WO2008143876A2 - Agents et essais pour moduler une neurodégénérescence - Google Patents

Agents et essais pour moduler une neurodégénérescence Download PDF

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WO2008143876A2
WO2008143876A2 PCT/US2008/006154 US2008006154W WO2008143876A2 WO 2008143876 A2 WO2008143876 A2 WO 2008143876A2 US 2008006154 W US2008006154 W US 2008006154W WO 2008143876 A2 WO2008143876 A2 WO 2008143876A2
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pdi
compound
disease
sup
group
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PCT/US2008/006154
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WO2008143876A3 (fr
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Brent R. Stockwell
Benjamin G. Hoffstrom
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The Trustees Of Columia University In The City Of New York
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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
    • A61K31/13—Amines
    • A61K31/131—Amines acyclic

Definitions

  • the present invention is related to co-owned and copending U.S.
  • the present invention is also related to and claims benefit of U.S. Patent Application Serial No. 60/930,200 filed May 14, 2007; U.S. Patent Application Serial No. 60/930,267 filed May 15, 2007; U.S. Patent Application Serial No.
  • the present invention relates to methods for modulating neurodegeneration in a patient in need of such modulation by administering, e.g., an effective amount of a compound that modulates protein disulfide isomerase (PDI)- induced cellular toxicity.
  • PDI protein disulfide isomerase
  • the present invention further relates to compounds and compositions that may be used to treat, prevent or modulate neurodegeneration, such as neurodegeneration caused by Huntingtin's Disease (HD). Screening assays for identifying such compounds are also provided.
  • Protein disulfide isomerase is a 53-57-kDa enzyme (depending on the isoform) expressed primarily in the endoplasmic reticulum (ER) of, but also in other locations throughout, eukaryotic cells.
  • ERp57 A related protein, ERp57, is also a protein disulfide isomerase.
  • PDI catalyzes both the oxidation and isomerization of disulfides on nascent polypeptides.
  • PDI catalyzes the reduction of protein disulfides under certain cellular conditions and has been shown to have activity in subcellular compartments such as the cytosol, and mitochondria as well as on the cell surface (55).
  • Disulfide formation can occur rapidly (at times, before the completion of synthesis) or may be delayed until after translation is complete.
  • PDI catalyzes disulfide formation and rearrangement by thiol/disulfide exchange.
  • PDI In addition to its role in the processing and maturation of secretory proteins in the endoplasmic reticulum, PDI and its homologs have been implicated in multiple important cellular processes. These include cellular insulin degradation, processing and maturation of various secretory and cell surface proteins in the ER following their synthesis, and functioning as chaperones to assist protein folding. These observations suggest that PDI is involved in protection of cells under stress or pathological conditions.
  • PDI is also found on the surface of other cell types such as endothelial cells, platelets, lymphocytes, hepatocytes, pancreatic cells and fibroblasts.
  • the reductive activity of plasma membrane PDI is required for endocytosis of certain exogenous macromolecules.
  • the cytotoxicity of diphtheria toxin is blocked by PDI inhibitors, which block the reductive cleavage of the interchain disulfide bonds in the toxin.
  • PDI-mediated reductive cleavage of disulfide bonds in human immunodeficiency virus envelope glycoprotein 120 is essential for infectivity.
  • the entry of the virus into cells can be largely prevented by PDI inhibitors. Because of these functional activities, PDI and its homologous enzymes are potentially interesting drug targets.
  • PDI has been implicated as a chaperone in ER processing and has been suggested to play a role in the formation of Lewy inclusion bodies in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and ALS, however, to our knowledge, it has not been demonstrated that inhibiting PDI activity suppresses cytotoxicity in any model of neurodegenerative disease. (25-28).
  • PDI activity in association with polyglutamine disease, including, for example, Huntington Disease.
  • the inventors are unaware of any literature that discloses or suggests a pro-apoptotic function for PDI. Introduction to Huntington Disease
  • HD Huntington Disease
  • HD high-glutamine-containing huntingtin protein
  • Htt polyglutamine-containing huntingtin protein
  • neuronal dysfunction may play a role in HD, neuronal loss is likely a component as well, especially in the late stages of the disease.
  • understanding how polyglutamine expression causes cell death is central to understanding the cascade of events that leads to extensive dysfunction of neuronal circuits and the resulting triad of cognitive, motor and emotional deficits.
  • polyglutamine-containing huntingtin protein can activate aberrant apoptosis, a specific and stereotypical form of cell death.
  • overexpression of polyglutamine-containing huntingtin protein in cell culture causes apoptosis in both neuronal (1-3) and non-neuronal cells (4, 5).
  • Apoptosis is an elaborate cell death program essential for neuronal pruning during development, and for the clearance of cells that become dysfunctional (70, 71).
  • the most common form of apoptosis proceeds via the intrinsic pathway through mitochondria.
  • an initiation event triggers mitochondrial outer membrane permeabilization (MOMP), which leads to the release of proteins (e.g., cytochrome c and Smac) from the mitochondrial intermembrane space (72).
  • proteins e.g., cytochrome c and Smac
  • caspase enzymes that degrade key structural and functional components of the cell (73).
  • Endoplasmic reticulum DNA damage, loss of cell adhesion, growth factor withdrawal, and endoplasmic reticulum (ER) stress (72, 74).
  • the endoplasmic reticulum is an important site of protein folding, dysregulation of which can activate a cell death cascade (75).
  • the aberrant protein accumulates in the cytosol, suggesting additional mechanisms exist to monitor protein folding and control cell homeostasis.
  • one object of the invention is to understand the mechanism(s) involved in neurodegeneration caused by PDI-induced cellular toxicity.
  • Another object of the invention is to develop screening assays for compounds that can modulate such mechanisms and to develop methods for treating, preventing, and/or ameliorating the symptoms of such diseases using the identified compounds or compositions containing same.
  • the present invention is directed to meeting these and other objects.
  • one embodiment of the invention is a method for modulating neurodegeneration.
  • This method comprises administering to a patient in need thereof an effective amount of a compound that modulates protein disulfide isomerase (PDI)-induced cellular toxicity.
  • the compound is preferably administered as part of a pharmaceutical composition.
  • Another embodiment of the invention is a method of modulating neuronal apoptosis associated with a polyglutamine disease.
  • This method comprises administering to a patient in need thereof an effective amount of a compound that modulates protein disulfide isomerase (PDI)-induced cellular toxicity.
  • the compound is preferably administered as part of a pharmaceutical composition.
  • Another embodiment of the invention is a method for modulating mutant-huntingtin-induced neuronal apoptosis.
  • This method comprises administering to a patient in need thereof an effective amount of a compound that modulates protein disulfide isomerase (PDI)-induced cellular toxicity.
  • PDI protein disulfide isomerase
  • it is preferred that the compound is administered as part of a pharmaceutical composition.
  • Another embodiment of the invention is a method for treating, preventing, or ameliorating the effects of Huntington's disease (HD) in a patient.
  • This method comprises administering to a patient in need thereof an amount of a compound that modulates protein disulfide isomerase (PDI)-induced cellular toxicity.
  • PDI protein disulfide isomerase
  • the compound is administered as part of a pharmaceutical composition.
  • Another embodiment of the invention is a method for reducing or suppressing misfolded protein-induced cytotoxicity, which is associated with a neurodegenerative disease.
  • This method comprises administering to a patient in need thereof an amount of a compound that is sufficient to reduce or suppress the misfolded protein-induced cytotoxicity.
  • the compound is administered as part of a pharmaceutical composition.
  • Another embodiment of the invention is a method of modulating caspase activation in a cell. This method comprises contacting the cell with a caspase-modulating amount of a compound that modulates protein disulfide isomerase (PDI)-induced cellular toxicity.
  • PDI protein disulfide isomerase
  • Another embodiment of the invention is a method of modulating mitochondrial outer membrane permeabilization (MOMP) in a cell.
  • This method comprises contacting the cell with a MOMP-modulating amount of a compound that modulates protein disulfide isomerase (PDI)-induced cellular toxicity.
  • PDI protein disulfide isomerase
  • Another embodiment of the invention is a method for identifying a target of a candidate compound identified in an assay as modulating a cellular phenotype of interest.
  • This method comprises derivatizing a candidate compound to make it Huisgen cycloaddition chemistry (HCC) compatible and contacting the derivatized candidate compound with a sample suspected of containing a target for the candidate compound under suitable conditions for binding of the derivatized candidate compound to the target, wherein if the target is present in the sample it will bind to the candidate compound, carrying out HCC to covalently attach a detectable label to the derivatized candidate compound, and determining whether the labeled derivatized candidate compound is bound to the target.
  • HCC Huisgen cycloaddition chemistry
  • Another embodiment of the invention is a compound for treating, preventing, or ameliorating the effects of a neurodegenerative disease identified according to any of the methods of the present invention.
  • the present invention also includes pharmaceutical compositions comprising compounds identified according to any of the methods of the present invention.
  • Another embodiment of the present invention is a method of identifying a compound useful for the treatment of a neurodegenerative disease.
  • This method comprises determining whether the compound binds to a protein disulfide isomerase (PDI), wherein the ability to bind to PDI indicates that the compound may be used to treat a neurodegenerative condition.
  • the determining step preferably comprises carrying out virtual high throughput screening to identify compounds that bind to at least one PDI.
  • Another embodiment of the present invention is a method of identifying a compound useful for the treatment of a neurodegenerative disease.
  • This method comprises determining whether the compound inhibits a protein disulfide isomerase (PDI), wherein the ability to inhibit PDI indicates that the compound may be used to treat a neurodegenerative condition.
  • the determining step preferably comprises carrying out virtual high throughput screening to identify compounds that inhibit at least one PDI.
