WO2008143876A2 - Agents and assays for modulating neurodegeneration - Google Patents

Abstract

The present invention is directed to methods for modulating neurodegeneration. These methods include administering to a patient in need thereof an effective amount of a compound that modulates protein disulfide isomerase (PDI)-induced cellular toxicity. Compounds and compositions for treating such patients are also provided. The invention further provides assays for identifying such compounds.

Description

AGENTS AND ASSAYS FOR MODULATING NEURODEGENERATION

RELATED APPLICATIONS

[0001] The present invention is related to co-owned and copending U.S.

Patent Application Serial No. 11/498,110 filed on August 2, 2006, which is a continuation-in-part of U.S. Patent Application Serial No. 11/349,653 filed on February 7, 2006, which is a continuation-in-part of U.S. patent application no. 10/837,360 filed on April 30, 2004, which is a continuation-in-part of U.S. Patent Application Serial No. 10/767,591 filed on January 29, 2004 (now abandoned), as well as to the underlying provisional patent applications to which these utility applications claim benefit. 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. 60/958,748 filed July 9, 2007; and U.S. Patent Application Serial No. 61/063,505 filed February 4, 2008. The entire contents of each of these U.S. patent applications and provisional patent applications are incorporated by reference as if recited in full herein.

FIELD OF THE INVENTION

[0002] 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. 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.

BACKGROUND OF THE INVENTION

Protein Disulfide lsomerase

[0003] Protein disulfide isomerase (PDI) 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. A related protein, ERp57, is also a protein disulfide isomerase. In the endoplasmic reticulum, 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).

[0004] PDI accelerates slow chemical steps that accompany protein folding

(for a review, see references 58 and 59). Disulfide formation can occur rapidly (at times, before the completion of synthesis) or may be delayed until after translation is complete. During protein folding in the ER, PDI catalyzes disulfide formation and rearrangement by thiol/disulfide exchange.

[0005] 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.

[0006] 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. Similarly, PDI-mediated reductive cleavage of disulfide bonds in human immunodeficiency virus envelope glycoprotein 120 is essential for infectivity. Thus, 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.

[0007] In view of the functional activities of PDI and homologous enzymes, assessing enzyme activity in various clinical and research settings is of interest. 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). In addition, prior to the present invention, there have been no reports of PDI activity in association with polyglutamine disease, including, for example, Huntington Disease. Moreover, the inventors are unaware of any literature that discloses or suggests a pro-apoptotic function for PDI. Introduction to Huntington Disease

[0008] There are numerous inherited neurodegenerative diseases, including

Huntington Disease (HD), caused by expression of an expanded polyglutamine- containing protein. HD is an autosomal dominant neurodegenerative disease. Typically, onset of the disease occurs in the fourth or fifth decade of life. The mean survival rate for HD patients is 15-20 years. To date, there is no known cure for this insidious disease.

[0009] In the case of HD, expansion of a CAG repeat in the huntingtin gene causes expression of a polyglutamine (polyQ)-containing huntingtin protein (Htt); expansion to more than 35Q causes a significant risk of developing HD. In patients with HD, neuronal loss is most pronounced in the striatum, although other regions, including the cortex, are affected. HD manifests with motor, cognitive and emotional abnormalities.

[0010] Although neuronal dysfunction may play a role in HD, neuronal loss is likely a component as well, especially in the late stages of the disease. Thus, 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. Moreover, several lines of evidence suggest that polyglutamine-containing huntingtin protein can activate aberrant apoptosis, a specific and stereotypical form of cell death. First, overexpression of polyglutamine-containing huntingtin protein in cell culture causes apoptosis in both neuronal (1-3) and non-neuronal cells (4, 5). In addition, there is evidence for apoptosis in mouse and nematode models of HD (6-9), and minocycline, a potential caspase-1 inhibitor, partially prevents HD-associated phenotypes in mice (10, 11). Finally, markers of apoptosis are found in post-mortem analysis of human HD patient brains (12-15). Together, these data suggest that mutant-huntingtin-induced apoptosis plays a role in HD pathophysiology.

[0011] 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. In this pathway, 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). These pro-apoptotic factors in turn activate caspase enzymes that degrade key structural and functional components of the cell (73).

[0012] Several upstream triggers of MOMP have been reported, including

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). However, in some neurodegenerative diseases (e.g. HD and PD) the aberrant protein accumulates in the cytosol, suggesting additional mechanisms exist to monitor protein folding and control cell homeostasis.

