. In each cell the aggregation of PHD3 preceded cell death. In contrast, none of the untransfected or non-aggregating cells (4/4) demonstrated signs of cell death.

Claims

1 . Use of a prolyl hydroxylase inhibitor for the manufacture of a medicament for inhibiting PHD3-induced protein aggregation in a disease selected from a group consisting of cytotoxicity or mechanical trauma induced tissue damage, reperfusion associated disorders, ischemic diseases, myocardiopathies, delayed or impaired wound healing, rejection or tissue damage in organ transplantation, alcoholic liver disease, and neurodegenerative diseases.
2. The use according to claim 1 wherein said inhibitor is selected from a group consisting of 2-oxoglutarate analogs, oxalo-amino acids, hydroxamic acids, hydroxylated aromatic compounds, pyridine N-oxide derivatives, 3-hydroxyquinolone 2-carboximide derivatives and pyridylcarbonyl glycines, malonic acid, 3-nitroproprionic acid, and theonyl trifluoroacetate.
3. The use according to claim 1 or 2, wherein said neurodegenerative disease is a frontotemporal dementia with motoneuron disease and said inhibitor is a 2-oxoglutarate analog.
4. A method of treating, preventing and/or alleviating a condition associated with PHD3-induced protein aggregation, said method comprising administering to a patient in need thereof a therapeutically effective amount of a prolyl hydroxylase inhibitor selected from a group consisting of 2- oxoglutarate analogs, oxalo-amino acids, hydroxamic acids, hydroxylated aromatic compounds, pyridine N-oxide derivatives, 3-hydroxyquinolone 2- carboximide derivatives and pyridylcarbonyl glycines, malonic acid, 3- nitroproprionic acid, and theonyl trifluoroacetate.
5. The method according to claim 4, wherein said condition is selected from a group consisting of cytotoxicity or mechanical trauma induced tissue damage, reperfusion associated disorders, ischemic diseases, myocardiopathies, delayed or impaired wound healing, rejection or tissue damage in organ transplantation, alcoholic liver disease, and neurodegenerative diseases.
6. A method for identifying a therapeutic lead compound comprising the following steps: a) providing a cell culture comprising cells expressing PHD3, b) treating said cells with a compound suspected to inhibit PHD3-induced protein aggregation, c) analysing said cells for the inhibition of said protein aggregation, and d) identifying said compound as a therapeutic lead compound if the analysis in step c) is positive.
7. The method according to claim 6, wherein the cells have been treated to induce upregulation of PHD3 expression.
8. The method according to claim 7, wherein said treatment comprises transfecting the cells with a DNA encoding PHD3 protein.
9. The method according to claim 7, wherein said treatment comprises exposing the cells to stress to induce endogenous PHD3 expression.
10. The method according to claim 9, wherein said stress exposure is selected from the group consisting of hypoxia, reoxygenation and NGF withdrawal.
11. A method for the production of a pharmaceutical composition, wherein a therapeutic lead compound is identified by a method according to claim 4, and further mixed with a pharmaceutically acceptable excipient.
12. A compound capable of inhibiting PHD3-induced protein aggregation, identified by a method according to any one of claims 6 - 10.
13. Use of a compound capable of inhibiting PHD3 -induced protein aggregation, identifiable by a method according to any one of claims 6 - 10, for the manufacture of a medicament for treating, preventing and/or alleviating a condition associated with aggresome formation.
14. The use according to claim 13, wherein said condition is selected from the group consisting of cytotoxicity or mechanical trauma induced tissue damage, reperfusion associated disorders, ischemic diseases, myocardiopathies, rejection or tissue damage in organ transplantation, delayed or impaired wound healing, and alcoholic liver disease, neurodegenerative disorders.
15. A method for treating, preventing and/or alleviating a condition associated with PHD3-induced protein aggregation, said method comprising administering to a patient in need thereof a therapeutically effective amount of a compound capable of inhibiting said protein aggregation, identifiable by a method according to any one of claims 6 - 10.
16. A method of inhibiting PHD3-induced protein aggregation in a mammal, said method comprising administering to said mammal a compound capable of inhibiting said protein aggregation, identifiable according to any one of claims 6 - 10.
17. A method of diagnosing a neurodegerative disease comprising the following steps: a) staining a tissue sample obtained from a mammal, said mammal suspected i to suffer from a neurodegenerative disorder, using an antibody against PHD3 b) determining the amount of PHD3-induced protein aggregates in said samples, increased amount of protein aggregates being indicative of a neurodegenerative disorder.
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EP1912637A2 (en) * 2005-07-13 2008-04-23 Dana-Farber Cancer Institute, Inc. Inhibitors of egln3 activity for the treatment of neurodegenerative disorders

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