  • Another embodiment of the invention is a method for identifying candidate compounds for use in treating a neurodegenerative disease.
  • This method comprises (a) screening a library of test compounds in a cell viability assay, which assay comprises cells that are capable of undergoing huntingtin-modulated cell death, (b) selecting those test compounds from step (a) that have a %Rescue >50%,
  • PDI disulfide isomerase
  • a cell viability assay which assay comprises cells that are capable of undergoing huntingtin-modulated cell death
  • test compounds from step (c) that (i) exhibit PDI inhibition in the PDI in vitro inhibition assay and (ii) have a %Rescue ⁇ 50% in the cell viability assay as candidate compounds for use in treating a neurodegenerative disorder.
  • the present invention provides for a method of identifying a compound useful for the treatment or prevention of a neurodegenerative disease by determining whether the compound binds to and/or inhibits PDI, where the ability to bind to and/or inhibit PDI is consistent with the ability of the compound to treat and/or prevent neurodegeneration.
  • test compounds which bind to
  • PDI may be identified by a method in which HCC is used to link a detector compound with derivatized test compound bound to its receptor. According to one non-limiting subset of such embodiments, the invention provides for a method comprising:
  • test compound is derivatized with an alkyne and the detectable label is derivatized with an azide, and used in the same general method as described above.
  • HCC-based methods are amenable to high-throughput screening of test compounds.
  • the inventive technique may be used generally to identify a hitherto unknown target receptor for an "orphan ligand" with desirable biological activity.
  • the present invention provides for a method for identifying the target receptor comprising: a. providing an orphan ligand derivatized with an azide (or alkyne) to obtain an azide (or alkyne) orphan ligand;
  • the label may be an affinity label which may be used to collect orphan ligand-bound target receptor (for example, using immunoglobulin-based affinity chromatography).
  • the step of exposing derivatized orphan ligand to its receptor target may be performed by introducing the derivatized orphan ligand into a cell (thereby using the cellular environment to provide natural conditions for binding); subsequently, HCC, in the presence of copper ions and under denaturing conditions, is performed in vitro.
  • Figure 1 illustrates the modeling of Htt-polyQ neurotoxicity in PC 12 cells.
  • Figure 1A shows an inducible construct for production of Htt-EGFP fusion proteins.
  • Rat neuronal PC12 cells are transfected with Htt-exon-1 constructs containing either 25 (Q25) or 103 (Q103) polyglutamine repeats (mixed CAG/CAA).
  • Figure 1 B is a cartoon of the Htt-exon-1 expression in PC12 cells and the screening assay for cell viability using Alamar Blue. Briefly, induction of Htt-Q103 expression leads to the formation of perinuclear cytoplasmic inclusions (or aggresomes) of the fusion protein followed by cytotoxicity after 48 hours.
  • FIG. 1C is a graph showing the quantification of Htt-Q25 and Htt-Q103 cell viability as a measure of Alamar Blue fluorescence.
  • Figure 2 shows dose-response curves for eight of the best suppressors of Htt-Q103 toxicity (SUP-1 , -2, -3, -4, -5, -6, -7, and -8 respectively).
  • the viability of uninduced Q103 (upper curves) and tebufenozide-induced Q103-expressing cells (lower curves) was detected by Alamar Blue fluorescence at 72 hours post-induction (each data point is the average of 4 trials).
  • the inset depicts the structure of each SUP compound.
  • Figure 3 shows the results of a fluorometric assay for caspase activity in Htt-Q25 and Htt-Q103 expressing cells.
  • Figure 3A is a bar graph showing caspase-3 activity measured at 15 hours post-Htt induction. As shown, cells expressing Htt-Q103 exhibit elevated levels of caspase-3 activity over uninduced Htt- Q103 or induced Htt-Q25 expressing cells (third, first and second bars, respectively). Suppressors of Htt-toxicity, SUP-2 and SUP-3, suppress caspase-3 activity when added to the cells in culture (fourth, fifth, and sixth bars).
  • FIG. 3B is a Western blot detection of active caspase-3, -6, and -7. Caspases-3, -6, and -7 are differentially activated in Htt-Q103 expressing cells and this activity is suppressed by SUP-2 and SUP-3.
  • the general caspase inhibitor (BOC) rescues cell survival by directly inhibiting the active enzymes.
  • the initiation factor, elF4E is shown as a loading control. All proteins were detected from the same blot that was stripped and re- probed. Drug concentrations for both assays: SUP-2 (5 ⁇ M), SUP-3 (10 ⁇ M), and BOC-D-FMK (50 ⁇ M).
  • FIG. 5 is a graphic summarizing the Huisgen cycloaddition chemistry reaction used to identify target proteins. Briefly, we adapted the copper-mediated cycloaddition of an azide and alkyne (i.e. "click" chemistry, developed by Sharpless, Finn and Cravatt), to target identification for small molecule hits from phenotypic screens. First, a hit compound should covalently label the target protein, as is the case with SUP-2, which contains a chloromethylcarbonyl moiety; the hit compound is then derivatized with an alkyne. This compound is added to, e.g., cells or cell extracts, where the target protein is covalently bound through the chloromethylcarbonyl moiety.
  • SUP-2 which contains a chloromethylcarbonyl moiety
  • the covalent compound- target adduct is then coupled to a fluorescent tag, such as an azido rhodamine (or fluorescein) tag, using the copper-mediated cycloaddition reaction.
  • a fluorescent tag such as an azido rhodamine (or fluorescein) tag
  • the entire adduct protein-hit-fluorescent tag
  • the target protein can then be sequenced using mass spectrometry and identified by matching the sequences obtained to the protein databases.
  • Figures 6A and B show the protein sequences of PDI and ERp57, respectively.
  • the target protein for SUP-1 was purified according to the scheme shown in Figure 5.
  • the resulting target protein was sequenced at the Gygi lab/Taplin Biological Mass Spectrometry Facility at Harvard Medical School. Two related proteins were identified, protein disulfide isomerase (PDIA1 ( Figure 6A) (SEQ ID NO: 1)) and ERp57 (PDIA3 ( Figure 6B) (SEQ ID NO: 2)).
  • PDIA1 Figure 6A
  • Figure 6B Figure 6B
  • the specific peptide sequences obtained by mass spectrometry are underlined in the database sequences of each protein.
  • Figure 7 illustrates the identification of SUP-2 target protein in PC12 cells.
  • Figure 7A is a Western blot showing that SUP-2A labels a ⁇ 53 kD protein from a PC12 extract following copper (I) catalyzed cycloaddition of a rhodamine azide tag. Pre-treatment of the cell extract with either SUP-2, SUP-3, SUP-4, SUP-1 , or cystamine blocks SUP-2A target binding. Inactive analogs SUP-1A and hypotaurine do not compete for SUP-2A target binding. SUP-2A also tags commercial bovine PDI (right lane).
  • Figure 7B is an anti-protein disulfide isomerase Western blot of affinity purified SUP-2A target protein, which confirms the sequence data.
  • EXTRACT PC12 extract
  • SUP-2A affinity purified SUP-2A fluorescein tagged target from PC12 extract
  • SUP-2A COMP pretreatment of PC12 extract with 2Ox SUP-2 followed by SUP-2A labeling, fluorescein coupling, and affinity purification.
  • Figure 8 illustrates that compounds that bind to PDI rescue Htt-Q103 induced toxicity and inhibit PDI enzymatic reductase activity in vitro.
  • Figure 8A shows SUP-2 (16F16) and its alkyne-modified analogs, SUP-2A (16F16-propargyl), which is active, and SUP-2ADC, which is inactive.
  • Figure 8B shows SUP-1 and its inactive analog muscimol (SUP-1A), and Figure 8C shows cystamine and an inactive analog hypotaurine.
  • Figure 9 shows PDI assay results using various compounds identified according to the present invention.
  • Figure 10 shows the structures of hit compounds according to the present invention identified in the PDI and cell viability assays.
  • Figures 11A-P show the results of Alamar Blue cell viability assays for certain hit compounds according to the present invention.
  • Figures 12A-I show the results of various in vitro PDI binding assays for certain hit compounds according to the present invention.
  • Figure 12J shows the enzyme and substrate signal.
  • Figures 13A and B show the results of various HCC competition assays for certain hit compounds according to the present invention.
  • Figure 14 is a summary of the results of the cell viability, in vitro PDI binding, and HCC binding assays according to the present invention.
  • Figure 15 is a flow chart showing a proposed mechanism for certain compounds according to the present invention. As shown, the active compounds act upstream of MOMP.
  • Figure 16 illustrates that PDI causes release of apoptogenic factors from mitochondria.
  • Figure 16A shows that PDI induces MOMP.
  • Figure 16B shows that PDI inhibitors prevent the PDI-induced MOMP.
  • Figure 16C shows that PDI- driven MOMP is independent of BAX.
  • Figure 17 is a schematic showing a model for the mHTT-PDI-MOMP pathway.
  • Figure 18 is a Western blot, which shows the results of a test for suppression of caspase-2 activation by serum withdrawal. Neither Arteannuin B (SUP-3) nor 16F16 (SUP-2) suppressed the appearance of caspase-2 active fragments by Western blot of active caspase-2 fragments.
  • Figure 19 is a comparison of the nuclear magnetic resonance (NMR) spectra of Compound 141 and securinine.
  • Compound 141 and securinine have identical 13 C-NMR (upper panels) and 1 H-NMR (lower panels) spectra.
  • Figure 2OA is a dose-response curve for Compound 141 in the cell viability assay.
  • the viability of tebufenozide-induced htt-Q25 (blue) and htt-Q103 (red)-expressing cells was detected by Alamar Blue fluorescence and plotted as a percentage of uninduced cells at 48 hours post-induction.