[0013] In view of the foregoing, 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. SUMMARY OF THE INVENTION

[0014] Accordingly, 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. In this embodiment, the compound is preferably administered as part of a pharmaceutical composition.

[0015] 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. In this embodiment, the compound is preferably administered as part of a pharmaceutical composition.

[0016] 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. In this embodiment, it is preferred that the compound is administered as part of a pharmaceutical composition.

[0017] 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. In this embodiment, it is preferred that the compound is administered as part of a pharmaceutical composition. [0018] 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. In this embodiment, it is preferred that the compound is administered as part of a pharmaceutical composition.

[0019] 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.

[0020] 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.

[0021] 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. [0022] 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.

[0023] 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. In this embodiment, the determining step preferably comprises carrying out virtual high throughput screening to identify compounds that bind to at least one PDI.

[0024] 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. In this embodiment, the determining step preferably comprises carrying out virtual high throughput screening to identify compounds that inhibit at least one PDI.

[0025] 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%,

wherein

(Average Induced Value) - (Average Background Value)

%Rescue = -r^ ^-^-7 -^ x 100,

(Average Uninduced Value)- (Average Background Value;

(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% in the cell viability assay as candidate compounds for use in treating a neurodegenerative disorder.

[0026] In yet another embodiment, 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.

[0027] In particular non-limiting embodiments, 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:

a. providing a test compound derivatized with an azide to obtain an azide test compound; b. exposing the azide test compound to PDI under conditions suitable for binding;

c. providing a detectable label derivatized with an alkyne under conditions suitable for HCC to operate on the alkyne in the detectable label and any azide test compound bound to PDI to thereby indirectly bind the detectable label to PDI; and d. determining whether detectable label is bound to PDI,

where the presence of detectable label bound to PDI indicates that the test compound binds to PDI.

[0028] In a reciprocal embodiment, the 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. These HCC-based methods are amenable to high-throughput screening of test compounds.

[0029] Alternatively or additionally, other methods may be used to identify compounds which bind to and/or inhibit PDI, and which therefore may be useful as neuroprotective agents.

[0030] Because HCC operates to join azide and alkyne groups, and the incorporation of either azide or alkyne into a test compound may be designed so as to minimally perturb the binding affinity between a ligand for its receptor, the inventive technique may be used generally to identify a hitherto unknown target receptor for an "orphan ligand" with desirable biological activity. According to such non-limiting embodiments, 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;

b. exposing the derivatized orphan ligand to a purported target receptor or a mixture of proteins which are expressed in a cell in which the orphan ligand exhibits biological activity;

c. providing a label derivatized with an alkyne (or azide) under conditions suitable for HCC to operate on the alkyne or azide in the label and the derivatized orphan ligand to thereby indirectly bind the label to the target receptor via bound orphan ligand; and

d. identifying the target receptor bound to the label.

[0031] In a non-limiting subset of embodiments, 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). Advantageously, in non- limiting embodiments, 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.

BRIEF DESCRIPTION OF THE FIGURES

[0032] 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. Expression of Htt-Q25 remains diffuse throughout the cytoplasm and is not cytotoxic. Figure 1C is a graph showing the quantification of Htt-Q25 and Htt-Q103 cell viability as a measure of Alamar Blue fluorescence. The addition of the general caspase inhibitor (Boc-D-fmk, 50μM) rescues Htt-Q103 toxicity after a 72 hour induction with tebufenozide (Z-statistics calculated for 15000, 7500, and 3250 cells, yellow box).

[0033] 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.

[0034] 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). Unlike the general caspase inhibitor, Boc-D-fmk (BOC), SUP-2 and SUP-3 do not directly inhibit caspase-3 activity post-extraction (seventh, eighth, and ninth bars). Figure 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 μM), SUP-3 (10 μM), and BOC-D-FMK (50 μM).

[0035] Figures 4A-D are photographs characterizing drug effects on PC12 cell morphology, Htt protein expression, and aggregate formation. The photographs show merged DIC-fluorescence and fluorescence micrographs of suppressor treated Q103- and Q25-expressing cells. Following a 42 hour induction with tebufenozide, control (untreated) Q 103 cells (Figure 4A) show a rounded detached morphology whereas SUP-1 or SUP-2-treated (Figures 4B and 4C respectively, 2.5 μg/ml in DMSO) remain spread and attached to the substrate. Treatment with SUP-1 or SUP- 2 does not suppress Htt-Q103 aggregate formation (Figures 4B and 4C). Treatment with SUP-1 or SUP-2 does not suppress Htt-Q25 expression (SUP-1 treatment shown in Figure 4D, bar=20μm). As can be seen, the active compounds reverse morphology of dying cells.