  • Figure 2OB is a graph showing the results of securinine in the in vitro PDI assay.
  • Figure 21 are two plots showing the results of compounds BBC7M13
  • Figure 22 is a laser scanned gel of 16F16A-rhodamine tagged proteins
  • PDI 53 kDa doublet
  • the inactive analog hypotaurine (HYPT) does not compete for 16F16A target binding.
  • Purified bovine PDI (B-PDI) is covalently labeled by 16F16A-rhodamine (arrowheads).
  • Figure 23 is a series of graphs and cartoons showing the localization and regulation of PDI in Q25 and Q103 cells.
  • Figures 23a-c are bar graphs showing the quantification of PDI in mitochondrial, cytosolic, and ER/microsomal cell fractions, by IR-Western blot and LI-COR infrared imaging.
  • Time-course analysis shows a 2.8- fold increase of Q103 mitochondrial PDI over Q25 mitochondrial PDI at 24 hours post induction.
  • Induced Q103 cells rescued with 16F16 (7.8 ⁇ M) show a 5.3-fold increase of mitochondrial PDI over induced Q25 cells at 24 hours.
  • Fig. 23d is a Western blot of PDI from ER and mitochondrial fractions before and after protease shaving with trypsin (30 min, 25 0 C) followed by quenching with SBTI.
  • Calnexin a transmembrane protein localized to intact ER and mitochondrial-associated-ER-membranes (MAM), is degraded by trypsin due to exposure of its cytosolic domain.
  • FIG 24 is a series of bar graphs demonstrating that PDI inhibitors suppress MOMP in purified mitochondria and in cells,
  • PDI inhibitors 16F16, BBC7M13 (M13), BBC7E8 (E8), thiomuscimol (THIOM), and cystamine (CYS) suppress PDI-driven MOMP, whereas inactive analogs muscimol (MUSC) and hypotaurine (HYPT) do not.
  • PDI inhibitors suppress cytochrome c release and caspase-3 activation in Q103-expressing cells. Cells were harvested at 19 hours post-induction; cytochrome c and caspase-3 were normalized to actin protein for each treatment. Similar experiments also showed that PDI inhibitors suppress activation of caspase-6 and caspase-7 (Figure 3). Abbreviations: uninduced (U) 1 induced (I).
  • Figure 25 is a graphic showing the set up for the rat brain slide HD assay.
  • rat brain slices are co-transfected with expression vectors for human htt exon-1 containing 73 glutamines as a Cyan Fluorescent fusion protein (htt-Q73-CFP) and a Yellow Fluorescent Protein (YFP) reporter to monitor morphology of transfected neurons.
  • DNA constructs were coated onto 1.6 micron elemental gold particles and delivered to the brain-slice explants using a biolistic device.
  • MSNs medium spiny neurons
  • a group of striatal neurons most affected in HD is induced over 4 to 7 days in htt-Q73-CFP expressing cells compared to CFP transfected cells.
  • MSN health is assayed by observing morphology and integrity of transfected MSNs at day 5.
  • Figure 25A is a graphic showing a gene gun shooting a coronal brain slice from rat that then expressed YFP in both cortex and striatum.
  • Figure 25B is a graphic showing an actual gold particle in the nucleus of a transfected neuron.
  • Figure 25C is a graphic showing a healthy YFP- transfected medium spiny neuron in the striatum.
  • Figure 26 is a series of bar graphs showing the results of the rat brain slice experiment. BBC7E8 (E8) and 16F16 were able to rescue the toxicity of Htt- Q73-CFP.
  • Figure 27 is a bar graph summarizing the results of the high-throughput assays for suppressors of mutant-huntingtin-induced apoptosis.
  • Figure 28 is a table summarizing the suppressors of mutant-huntingtin- induced apoptosis identified in the high-throughput assays, their known biological functions, and their ability to suppress the activation of caspase-3/7.
  • Figure 29 shows a set of connectents, which demonstrated that suppressors of Htt-Q103 toxicity, specifically SUP-1 , do not alter Htt-Q25 or Htt- Q103 protein expression.
  • Figure 30 shows dose-response curves of SUP-2 (16F16) and several peptide-coupled caspase inhibitors in suppressing the Htt-Q103 toxicity.
  • Figure 31 shows the characterization of Htt-Q103-induced cell death in
  • PC12 cells by examining the expression level of various proteins in these cells.
  • Figure 32A shows the structure of a derivative of SUP-2
  • Figure 32B shows a dose-response curve of 16F16- biotinamine in suppressing the Htt-Q103 toxicity (lighter curve).
  • One embodiment of the invention is a method for modulating neurodegeneration. This method comprises administering to a patient in need thereof an effective amount of a compound that modulates protein disulfide isomerase (PDI)-induced cellular toxicity.
  • the compound is preferably administered as part of a pharmaceutical composition.
  • neurodegeneration or a “neurodegenerative disease” refers to a disease state characterized by misfolded protein-induced cell death.
  • disease states include, e.g., a polyglutamine disease, a prion disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, and Alzheimer's disease.
  • ALS amyotrophic lateral sclerosis
  • Alzheimer's disease All polyglutamine diseases known or to be discovered are within the scope of the present invention.
  • polyglutamine diseases include at least the following: Huntington's Disease (HD), spinal and bulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA), and spinocerebellar ataxia (SCA) types 1 , 2, 3, 6, 7, and 17.
  • the polyglutamine disease is HD.
  • a prion disease means the family of rare progressive neurodegenerative disorders that affect both humans and animals, which are caused by prions, i.e., abnormal, transmissible agents that are able to induce abnormal folding of normal cellular prion proteins in the brain.
  • prions i.e., abnormal, transmissible agents that are able to induce abnormal folding of normal cellular prion proteins in the brain.
  • prion diseases according to the present invention that affect humans include Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease (vCJD), Gerstmann-Straussler-Scheinker Syndrome, and Fatal Familial Insomnia.
  • prion diseases according to the present invention that affect animals include Bovine Spongiform Encephalopathy (BSE), Chronic Wasting Disease (CWD), Scrapie, Transmissible mink encephalopathy, Feline spongiform encephalopathy, and Ungulate spongiform encephalopathy.
  • BSE Bovine Spongiform Encephalopathy
  • CWD Chronic Wasting Disease
  • Scrapie Scrapie
  • Transmissible mink encephalopathy Feline spongiform encephalopathy
  • Ungulate spongiform encephalopathy Ungulate spongiform encephalopathy.
  • Effective amount or "therapeutically effective amount” means the concentration of a compound that is sufficient to elicit the desired effect (e.g., treatment of a condition, the death of a neuronal cell). It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the individual. Other factors which influence the effective amount may include, but are not limited to, the severity of the individual's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention. Typically, for a human subject, an effective amount will range from about 0.001 mg/kg of body weight to about 50 mg/kg of body weight.
  • a larger total dose can be delivered by multiple administrations of the agent.
  • Methods to determine efficacy and dosage are known to those skilled in the art. See, for example, lsselbacher et at. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference.
  • the terms “about” or “approximately” mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend, in part, on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably up to 1%, 2%, 3%, or 4% of a given value. Alternatively, particularly with respect to biological systems or processes, the terms can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
  • PDI is an enzyme that can be targeted by small molecules, peptides, antibodies or other biologically active molecules to prevent or inhibit the onset and/or progression of neurodegeneration.
  • PDI is intended to refer to enzymes having protein disulfide isomerase activity, classified as EC 5.3.4.1. In this classification are included a number of enzymes, e.g.
  • ER38_NEUCR (Q92249); ER60_SCHMA (P38658); HUMER60P (BAA11928) EUG1_YEAST (P32474); MPD1_YEAST (Q12404); PDA2JHUMAN (Q13087); PDA3_BOVIN (P38657); PDA3JHUMAN (P30101); PDA3_MOUSE (P27773); PDA3_PAPHA (P81246); PDA3_RAT (P11598); PDA4_CAEEL (P34329); PDA4_HUMAN (P13667); PDA4_MOUSE (P08003); PDA4_RAT (P38659); PDA5_HUMAN (Q14554); PDA6_ARATH (022263); PDA6_CAEEL (Q11067); PDA6_HUMAN (Q15084); PDA6_MEDSA (P38661); PDA6_MESAU (P38660); PDA6_RAT (Q63081
  • PDI means human PDIA1 or PDIA3 (ERp57). Also included within the meaning of PDI is combinations or complexes of two or more PDIs. Additionally, PDI may be complexed with other polypeptides, which can modulate the enzymatic activity.
  • toxicity refers to the ability of an agent, such as a polyQ expanded mutant htt protein, to kill or inhibit the growth/proliferation of cells.
  • agent such as a polyQ expanded mutant htt protein
  • PDI-induced cellular toxicity means cell death or other diminished cellular capacity modulated by PDI or one or more agents upstream or downstream from PDI in the pathway leading to cellular toxicity.
  • modulates PDI-induced cellular toxicity may refer to the ability of a molecule to inhibit or decrease, e.g., mis-folded protein-mediated toxicity to cells, such as neurons, caused by an agent (e.g., a polyQ expanded mutant htt protein), thereby promoting cell viability (growth or proliferation).
  • agent e.g., a polyQ expanded mutant htt protein
  • the compound that modulates protein disulfide isomerase (PDI)- induced cellular toxicity may be a PDI inhibitor.
  • a target of the PDI inhibitor is at least one PDI.
  • PDI inhibitor means an agent, such as for example, a small molecule, that binds to and/or inhibits, in whole or in part, PDI activity and modulates neurodegeneration in a mammal, such as a human or an animal.
  • PDI inhibitors may be identified using the methods disclosed herein.