[0036] Figure 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. Under denaturing conditions, 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. This creates a stable triazole linkage between the hit compound and the fluorescent tag. The entire adduct (protein-hit-fluorescent tag) can then be purified using an antibody against the fluorescent tag immobilized on a solid-phase resin. The target protein can then be sequenced using mass spectrometry and identified by matching the sequences obtained to the protein databases.

[0037] 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)). The specific peptide sequences obtained by mass spectrometry are underlined in the database sequences of each protein.

[0038] 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, and SUP-2A COMP = pretreatment of PC12 extract with 2Ox SUP-2 followed by SUP-2A labeling, fluorescein coupling, and affinity purification.

[0039] 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.

[0040] Figure 9 shows PDI assay results using various compounds identified according to the present invention.

[0041] Figure 10 shows the structures of hit compounds according to the present invention identified in the PDI and cell viability assays.

[0042] Figures 11A-P show the results of Alamar Blue cell viability assays for certain hit compounds according to the present invention.

[0043] 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.

[0044] Figures 13A and B show the results of various HCC competition assays for certain hit compounds according to the present invention. [0045] 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.

[0046] 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.

[0047] 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.

[0048] Figure 17 is a schematic showing a model for the mHTT-PDI-MOMP pathway.

[0049] 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.

[0050] Figure 19 is a comparison of the nuclear magnetic resonance (NMR) spectra of Compound 141 and securinine. Compound 141 and securinine have identical ¹³C-NMR (upper panels) and ¹H-NMR (lower panels) spectra.

[0051] 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. [0052] Figure 21 are two plots showing the results of compounds BBC7M13

(M13) and BBC7E8 (E8) in the in vitro PDI assay. Legend: "E": Enzyme; "S": Substrate.

[0053] Figure 22 is a laser scanned gel of 16F16A-rhodamine tagged proteins

(PDI = 53 kDa doublet) in crude PC12 extract. Pretreatment of PC12 extract with BBC7M13 (M13), BBC7E8 (E8), or cystamine (CYS) blocks 16F16A target binding. The inactive analog hypotaurine (HYPT) does not compete for 16F16A target binding. Purified bovine PDI (B-PDI) is covalently labeled by 16F16A-rhodamine (arrowheads).

[0054] 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 μM) show a 5.3-fold increase of mitochondrial PDI over induced Q25 cells at 24 hours. The viability of induced Q103 cells at 24 hours is ~50% of the initial seeding number. Levels of PDI were normalized using antibodies to F1-ATPase, actin, and calnexin, for the mitochondria, cytosol, and ER₁ respectively. Fig. 23d is a Western blot of PDI from ER and mitochondrial fractions before and after protease shaving with trypsin (30 min, 25⁰C) 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. Proteins localized to mitochondrial intermembrane space (Smac) or inner mitochondrial membrane (F1- ATPase) are protected from proteolysis (the two upper arrowheads on the right side). PDI from the ER fraction is protected from proteolysis (arrowheads on the left side; 38 kDa PDI fragment is likely due to incomplete quenching with SBTI), whereas PDI associated with mitochondria is degraded (the two lower arrowheads on the right side). Quantification of protected PDI (53 kDa plus 38 kDa fragment) from protease shaved ER shows <2% digestion whereas the same analysis shows a 78% digestion of mitochondrial PDI (graph at left). The proteolytic susceptibility of mitochondrial PDI (d) supports a model of PDI being bound to MAM contact sites (f) or the outer mitochondrial membrane (g) as opposed to being lumenal-ER (e).

[0055] Figure 24 is a series of bar graphs demonstrating that PDI inhibitors suppress MOMP in purified mitochondria and in cells, (a) Mitochondrial MOMP assay via Western blot detection and LI-COR quantification of Smac protein released from purified mitochondria. Purified PC12 mitochondria were suspended in either cytosolic extract (S70 = 70,000 g clarified) or mitochondria isolation buffer and incubated (90 min, 37°C) with either BSA (1 mg/ml), or PDI (6U). The mitochondria were then pelleted and the degree of MOMP calculated as a percentage of Smac released from the mitochondrial pellet (P) into the supernatant (S). 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. (b-c) 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). [0056] Figure 25 is a graphic showing the set up for the rat brain slide HD assay. In this model, 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. Degeneration in medium spiny neurons (MSNs), 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. Finally, Figure 25C is a graphic showing a healthy YFP- transfected medium spiny neuron in the striatum.