  • Representative examples of compounds that mediate PDI-induced cellular toxicity and which are PDI inhibitors include SUP-1-8, cystamine, and certain of the compounds shown in the Figures, particularly Figures 2, 8, 10, 14, 20, and 32.
  • the PDI inhibitors are SUP-1-4, cystamine, compound C13 (BBC7M13), compound D13 (BBC7E8), compound J15, compound F14, or securinine.
  • the "PDI inhibitor” includes analogs, enantiomers, optical isomers, diastereomers, N-oxides, crystalline forms, hydrates, pharmaceutically acceptable salts, and combinations of the compounds disclosed herein.
  • the present invention also includes pharmaceutical formulations containing one or more of these compounds.
  • compositions of the invention refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., human).
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in mammals, and more particularly in humans.
  • Another embodiment of the invention is a method of modulating neuronal apoptosis associated with a polyglutamine disease.
  • This method comprises administering to a patient in need thereof an effective amount of a compound that modulates protein disulfide isomerase (PDI)-induced cellular toxicity.
  • the compound is preferably administered as part of a pharmaceutical composition.
  • the compound is a PDI inhibitor.
  • the PDI inhibitor and the polyglutamine disease are as previously defined herein.
  • the modulation is a decrease in neuronal apoptosis.
  • Another embodiment of the invention is a method for modulating mutant-huntingtin-induced neuronal apoptosis.
  • This method comprises administering to a patient in need thereof an effective amount of a compound that modulates protein disulfide isomerase (PDI)-induced cellular toxicity.
  • PDI protein disulfide isomerase
  • the compound is administered as part of a pharmaceutical composition.
  • the compound is a PDI inhibitor.
  • the PDI inhibitor is as previously defined herein.
  • mutant huntingtin means mutations in the huntingtin gene, e.g., mutations which result in the expression of a polyglutamine (polyQ)- containing huntingtin protein with more than 25 glutamine residues, prefereably more than 35 glutamine residues.
  • polyQ polyglutamine
  • Another embodiment of the invention is a method for treating, preventing, or ameliorating the effects of Huntington's disease (HD) in a patient.
  • This method comprises administering to a patient in need thereof an amount of a compound that modulates protein disulfide isomerase (PDI)-induced cellular toxicity.
  • PDI protein disulfide isomerase
  • the compound is administered as part of a pharmaceutical composition.
  • the compound is a PDI inhibitor.
  • the PDI inhibitor is as defined previously herein.
  • treat or “treating” is used herein to mean to relieve or alleviate or delay the progression of at least one symptom of a disease in a subject.
  • the term “treat” also denotes to arrest, delay the onset ⁇ i.e., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease.
  • prevent is used in terms of prophylactic administration of a compound or pharmaceutical composition prior to the onset of disease or to prevent recurrence of a disease.
  • Administration of the dosage form to prevent the disease need not absolutely preclude the development of symptoms.
  • Prevent can also mean to reduce the severity of the disease or its symptoms.
  • Another embodiment of the invention is a method for reducing or suppressing misfolded protein-induced cytotoxicity, which is associated with a neurodegenerative disease.
  • This method comprises administering to a patient in need thereof an amount of a compound that is sufficient to reduce or suppress the misfolded protein-induced cytotoxicity.
  • the compound is administered as part of a pharmaceutical composition.
  • the compound is preferably a PDI inhibitor as previously defined.
  • the neurodegenerative disease is as previously defined herein.
  • Another embodiment of the invention is a method of modulating caspase activation in a cell.
  • This method comprises contacting the cell with a caspase-modulating amount of a compound that modulates protein disulfide isomerase (PDI)-induced cellular toxicity.
  • PDI protein disulfide isomerase
  • the cell is a neuron.
  • the compound is preferably a PDI inhibitor as previously defined.
  • cystpase refers to a member of the family of cysteine proteases that are one of the main executors of the apoptotic process, including but not limited to caspase-1-10 and caspase-14, as well as other mammalian caspases.
  • the caspase is caspase-3, caspase-6, caspase-7, caspase-9, or combinations thereof.
  • the modulation of caspase activity is a decrease in caspase activation.
  • Another embodiment of the invention is a method of modulating mitochondrial outer membrane permeabilization (MOMP) in a cell.
  • This method comprises contacting the cell with a MOMP-modulating amount of a compound that modulates protein disulfide isomerase (PDI)-induced cellular toxicity.
  • the cell is a neuron.
  • the modulation is a decrease in MOMP.
  • the compound is preferably a PDI inhibitor as previously defined.
  • the MOMP-modulating amount of the compound is sufficient to prevent or reduce release of cytochrome c from mitochondria and to prevent activation of an apoptosome.
  • apoptosome refers to a multiprotein complex comprising cytochrome c that mediates the initiation of caspases.
  • the MOMP-modulating amount of the compound is sufficient to prevent or reduce caspase activation.
  • Another embodiment of the invention is a method for identifying a target of a candidate compound identified in an assay as modulating a cellular phenotype of interest.
  • This method comprises derivatizing a candidate compound to make it HCC compatible and contacting the derivatized candidate compound (in vivo or in vitro) with a sample suspected of containing a target for the candidate compound under suitable conditions for binding of the derivatized candidate compound to the target, wherein if the target is present in the sample it will bind to the candidate compound, carrying out HCC to covalently attach a detectable label to the derivatized candidate compound (in vivo or in vitro), and determining whether the labeled derivatized candidate compound is bound to the target.
  • the sample comprises a cell or a cellular extract.
  • this method further comprises identifying the target, if it is present, to which the derivatized candidate compound bound.
  • the target modulates protein disulfide isomerase (PDI)-induced cellular toxicity, or more preferably, the target is a PDI inhibitor.
  • PDI protein disulfide isomerase
  • modulating a cellular phenotype means altering an observed quality of a cell, including but not limited to cell fate.
  • a cellular phenotype that can be modulated is neuronal apoptosis, which may be induced by a neurodegenerative disease or any other disease characterized by misfolded protein-induced cell death.
  • the neurodegenerative disease is as previously defined; preferably, it is HD.
  • the term "derivatizing” means chemically modifying a compound such that it substantially retains the desired activity of the original compound but one or more new functional groups compatible for other chemical reactions, including but not limited to HCC, are introduced.
  • the derivatizing step introduces an alkyne group onto the candidate compound at a position that does not substantially interfere with the derivatized candidate compound's ability to bind to its target.
  • the derivatizing step introduces an azide group onto the candidate compound at a position that does not substantially interfere with the derivatized candidate compound's ability to bind to its target.
  • candidate compounds encompass numerous chemical classes, although typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons.
  • Candidate compounds comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of these functional chemical groups.
  • the candidate compounds may also comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • candidate compounds are also found among biomolecules including but not limited to peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • Candidate compounds are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. In this embodiment, the candidate compound may be a PDI inhibitor as previously defined herein.
  • HCC Human cycloaddition chemistry
  • a Cu(I) or Cu(ll)-based catalyst may be used to accelerate the rate of cycloaddition between azides and alkynes by about 106-fold (115-116).
  • This copper-catalyzed reaction proceeds readily at physiological temperatures and in the presence of biological materials to provide 1 , 4-disubstituted triazoles with nearly complete regioselectivity (40).
  • the copper-mediated reaction has been used to tag azides installed within virus particles (110), nucleic acids (111) and proteins from complex tissue lysates (43) with virtually no background labeling.
  • HCC compatible means suitable for the HCC reaction.
  • the step of carrying out HCC comprises reacting the alkyne-derivatized candidate compound with a detectable label comprising an azide under conditions suitable for Cu(l)-mediated cycloaddition between the alkyne and the azide.
  • a label can be any composition which is detectable. As used herein,
  • the label refers to anything that is identifiable.
  • the label may be radioactive, fluorescent, chromogenic, enzymatic, or a target antigen for labeled antibody.
  • Any analytical means known in the art can be used for determining or detecting the detection antibody. These means include the use of spectroscopy, chemistry, photochemistry, biochemistry, immunochemistry, or optics.
  • the label can be, for example, an enzyme (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and others commonly used in an ELISA), a radiolabel, a chemiluminescent compound (e.g.
  • luciferin and 2,3-dihydrophthalazinediones, luminol, etc.
  • a fluorescent dye e.g., fluorescein isothiocyanate, Texas red, rhodamine, etc.
  • any other dye known in the art e.g., fluorescein isothiocyanate, Texas red, rhodamine, etc.
  • the label may be coupled directly or indirectly (e.g., via binding pairs such as biotin and avidin) to the detection antibody according to methods well known in the art. As indicated above, a wide variety of labels may be used. The choice of label may depend on the sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, or disposal provisions. For a review of various labeling or signal producing systems which may be used, see U.S. Pat. No. 4,391 ,904.
  • Detection antibodies can be detected or determined by any suitable method known in the art.
  • a label on an antibody can be detected by a gamma counter if the label is a radioactive gamma emitter, or by a fluorimeter, if the label is a fluorescent emitter.
  • the label can be detected colorimetrically employing a substrate for the enzyme.
  • the detection antibody is detected using alkaline phosphatase-conjugated, species- specific immunoglobulin. Any substrate of alkaline phosphatase can be used.
  • pNPP p-nitrophenylphosphate
  • pNPP can be the substrate and the reaction product, p-nitrophenol, can be detected optically.
  • the step of carrying out HCC comprises reacting the azide-derivatized candidate compound with a detectable label comprising an alkyne under conditions suitable for Cu(l)-mediated cycloaddition between the alkyne and the azide.
  • the target is bound to a solid substrate.
  • the method is a high throughput screening assay.
  • High Throughput Screening as used herein defines a process in which large numbers of compounds are tested rapidly and in parallel for binding activity or biological activity against target molecules.
  • the test compounds may act as, for example but not limited to, inhibitors of target enzymes, as competitors for binding of a natural ligand to its receptor, or as agonists/antagonists for receptor-mediated intracellular processes.
  • "large numbers of compounds” may be, for example, more than 100 or more than 300 or more than 500 or more than 1 ,000 compounds.
  • the process is an automated process. HTS is a known method of screening to those skilled in the art.
  • virtual high throughput screening may be used.
  • virtual high throughput screening means a rapid filtering of large databases or libraries of candidate compounds though the use of computational approaches based on discrimination functions that permit the selection of compounds to be tested for biological activity. Such approaches are within the skill of the art. See, e.g., Plewczyski et al., Chem. Biol. Drug. Res., 69(4):269-79 (2007), Lu et al., J. Med. Chem., 49(17):5154-61 (2006), Nicolazzo et al., J. Pharm.
  • candidate compounds such as candidate PDI-inhibitors
  • a cell viability assay such as, e.g., the PC 12 Assay, described in further detail in Example 1.
  • candidate compounds are selected that have a %Rescue of greater
  • %Rescue is calculated as follows:
  • %Rescue j 1 - ⁇ 1 - ⁇ x 100.
  • Another embodiment of the invention is a compound for treating
  • the present invention also includes pharmaceutical compositions comprising compounds identified according to any of the methods of the present invention.
  • Another embodiment of the present invention is a method of identifying
  • This method comprises determining whether the compound binds to a protein disulfide isomerase (PDI), wherein the ability to bind to PDI indicates that the compound may be used to treat a neurodegenerative condition.
  • the determining step preferably comprises carrying out virtual high throughput screening to identify compounds that bind to at least one PDI.
  • Another embodiment of the present invention is a method of identifying a compound useful for the treatment of a neurodegenerative disease.
  • This method comprises determining whether the compound inhibits a protein disulfide isomerase (PDI), wherein the ability to inhibit PDI indicates that the compound may be used to treat a neurodegenerative condition.
  • the determining step preferably comprises carrying out virtual high throughput screening to identify
  • Another embodiment of the invention is a method for identifying candidate compounds for use in treating a neurodegenerative disease.
  • This method comprises (a) screening a library of test compounds in a cell viability assay, which assay comprises cells that are capable of undergoing huntingtin-modulated cell death, (b) selecting those test compounds from step (a) that have a %Rescue >50%, or as previously defined, wherein
  • %Rescue ⁇ 1 - ⁇ - ⁇ -, ⁇ r x 100
  • step (c) screening the test compounds selected in step (b) in (i) an in vitro protein disulfide isomerase (PDI) inhibition assay and (ii) a cell viability assay, which assay comprises cells that are capable of undergoing huntingtin-modulated cell death, (d) selecting those test compounds from step (c) that (i) exhibit PDI inhibition in the PDI in vitro inhibition assay and (ii) have a %Rescue >50%, or as previously defined, in the cell viability assay as candidate compounds for use in treating a neurodegenerative disorder.
  • at least one of the assays set forth above is an HTS.
  • the method for identifying candidate compounds for use in treating a neurodegenerative disease further comprises carrying out the following additional assays on the candidate compounds selected from step (d) above: (a) the cell viability assay, (b) the in vitro PDI inhibition assay, and (c) a direct PDI binding assay, wherein in each assay, each candidate compound is tested in an appropriate dilution series.
  • at least one of the assays set forth above is an HTS.
  • libraries may be obtained from a wide variety of sources.
  • libraries may be combinatorial chemical libraries, biological libraries; spatially addressable parallel solid phase or solution phase libraries; libraries prepared by synthetic library methods requiring deconvolution; the "one- bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach may be to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (83).
  • Such compounds/libraries may be prepared using methods known in the art or purchased or otherwise acquired from commercial, academic or not-for-profit sources.
  • the library of test compounds is comprised of compounds filtered in silico to be able to penetrate the blood brain barrier and to lack toxic or reactive functionalities.
  • the phrase "cell viability assay” means any suitable assay, preferably an HTS assay, which measures a candidate compound's ability to modulate misfolded protein mediated cell toxicity.
  • a non-limiting example of such a cell viability assay includes the PC 12 neuronal assay disclosed in Example 1.
  • the PC 12 neuronal assay is an HTS.
  • in vitro PDI inhibition assay means any assay, preferably an HTS assay, which identifies candidate compounds that bind to and/or inhibit PDI.
  • a non-limiting example of an in vitro PDI assay is disclosed in Example 5. Although the assay in Example 5 is described in terms of a 384 well plate, the assay is easily adapted to a 1536-well, or higher, format.
  • direct PDI binding assay means any assay, which identifies candidate compounds that are able to be covalently linked to PDI, as well as other assays that provide the same or substantially the same information.
  • a non-limiting example of such a direct PDI binding assay is disclosed in Example 4.
  • the direct PDI binding assay may comprise: (a) derivatizing a candidate compound selected from step (d) of the method for identifying candidate compounds for use in treating a neurodegenerative disease, as disclosed above, to make such a candidate compound HCC compatible; (b) contacting the derivatized candidate compound with a PDI under suitable conditions for binding of the derivatized candidate compound to the PDI; (c) carrying out HCC to covalently attach a detectable label to the derivatized candidate compound; and (d) determining whether the labeled derivatized candidate compound binds to the PDI.
  • Such an assay may be an HTS assay.
  • kits for screening methods, including, but not limited to, in vivo cell-based assays, in vitro methods, combined in vivo I in vitro assays, and high throughput screening ("HTS") assays, for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs), which bind to PDI proteins and/or, have an inhibitory effect on PDI expression or PDI activity.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs)
  • the present invention provides for screening compounds which are known or believed to bind and/or inhibit PDI, either from the prior art or as determined using screening methods described herein, for neuro-protective activity in a model system for neurodegeneration.
  • assays which directly determine whether a candidate compound binds to PDI.
  • the assay includes providing a test compound linked to a detectable label, exposing the test compound to PDI under conditions that permit binding, and then detecting the bound test compound via its label.
  • any detectable label may theoretically be used, the presence of the label could interfere with the native ability of the candidate compound to bind to PDI.
  • this problem may be avoided, as disclosed above, by derivatizing the candidate compound with an azide and a detectable label with an alkyne (or vice versa), allowing the derivatized test compound to bind to PDI (in vitro or in vivo), and then, in vitro, reacting the bound, derivatized test compound with the derivatized label using HCC in the presence of CU(II) under denaturing conditions.
  • SUP-2 is used as an example of a candidate compound
  • SUP-2A binds to 53 kD PDI and, following, e.g., fluorescein coupling, detectably labels the protein.
  • the present invention provides for in vitro (i.e., cell-free) assays which may be used to determine whether a test compound binds to PDI.
  • the present invention provides for an in vitro competitive binding assay which comprises contacting a PDI protein or biologically active portion thereof with a compound known to bind to the PDI (a "PDI ligand") as well as a test compound, and then determining whether and/or to what extent the PDI ligand binds to PDI in the presence, relative to in the absence, of a test compound.
  • a test compound to decrease the amount of PDI ligand bound indicates that the test compound binds to PDI.
  • the PDI ligand is directly or indirectly labeled.
  • PDI may be exposed to PDI ligand and test compound simultaneously, (ii) PDI may be first exposed to PDI ligand, followed by test compound, or (iii) PDI may be first exposed to test compound, followed by PDI ligand.
  • PDI ligand include cystamine, SUP-2, SUP-3, or SUP-1 (as defined herein). This assay could be used in high-throughput methods in which each reaction chamber contained PDI bound to solid support, labeled ligand, and a test compound, whereby the readout provides a measure of the candidate compound's ability to compete with the PDI ligand for its binding site on the PDI.
  • the invention provides for an in vitro assay in which a PDI protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to (interact with) the PDI protein or biologically active portion thereof is determined.
  • Determining the ability of the test compound to bind to/interact with a PDI protein can be accomplished, for example, by determining the ability of the PDI protein to bind to a PDI target molecule by one of the methods described above for determining direct binding. Determining the ability of the PDI protein to bind to a PDI target molecule can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). (84, 85).
  • BIOS Biomolecular Interaction Analysis
  • BIOA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
  • SPR surface plasmon resonance
  • the present invention provides for an in vitro method to screen for compounds that suppress or inhibit (reversibly or irrepressibly) the reductase activity of PDI.
  • Determining the ability of the test compound to modulate PDI activity can be accomplished by monitoring, for example, changes in intracellular compound concentrations by, e.g., flow cytometry, or by the activity of a PDI-regulated transcription factor.
  • such methods are performed as HTS assays.
  • the present invention provides for in vivo (i.e., cell-based) assays which may be used to determine whether a test compound modulates the function or expression of PDI.
  • the assay is a cell- based assay in which a cell which expresses a PDI protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate PDI activity is determined.
  • the cell-based assay may be used to screen for compounds that suppress an unfolded protein response (UPR) elicited by polyQ-expanded huntingtin protein.
  • URR unfolded protein response
  • the present invention provides for a cell-based assay that includes contacting a cell expressing a PDI target molecule (e.g., a PDI ligand or substrate such as choline and/or an acceptor molecule to be reduced or oxidized) with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the PDI target molecule. Determining the ability of the test compound to modulate the activity of a PDI target molecule can be accomplished, for example, by determining the ability of the PDI protein to bind, interact with, or modify the PDI target molecule.
  • a PDI target molecule e.g., a PDI ligand or substrate such as choline and/or an acceptor molecule to be reduced or oxidized
  • determining the ability of the test compound to modulate e.g., stimulate or inhibit
  • Determining the ability of the PDI protein or a biologically active fragment thereof, to bind to, interact with, or modify a PDI target molecule or ligand can be accomplished by one of the methods described above for determining direct binding. Alternatively, determining the ability of the PDI protein to bind to or interact with a PDI target molecule or ligand can be accomplished by determining the activity of the target molecule.