[0057] 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.

[0058] Figure 27 is a bar graph summarizing the results of the high-throughput assays for suppressors of mutant-huntingtin-induced apoptosis.

[0059] 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. [0060] Figure 29 shows a set of expriments, which demonstrated that suppressors of Htt-Q103 toxicity, specifically SUP-1 , do not alter Htt-Q25 or Htt- Q103 protein expression.

[0061] Figure 30 shows dose-response curves of SUP-2 (16F16) and several peptide-coupled caspase inhibitors in suppressing the Htt-Q103 toxicity.

[0062] 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.

[0063] Figure 32A shows the structure of a derivative of SUP-2

(16F16),16F16-biotinamine. Figure 32B shows a dose-response curve of 16F16- biotinamine in suppressing the Htt-Q103 toxicity (lighter curve).

DETAILED DESCRIPTION

[0064] 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. In this embodiment, the compound is preferably administered as part of a pharmaceutical composition.

[0065] As used herein, "neurodegeneration" or a "neurodegenerative disease" refers to a disease state characterized by misfolded protein-induced cell death. In the present invention, such disease states include, e.g., a polyglutamine disease, a prion disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, and Alzheimer's disease. [0066] All polyglutamine diseases known or to be discovered are within the scope of the present invention. For example, 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. In a preferred embodiment, the polyglutamine disease is HD.

[0067] As used herein, "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. Representative, non-limiting examples of 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. Representative, non-limiting examples of 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.

[0068] "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.

[0069] 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.

[0070] 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. As used herein, the term "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); PDI1_ARATH (Q9XI01); PDI1_CAEEL (Q17967); PDI1_SCHPO (Q10057); PDI2_ARATH (Q9SRG3); PDI2_CAEEL (Q17770); PDI2_SCHPO (O13811); PDI_ASPNG (Q12730); PDI_ASPOR (Q00248); PDI_BOVIN (P05307); PDI_CHICK (P09102); PDI_DATGL (Q9XF61); PDI_DROME (P54399); PDI_HORVU (P80284); PDIJHUMAN (P07237); PDI_HUMIN (P55059); PDI_MAIZE (P52588); PDI_MEDSA (P29828); PDI_MOUSE (P09103); PDI_RABIT (P21195), PDI_RAT (P04785); PDI_RICCO (Q43116), PDI_WHEAT (P52589); PDI_YEAST (P17967); TIGA_ASPNG (Q00216); and YIA5_YEAST (P40557). Preferably, "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.

[0071] As used herein, "toxicity," "cytotoxicity," or "cellular 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. The phrase "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. Thus, "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). [0072] The compound that modulates protein disulfide isomerase (PDI)- induced cellular toxicity may be a PDI inhibitor. Preferably, a target of the PDI inhibitor is at least one PDI. In the present invention, "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. Preferably, the PDI inhibitors are SUP-1-4, cystamine, compound C13 (BBC7M13), compound D13 (BBC7E8), compound J15, compound F14, or securinine. In the present invention, where appropriate, 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.

[0073] The phrase "pharmaceutically acceptable" as used in connection with compounds and 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). Preferably, as used herein, the term "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. [0074] 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. In this embodiment, the compound is preferably administered as part of a pharmaceutical composition. Preferably, the compound is a PDI inhibitor. In this embodiment, the PDI inhibitor and the polyglutamine disease are as previously defined herein. Preferably, the modulation is a decrease in neuronal apoptosis.

[0075] 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. In this embodiment, it is preferred that the compound is administered as part of a pharmaceutical composition. Preferably, the compound is a PDI inhibitor. In this embodiment, the PDI inhibitor is as previously defined herein.

[0076] As used 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.

[0077] 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. In this embodiment, it is preferred that the compound is administered as part of a pharmaceutical composition. Preferably, the compound is a PDI inhibitor. In this embodiment, the PDI inhibitor is as defined previously herein.

[0078] The term "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. Within the meaning of the present invention, 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.

[0079] The term "prevent," "preventing," or "prevention" 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.

[0080] 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. In this embodiment, it is preferred that the compound is administered as part of a pharmaceutical composition. In this embodiment, the compound is preferably a PDI inhibitor as previously defined. Further, in this embodiment, the neurodegenerative disease is as previously defined herein.