  • the activity of the target molecule can be determined by monitoring the effect of the target on an appropriate substrate, detecting the induction of a reporter gene (comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker), or detecting a target-regulated cellular response such as changes in cellular component levels or changes in cellular proliferation responses.
  • a reporter gene comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker
  • a target-regulated cellular response such as changes in cellular component levels or changes in cellular proliferation responses.
  • determining the ability of the test compound to modulate the activity of a PDI protein can be accomplished by determining the ability of the PDI protein to further modulate the activity of a downstream effector of a PDI target molecule. For example, the activity of the effector molecule on an appropriate target can be determined or the binding of the effector to an appropriate target can be determined as previously described.
  • modulators of PDI expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of PDI mRNA or protein in the cell is determined.
  • the level of expression of PDI mRNA or protein in the presence of the candidate compound is compared to the level of expression of PDI mRNA or protein in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of PDI expression based on this comparison. For example, when expression of PDI mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of PDI mRNA or protein expression. Alternatively, when expression of PDI mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of PDI mRNA or protein expression.
  • the level of PDI mRNA or protein expression in the cells can be determined by methods described herein for detecting PDI mRNA or protein.
  • PDI protein can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; and WO94/10300; see also references 86-89), to identify other proteins, which bind to or interact with PDI ("PDI-binding proteins" or "PDI-bp") and are involved in PDI activity.
  • PDI-binding proteins are also likely to be involved in the propagation of signals by the PDI proteins or PDI targets as, for example, downstream elements of a PDI- mediated signaling pathway.
  • such PDI-binding proteins are likely to be PDI inhibitors.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • the gene that codes for a PDI protein is fused to a gene encoding the DNA binding domain of a known transcription factor.
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey" or "sample”) is fused to a gene that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene, which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the PDI protein.
  • test compound Once a test compound has been identified as binding to, interacting with, or modulating PDI using an assay as set forth above, further tests may be performed to confirm the anti-neurodegenerative activity of the test compound.
  • a model system for neurodegeneration may be exposed to the test compound at various concentrations and for various periods of time, and the system may be examined and compared to one or more control(s) (for example, a positive or negative control experiment conducted in parallel or a pre-determined value) to determine the effect of the test compound on neurodegeneration in the model system.
  • the model system may be a cell culture or an organism.
  • suitable cell culture systems include rat neuronal PC12 cells and rat striatal neuronal ST14A cells as described in the Examples below.
  • PC12 cells or ST14A cells can be transfected with exon-1 of the human expanded huntingtin gene containing expanded polyQ repeats (e.g., Q 103) at the N- terminal region.
  • expanded polyQ repeats e.g., Q 103
  • Htt-Q103 polyQ-expanded human expanded huntingtin exon-1
  • Htt-Q25 wild-type e.g., Htt-Q25
  • apoptotic disease cellular phenotypes has been altered to resemble a more normal or more wild type, or non-apoptotic disease phenotype.
  • Cellular phenotypes that are associated with apoptotic disease states include aberrant DNA fragmentation, membrane blebbing, caspase activity, cytochrome c release from mitochondria, and translocation or accumulation of PDI or other chaperone proteins on or within the mitochondria. In a specific embodiment of the present invention, caspase activation is measured.
  • an agent identified as described herein e.g., a PDI modulating agent, an antisense PDI nucleic acid molecule, a PDI-specific antibody, or a PDI-binding partner
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
  • compositions and methods of the invention directed toward treatment and/or prophylaxis of neurodegenerative disorders animal models based on the effects induced by 1-methyl-4-phenyl-1 ,2,3,6-tetrahydropyridine (MPTP) are relevant (see references 90-94, all incorporated fully herein by reference).
  • Models based on MPTP-induced effects include chronic hemi-
  • Parkinsonian monkeys (95, incorporated fully herein by reference), degeneration of nigrostriatal dopamine neurons in mice (96, incorporated fully herein by reference), evaluations of cognitive function in MPTP-treated animals (97, incorporated fully herein by reference), and measurement of striatal levels of 1-methyl-4- phenylpyridinium (MPP+) (98, incorporated fully herein by reference).
  • MPP+ 1-methyl-4- phenylpyridinium
  • kainic acid-induced effects 99, incorporated fully herein by reference
  • 6-hydroxydopamine (6-OHDA) lesion in rat 100-102, all incorporated fully herein by reference
  • quinolinic j acid-induced hippocampal neurodegeneration (112, incorporated fully herein by reference)
  • murine models of neonatal excitotoxic brain injury 113, incorporated fully herein by reference
  • reserpine-induced striatal dopamine deficiency 114.
  • Effects on the age-associated loss of nigrostriatal dopaminergic neurons may also be evaluated to determine the potential for preventing or alleviating neurodegenerative disease (See, e.g., 103-104, all incorporated fully herein by reference).
  • Several animal models of Parkinson's disease have been generated in which effective therapies are indicative of therapeutic efficacy in humans. These animal models include three rat models (the rats having lesions in substantia nigral dopaminergic cells caused by treatment with 6-hydroxydopamine, 1-methyl-4- phenyl-1 ,2,3,6-tetrahydropyridine (MPTP), or surgical transection of the nigral striatal pathway) (See, e.g., 105), a rhesus monkey model (the monkeys having lesions in substantia nigral dopaminergic cells caused by treatment with MPTP) (See, e.g., 106-108), and a sheep model (the sheep having lesions in substantia nigral dopaminergic cells caused by treatment with MPTP) (109). Therapeutic efficacy in any one of these models of Parkinson's disease is predictive of therapeutic efficacy in humans.
  • rat models the rats having lesions in substantia nigral dopaminergic cells caused by treatment with 6-
  • binding of a test compound to a PDI protein, or interaction of a PDI protein with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • PDI fusion proteins or target fusion proteins can be adsorbed onto beads or derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or PDI protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above.
  • the complexes can be dissociated from the matrix, and the level of PDI binding or activity determined using standard techniques.
  • a PDI protein or a PDI target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated PDI protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, III.), and immobilized in the wells of streptavid in-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with PDI protein or target molecules but which do not interfere with binding of the PDI protein to its target molecule can be derivatized to the wells of the plate, and unbound target or PDI protein trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the PDI protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the PDI protein or target molecule.
  • the present invention provides for an assay system for identifying a (hitherto unappreciated) target receptor for a ligand of interest (an "orphan ligand"), which utilizes HCC under denaturing conditions.
  • an assay system for identifying a (hitherto unappreciated) target receptor for a ligand of interest (an "orphan ligand"), which utilizes HCC under denaturing conditions.
  • HCC was used to identify PDI as the target receptor for "orphan ligand" SUP-2, is described herein.
  • the present invention provides for a method for identifying the target receptor of an orphan ligand of interest, comprising:
  • one HCC-compatible member is derivatized to contain an alkyne group and the other is derivatized to contain an azide group
  • the derivatization of the orphan ligand does not prevent its binding to its target receptor (for example, but not by way of limitation, the derivatized orphan ligand is not prevented from binding to its target receptor if it retains its biological activity);
  • the target receptor may be contained in a cell lysate, a partially purified mixture of proteins, a mixture of proteins as contained in or bound to a solid support, or, preferably, an intact cell, to form a derivatized orphan ligand/target receptor complex;
  • the treatment methods of the invention in general comprise administration of a therapeutically effective amount of one or more compounds of the invention to an animal, including a mammal, particularly a human.
  • the present invention provides a method of treating a subject suffering from a neurodegenerative condition comprising providing to said subject an agent in an amount effective to inhibit PDI in neurons of the subject.
  • One aspect of the present invention is directed to the treatment of neurodegenerative diseases by administering to an individual a therapeutically effective amount a PDI inhibitor, which amount is suitable for prophylaxis and/or treatment of the particular neurodegenerative disease.
  • Compounds of the invention are useful to treat and/or prevent various neurodegenerative diseases such as Parkinson's disease, Huntington's disease, Amyotrophic Lateral Sclerosis, Alzheimer's disease, Down's Syndrome, Korsakoff's disease, cerebral palsy and/or age-dependent dementia.
  • the compound can be a known inhibitor of PDI or an inhibitor discovered by the assays of the invention.
  • Non-limiting examples of PDI inhibitors include antisense RNA or RNAi at least in part complementary to the PDI gene, cystamine, and SUP-2. These agents may preferably be administered so as to selectively be present in the nervous system or, if appropriate, a specific location within the nervous system (e.g., basal ganglia).
  • the blood-brain barrier may, for example, afford sufficient selectivity if the agent or a combination thereof is delivered intrathecally.
  • the compounds and compositions of the present invention may be administered in any appropriate manner.
  • candidate compounds can be profiled in order to determine their suitability for inclusion in a pharmaceutical composition.
  • One common measure for such agents is the therapeutic index, which is the ratio of the therapeutic dose to a toxic dose.
  • the thresholds for therapeutic dose (efficacy) and toxic dose can be adjusted as appropriate (e.g., the necessity of a therapeutic response or the need to minimize a toxic response).
  • a therapeutic dose can be the therapeutically effective amount of a candidate compound (relative to treating one or more conditions) and a toxic dose can be a dose that causes death (e.g., an LD 50 ) or causes an undesired effect in a proportion of the treated population.
  • the therapeutic index of a compound, agent, or composition according to the present invention is at least 2, more preferably at least 5, and even more preferably at least 10.
  • Profiling a candidate compound can also include measuring the pharmacokinetics of the compound, to determine its bioavailability and/or absorption when administered in various formulations and/or via various routes.
  • a compound of the present invention such as a compound that mediates PDI-induced cellular toxicity, e.g., a PDI inhibitor, may be administered to an individual in need thereof.
  • the individual is a mammal such as a human, or a non-human mammal.
  • a compound of the invention can be administered as a pharmaceutical composition containing, for example, the compound and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters.
  • the aqueous solution is pyrogen free, or substantially pyrogen free.
  • Excipients may be selected and incorporated into such compositions, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs.
  • a pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize or to increase the absorption of a compound, such as, a PDI inhibitor.
  • physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients.
  • the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent depends, for example, on the route of administration of the composition.
  • the pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention. Liposomes, for example, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
  • a pharmaceutical composition (preparation) containing a compound of the invention can be administered to an individual by any of a number of routes of administration including, for example, orally; intramuscularly; intravenously; anally; vaginally; parenterally; nasally; intraperitoneal ⁇ ; subcutaneously; and topically.
  • the composition can be administered by injection or by incubation.
  • compositions suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules, a solution or a suspension in an aqueous or non-aqueous liquid, an oil-in-water or water-in-oil liquid emulsion, an elixir or syrup, a pastille, a bolus, an electuary or a paste.
  • These formulations may be prepared by methods known in the art, e.g., by means of conventional pan-coating, mixing, granulation or lyophilization processes.
  • Solid dosage forms for oral administration may be prepared by mixing the active ingredient(s) with one or more pharmaceutically-acceptable carriers and, optionally, one or more fillers, extenders, binders, humectants, disintegrating agents, solution retarding agents, absorption accelerators, wetting agents, absorbents, lubricants, and/or coloring agents.
  • Solid compositions of a similar type maybe employed as fillers in soft and hard-filled gelatin capsules using a suitable excipient.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using a suitable binder, lubricant, inert diluent, preservative, disintegrant, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine.
  • the tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein. They may be sterilized by, for example, filtration through a bacteria-retaining filter.
  • compositions may also optionally contain opacifying agents and may be of a composition such that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • the active ingredient can also be in microencapsulated form.
  • Liquid dosage forms for oral administration include pharmaceutically- acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain suitable inert diluents commonly used in the art.
  • the oral compositions may also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions may contain suspending agents.
  • compositions for rectal or vaginal administration may be presented as a suppository, which maybe prepared by mixing one or more active ingredient(s) with one or more suitable nonirritating carriers which are solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • Pharmaceutical compositions which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such pharmaceutically-acceptable carriers as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants.
  • the active compound may be mixed under sterile conditions with a suitable pharmaceutically-acceptable carrier.
  • the ointments, pastes, creams and gels may contain excipients.
  • Powders and sprays may contain excipients and propellants.
  • compositions suitable for parenteral administrations comprise one or more modulators in combination with one or more pharmaceutically- acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.
  • suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.
  • Proper fluidity can be maintained, for example, by the use of coating materials, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain suitable adjuvants, such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption.
  • the rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form.
  • delayed absorption of a parenterally-administered drug may be accomplished by dissolving or suspending the drug in an oil vehicle.
  • injectable depot forms may be made by forming microencapsule matrices of the active ingredient in biodegradable polymers. Depending on the ratio of the active ingredient to polymer, and the nature of the particular polymer employed, the rate of active ingredient release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use.
  • sterile liquid carrier for example water for injection
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.
  • a compound (e.g., PDI inhibitor) of the present invention may be used alone or conjointly administered with another type of agent designed to mediate neurodegeneration.
  • the phrase "conjoint administration” refers to any form of administration in combination of two or more different therapeutic compounds such that the second compound is administered while the previously administered therapeutic compound is still effective in the body (e.g., the two compounds are simultaneously effective in the patient, which may include synergistic effects of the two compounds).
  • the different therapeutic compounds can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially.
  • an individual who receives such treatment can benefit from a combined effect of different therapeutic compounds.
  • the compound (e.g., PDI inhibitors) of the present invention will be administered to an individual (e.g., a mammal, preferably a human) in a therapeutically effective amount (dose).
  • an individual e.g., a mammal, preferably a human
  • a therapeutically effective amount dose
  • the construct contains alternating CAG/CAA repeats.
  • An EGFP tag is used to monitor expression, aggregation and localization of the Q 103 and Q25 proteins.
  • Both constructs are under the control of the Bombyx mori ecdysone receptor; hence expression is induced by addition of the ecdysone analog tebufenozide to the culture medium.
  • the mutant cells (Q103 cells), but not the wild-type cells (Q25 cells) display peri-nuclear inclusion bodies and apoptotic cell death in response to tebufenozide treatment (Figure 1C and data not shown).
  • Previous studies determined that expression of these constructs in an astrocyte cell line (BAS 8.1) did not cause apoptosis, suggesting a degree of neuronal selectivity (34).
  • BOC-D-fmk appears to prevent cell death at a point much further downstream — at the point of blocking effector caspases — than the hit compounds, which act upstream of caspase cleavage.
  • hit compounds treating induced Q103 cells with hit compounds suppresses mitochondrial release of cytochrome c and SMAC into the cytosol, placing the compounds' mechanism of action upstream of mitochondrial outer membrane permeabilization (MOMP) (See, e.g., Figures 15, 16A-C, and 24B-C).
  • MOMP mitochondrial outer membrane permeabilization
  • SUP-2 was particularly attractive as a probe reagent because it has a chloromethylcarbonyl functionality. Chloromethylcarbonyls are frequently used as moieties for covalently inhibiting enzymes: a nucleophilic residue on the enzyme displaces the chloro substituent in an S N 2 reaction, leading to a covalent bond between the inhibitor and the enzyme (37). Thus, we speculated that SUP-2, with its chloromethylcarbonyl functionality, was covalently labeling a target protein. Indeed, we found that an analog of SUP-2 lacking the chloro substituent (SUP-2ADC) was completely inactive (Figure 8).
  • a covalent interaction has the attractive feature of increasing the facility with which we can identify the protein target of a compound. Therefore, we focused on making affinity reagents based on SUP-2. We tried modifying the compound to see where we could introduce an affinity tag without losing the activity. We found that we could only make small changes in the structure without losing activity (data not shown). For example, if we modified the ester functionality from methyl to ethyl, activity was preserved, but further increasing the size of the alcohol, for example to a biotin-l inked alcohol, caused a loss of activity. Thus, we were able to identify a site that could tolerate small changes, but the traditional affinity strategy of introducing a linked biotin moiety, as an affinity handle, was not viable in this case.
  • HCC Huisgen cycloaddition chemistry
  • HCC 1 sometimes also called
  • Cell lysate was prepared by trypsinizing PC12 cells, washing with media followed by PBS 1 and then swelling in lysis buffer on ice for 15 minutes (10OmM sodium phosphate buffer pH 7.0, cell density at 50 million cells per ml). The cells were lysed by passing through a 30 ga needle (10 passes) using a 1ml syringe. The lysate was centrifuged (15 minutes, 10,000xg, 4°C) and the supernatant (lysate) separated from the pellet and stored on ice for HCC reaction.
  • the cell lysate was probed with a candidate compound alkyne analog, such as, e.g., the SUP-2A alkyne analog, as follows: 43 ⁇ l of cell lysate (2 million cell equivalents) was incubated with 35 ⁇ M SUP-2A (0.6 ⁇ l of 2.5mg/ml SUP-2A stock in DMSO).
  • a candidate compound alkyne analog such as, e.g., the SUP-2A alkyne analog
  • the probed lysate was then tagged with rhodamine azide under the following HCC reaction conditions: 10OuM rhodamine azide tag (from 2.5 mg/ml DMSO stock), 1mM TCEP (Tris (2- carboxyethyl) phosphine hydrochloride from 5OmM stock in dH 2 O), 0.1mM ligand (from 1.7mM in DMSO:t-BuOH 1 :4), 1mM Cu(II) sulfate (from 5OmM stock in dH 2 O).
  • the reaction mix was vortexed for 5 seconds and incubated for 1hr at room temperature.
  • Rhodamine tagged proteins were analyzed by standard SDS PAGE electrophoreses and scanning on a Molecular Devices laser scanner with the appropriate excitation and emission filters for rhodamine detection.
  • the competing molecule was added prior to the candidate compound alkyne analog, e.g., SUP-2A, addition (described above) at a 15x excess (525 uM final concentration from 25mg/ml DMSO stocks) and incubated for 1hr at room temperature.
  • the rhodamine azide tag was synthesized using the method of Speers and Cravatt (43) with minor modifications based on personal communications with A.E. Speers.
  • the structure of the HCC ligand and the rhodamine azide tag are shown below. In the present invention, however, any appropriate HCC ligand and azide tag may be used.
  • Protein disulfide isomerases as novel regulators of neuronal apoptosis
  • the in vitro PDI assay was carried out using the following modifications to the methods published by Raturi et al (46). Assay volumes were adjusted for 50 ⁇ l total volume and performed on a 384 well plate. Daughter plates were prepared at 60 ⁇ g/ml inhibitor compound in PBS. The enzyme assay buffer (PBS / 1.5 mM EDTA / 60 ⁇ M DTT) was prepared. For DTT, 3 ⁇ l of 10OmM DTT stock in water was added to 5ml PBS. Next, enzyme was added by hand to the plate, 20 ⁇ l/well at a dilution of 340 ⁇ l (stock 1mg/ml) -> 3060 ⁇ l assay buffer for 3.4 mis total was about 0.7 ⁇ M.
  • PDI and ERp57 may be targeted by cystamine, a simple organic disulfide that has been demonstrated to have activity in a mouse model of HD in multiple animal trials (47-52). Indeed, we then found that cystamine inhibits PDI activity in vitro and protects PC 12 cells from Q103-induced apoptosis, suggesting its in vivo activity in HD model mice may be due to targeting PDIs ( Figure 8C). Together, these results suggest that PDIs may play a pivotal role in the generation of HD neuronal apoptosis and the resulting pathophysiology.