[0081] 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. Preferably, the cell is a neuron. In this embodiment, the compound is preferably a PDI inhibitor as previously defined.

[0082] "Caspase," as used herein, 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. Preferably, the caspase is caspase-3, caspase-6, caspase-7, caspase-9, or combinations thereof. In one aspect, the modulation of caspase activity is a decrease in caspase activation.

[0083] 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. Preferably, the cell is a neuron. Preferably, the modulation is a decrease in MOMP. In this embodiment, the compound is preferably a PDI inhibitor as previously defined.

[0084] In one aspect, 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. As used herein, "apoptosome" refers to a multiprotein complex comprising cytochrome c that mediates the initiation of caspases.

[0085] In another aspect, the MOMP-modulating amount of the compound is sufficient to prevent or reduce caspase activation. [0086] 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. Preferably, the sample comprises a cell or a cellular extract.

[0087] In one aspect, this method further comprises identifying the target, if it is present, to which the derivatized candidate compound bound. Preferably, the target modulates protein disulfide isomerase (PDI)-induced cellular toxicity, or more preferably, the target is a PDI inhibitor.

[0088] As used herein, "modulating a cellular phenotype" means altering an observed quality of a cell, including but not limited to cell fate. One example of 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. In this embodiment, the neurodegenerative disease is as previously defined; preferably, it is HD.

[0089] 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. Preferably, 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. Alternatively, 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.

[0090] As used herein, "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.

[0091] 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.

[0092] "Huisgen cycloaddition chemistry" or "HCC" refers to a cycloaddition reaction between a compound with an azide group and a compound with an alkyne group. The alkyne may be internal or terminal. In HCC, 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.

[0093] In one aspect, 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.

[0094] A label can be any composition which is detectable. As used herein,

"detectable" refers to anything that is identifiable. For example, 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.), or any other dye known in the art.

[0095] 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.

[0096] 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. In the case of an enzyme, the label can be detected colorimetrically employing a substrate for the enzyme. In a preferred embodiment, the detection antibody is detected using alkaline phosphatase-conjugated, species- specific immunoglobulin. Any substrate of alkaline phosphatase can be used. For example, p-nitrophenylphosphate (pNPP) can be the substrate and the reaction product, p-nitrophenol, can be detected optically. [0097] In another aspect, 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.

[0098] In yet another aspect, the target is bound to a solid substrate.

[0099] . Preferebly, the method is a high throughput screening assay. The phrase "High Throughput Screening" (HTS) 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. In HTS, 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. In certain embodiments, "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. Preferably, the process is an automated process. HTS is a known method of screening to those skilled in the art.

[0100] In the present invention, virtual high throughput screening may be used. The phrase "virtual high throughput screening" (virtual HTS), as used herein, 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. Pharmacol., 58(3):281-93 (2006), and Langer and Wolber, Pure Appl. Chem., 76(5):991-996 (2004). [0101] In one HTS method according to the present invention, candidate compounds, such as candidate PDI-inhibitors, are selected in, e.g., a cell viability assay, such as, e.g., the PC 12 Assay, described in further detail in Example 1. In

such a method, candidate compounds are selected that have a %Rescue of greater

than or equal to (>) about 30%, such as for example 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In the present invention, %Rescue is calculated as follows:

(Average Induced Value) - (Average Background Value)

%Rescue = _j ¹ -^₁ -^ x 100.

(Average Uninduced Value)- (Average Background Value)

[0102] 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.

[0103] 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. In this embodiment, the determining step preferably comprises carrying out virtual high throughput screening to identify compounds that bind to at least one PDI.

[0104] 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. In this embodiment, the determining step preferably comprises carrying out virtual high throughput screening to identify

compounds that inhibit at least one PDI.

[0105] 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

„ . _ (Average Induced Value)- (Average Background Value) , _ΛΛ

%Rescue = γ¹ -^-÷-, ^r x 100,

(Average Uninduced Value)- (Average Background Value)

(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. Preferably, at least one of the assays set forth above is an HTS.

[0106] In one aspect, 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. Preferably, at least one of the assays set forth above is an HTS.

[0107] As noted above, libraries may be obtained from a wide variety of sources. For example, 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. Preferably, 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.

[0108] A number of different types of combinatorial libraries and methods for preparing such libraries have been described, including for example, PCT publications WO 93/06121 , WO 95/12608, WO 95/35503, WO 94/08051 and WO 95/30642, each of which is incorporated herein by reference.