  • PDI has recently been found to be upregulated in a transgenic rat model (G93A SOD1) of familial amyotrophic lateral sclerosis (ALS), another neurodegenerative disease (53).
  • G93A SOD1 familial amyotrophic lateral sclerosis
  • Mutant SOD1 has been shown to form intracellular aggregates, similar to the mutant huntingtin protein; in the case of mutant SOD1 , this leads to apoptotic neuronal death (53, 54).
  • PDI is mostly localized to the ER, it has been detected in other subcellular locations, including the plasma membrane, the nucleus, the cytosol, and the outer membrane of mitochondria (55, 56). The latter observation is most interesting, because this provides evidence that PDI could regulate the mitochondrial permeability transition, which causes release of apoptogenic factors from mitochondria and activation of the apoptosome and the intrinsic apoptotic cascade. Indeed, our data to date show that PDI localizes to mitochondria and this localization is increased in Q103-expressing cells compared to Q25-expressing cells. In addition, treatment with PDI inhibitors causes further accumulation of PDI, consistent with the notion that blocking apoptosis by inhibiting PDI allows these cells to survive and results in the detection of high PDI levels that would otherwise induce apoptosis.
  • this MOMP-inducing activity of PDI is relevant to the activity of the hit compounds from the PC 12 screen, then they should prevent this activity. Indeed, we found that these compounds suppressed the MOMP-inducing activity of PDI ( Figure 16B). In addition, cystamine also suppressed the MOMP-inducing activity of PDI. Moreover, analogs of these compounds that were not able to prevent mutant- huntingtin-induced apoptosis in PC12 cells also did not suppress this the MOMP- inducing activity of PDI. This suggests that in response to mutant huntingtin protein expression, PDI induces MOMP, which activates the intrinsic caspase cell death sequence in PC12 cells.
  • the initial four PDI inhibitors (i.e., SUP-1 , -2, -3, and -4) were not optimized for drug-like properties. Therefore, we used a high-throughput approach to screen for small molecule inhibitors of PDI having more drug-like properties.
  • the concentrations for compounds were from 160 ⁇ g/mL to 0.312 ⁇ g/mL.
  • Cystamine's concentration in the in-vitro PDI assay was from 200 ⁇ g/mL to 0.391 ⁇ g/mL. All 20 compounds were also tested in the HCC competition assay (according to the method of Example 4) against a 16F16 propargyl analog (SUP-2A) that has been shown to bind to PDI.
  • SUP-2A 16F16 propargyl analog
  • di (o-aminobenzoyl) glutathione disulfide contains a disulfide bond that can be reduced by PDI; two fluorphores self- quench when in close proximity to each other.
  • diabz-GSSG was incubated with bovine PDI, an increase of fluorescence was observed.
  • securinine was added to diabz-GSSG and bovine PDI, no increase in fluorescence was observed. Based on these data, we concluded that securinine can bind and inhibit PDI.
  • protein disulfide isomerases (EC 5.3.4.1) constitute a family of at least 17 enzymes of the thioredoxin superfamily that are involved in isomerization, reduction, and oxidation of disulfide bonds, primarily in the lumen of the ER (44, 76).
  • PDI proteins In addition to their well-described function in the ER, PDI proteins have been reported in the cytosol and on mitochondria, where their physiological function is less clear (44, 55, 77).
  • mitochondrial, cytosolic, and ER/microsomal fractions were prepared by differential centrifugation (4°C, 10 min at 10,000 g to pellet mitochondria followed by 3 hours at 70,000 g to pellet ER/microsomes) of PC 12 cell lysates (250 mM sucrose, 0.1% BSA, 10 mM Hepes pH 7.5, 5 mM KCI, 1.5 mM MgCI 2 , 1 mM EGTA, 1 mM EDTA) generated as described above and clarified (700 g, 5 min, 4°C) to remove nuclei and unlysed cells prior to fractionation. Quantification of PDI ( ⁇ 2.5 x10 5 cell eq.
  • mitochondria and ⁇ 5 x10 5 cell eq. for cytosol and ER/microsome was done by LI-COR scanning and image analysis of IR-Western blot for PDI normalized to F1-ATPase, actin, and calnexin for mitochondria, cytosol, and ER/microsome fractions respectively.
  • mitochondria and ER/microsomal fractions (5 x10 5 cell eq) were treated with trypsin (300 ⁇ g/ml, 30 min, 25 0 C) followed by quenching with SBTI (1 mg/ml).
  • rat brain slices were co-transfected with expression vectors for human htt exon-1 containing 73 glutamines as a Cyan Fluorescent fusion protein (htt-Q73-CFP) and a Yellow Fluorescent Protein (YFP) reporter to monitor morphology of transfected neurons.
  • htt-Q73-CFP Cyan Fluorescent fusion protein
  • YFP Yellow Fluorescent Protein
  • Brain slices were plated onto serum-supplemented culture medium and maintained at 32 0 C under 5% CO 2 as previously described (82); compounds were added to the culture medium at the time of plating.
  • DNA constructs encoding Yellow Fluorescent Protein (YFP) and Cyan Fluorescent Protein (CFP) or htt-Q73-CFP containing the full exon-1 domain of human htt, 73 polyQ repeats, and a CFP fusion at the C-terminal
  • YFP Yellow Fluorescent Protein
  • CFP Cyan Fluorescent Protein
  • htt-Q73-CFP htt-Q73-CFP containing the full exon-1 domain of human htt, 73 polyQ repeats, and a CFP fusion at the C-terminal
  • MSNs co-transfected with YFP + htt-Q73-CFP degenerated over the course of 4-7 days compared to control neurons transfected with YFP + CFP only. On day 5 after explantation and transfection, MSNs were identified based on their position
  • PDI can be dissociated from the reductase, oxidase and isomerase activity of PDI for
  • fusion proteins will be produced in BL21 cells and purified, as described for S. cerevisiae PDI purification from E. coli.
  • the pET vectors contain a thrombin-cleavage site, allowing optional removal of the affinity tag after purification. Purity will be assessed by SDS page, reactivity with both N- and C-terminally directed antibodies, MALDI-TOF MS, HPLC 1 CD and PDI enzymatic activity.
  • PDI and ERp57 are related ER chaperone proteins that have not been implicated in apoptosis directly.
  • one of our central aims is to define the mechanism by which PDI and ERp57 activate the intrinsic mitochondrial apoptotic pathway in response to Q103 expression. This will define a novel pathway for activating apoptosis in neuronal cells.
  • PDI and/or ERp57 represent a missing link between the ER unfolded protein response and permeabilization of mitochondria, allowing release of cytochrome c and activation of the caspase-9- containing apoptosome.
  • ERp57 translocation to mitochondria and what the effects of PDI and ERp57 are in mitochondria.

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Abstract

L'invention concerne des procédés pour moduler une neurodégénérescence. Ces procédés comprennent l'administration à un patient en ayant besoin d'une quantité efficace d'un composé modulant la toxicité cellulaire induite par une protéine disulfure isomérase (PDI). Des composés et des compositions pour traiter de tels patients sont également fournis. En outre, l'invention concerne des essais pour identifier de tels composés.
PCT/US2008/006154 2007-05-14 2008-05-14 Agents et essais pour moduler une neurodégénérescence WO2008143876A2 (fr)

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WO2010008860A1 (fr) * 2008-06-23 2010-01-21 President And Fellows Of Harvard College Modulation de maladie neurodégénérative par modulation de l’activité xbp-1
US9956236B2 (en) 2011-02-07 2018-05-01 Cornell University Methods for increasing immune responses using agents that directly bind to and activate IRE-1
WO2012113079A1 (fr) * 2011-02-23 2012-08-30 UNIVERSITé LAVAL Analogues de cystamine pour le traitement de la maladie de parkinson
RU2630583C2 (ru) * 2011-02-23 2017-09-11 Университет Лаваль Аналоги цистамина, применяемые для лечения болезни паркинсона
WO2013063243A1 (fr) * 2011-10-25 2013-05-02 New York University Activateurs à petites molécules d'aldolase-trap de la malaria et inhibiteurs du glidéosome
US9873661B2 (en) 2011-10-25 2018-01-23 New York University Small molecule malarial Aldolase-TRAP enhancers and glideosome inhibitors
CN102408431A (zh) * 2011-11-29 2012-04-11 中国中医科学院中药研究所 青蒿素b的制备方法及其新用途
US10655130B2 (en) 2012-03-09 2020-05-19 Cornell University Modulation of breast cancer growth by modulation of XBP1 activity
US10450566B2 (en) 2013-09-25 2019-10-22 Cornell University Compounds for inducing anti-tumor immunity and methods thereof
US10421965B2 (en) 2013-09-25 2019-09-24 Cornell University Compounds for inducing anti-tumor immunity and methods thereof
US9957506B2 (en) 2013-09-25 2018-05-01 Cornell University Compounds for inducing anti-tumor immunity and methods thereof
WO2015051284A3 (fr) * 2013-10-03 2015-06-04 Invenio Therapeutics Inc. Analogues de sécurinine et de norsécurinine à petites molécules et utilisation de ces derniers dans les cancers, les maladies inflammatoires et les infections
US9827229B2 (en) 2013-10-03 2017-11-28 Invenio Therapeutics Inc. Small molecule securinine and norsecurinine analogs and their use in cancers, inflammatory diseases and infections
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CN104761572A (zh) * 2015-03-11 2015-07-08 暨南大学 一叶萩型生物碱二聚体类化合物或其可药用盐及其制法和应用
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