[0109] In the present invention, 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. Preferably, the PC 12 neuronal assay is an HTS. [0110] As used herein, "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.

[0111] In the present invention, "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. In another non-limiting example, 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.

[0112] Other embodiments also provide 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. Additionally, 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.

[0113] In one embodiment of the invention, assays are provided 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. Although 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. In a specific non-limiting embodiment of the invention, 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. In one specific example set forth below, where SUP-2 is used as an example of a candidate compound, derivatized SUP-2 (referred to as SUP-2A) binds to 53 kD PDI and, following, e.g., fluorescein coupling, detectably labels the protein. This assay could be used in high- throughput methods in which each reaction chamber contained PDI bound to solid support and a labeled candidate compound (or derivatized candidate compound and label, using HCC), whereby the readout corresponds to the amount of bound label associated the candidate compound. [0114] In another set of non-limiting embodiments, 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.

[0115] For example, according to certain embodiments, 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. The ability of a test compound to decrease the amount of PDI ligand bound indicates that the test compound binds to PDI. In such embodiments, preferably, the PDI ligand is directly or indirectly labeled. In alternative embodiments, (i) 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. Non-limiting examples of 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.

[0116] In other non-limiting embodiments, 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). As used herein, "BIA" 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.

[0117] In certain other non-limiting embodiments, 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. Preferably, such methods are performed as HTS assays.

[0118] In another set of non-limiting embodiments, 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.

[0119] According to one such non-limiting embodiment, 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. For example, the cell-based assay may be used to screen for compounds that suppress an unfolded protein response (UPR) elicited by polyQ-expanded huntingtin protein. [0120] In another embodiment, 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. 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. For example, the activity of the target molecule (e.g., catalytic/enzymatic activity) 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.

[0121] In another alternative embodiment, 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. [0122] In yet another embodiment, 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.

[0123] In yet another embodiment, 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. Such 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. Alternatively, 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. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a PDI protein is fused to a gene encoding the DNA binding domain of a known transcription factor. In the other construct, 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. If the "bait" and the "prey" proteins are able to interact, in vivo, forming a PDI- dependent complex, 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.

[0124] 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. For example, 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. Specific non- limiting examples of suitable cell culture systems include rat neuronal PC12 cells and rat striatal neuronal ST14A cells as described in the Examples below. In particular, 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. Expressing polyQ-expanded human expanded huntingtin exon-1 (Htt-Q103) in these cells can lead to selective toxicity over wild-type (e.g., Htt-Q25) expressing cells. See U.S. Patent Application No. 11/498,110, filed August 2, 2006, the disclosure of which is incorporated by reference in its entirety.

[0125] Where a cell-based model system is used, after exposure to the test compound the cells are examined to determine whether one or more of the 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.

[0126] In an animal model of neurodegeneration, the effect of the test compound on the organismal manifestation(s) of neurodegeneration may be measured. For example, 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) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein. [0127] Regarding 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).

[0128] 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).

[0129] Other relevant animal models include 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); and reserpine-induced striatal dopamine deficiency (114).

[0130] 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).

[0131] 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.

[0132] In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either PDI or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. 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.

[0133] In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, 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. Alternatively, the complexes can be dissociated from the matrix, and the level of PDI binding or activity determined using standard techniques.

[0134] Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either 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). Alternatively, 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, in addition to those described above for the GST-immobilized 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.

[0135] In one set of non-limiting embodiments, 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. A non-limiting example, wherein HCC was used to identify PDI as the target receptor for "orphan ligand" SUP-2, is described herein. [0136] Accordingly, the present invention provides for a method for identifying the target receptor of an orphan ligand of interest, comprising:

(i) derivatizing the orphan ligand and derivatizing a detectable label to make it HCC compatible,

wherein one HCC-compatible member is derivatized to contain an alkyne group and the other is derivatized to contain an azide group, and

wherein 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);

(ii) exposing the derivatized orphan ligand to a composition suspected of containing its target receptor under conditions which permit selective binding of the derivatized orphan ligand to its target receptor (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;

(iii) exposing the derivatized detectable label to the derivatized orphan ligand and its target receptor, preferably in the form of a derivatized orphan ligand/target receptor complex;

(iv) providing conditions which permit the HCC reaction to occur (e.g., in the presence of Cu and as set forth herein and in the art) to form a labeled orphan ligand/receptor complex; and (v) identifying the target receptor to which the orphan ligand is bound (for example, but not by way of limitation, using an antibody directed to the label in an affinity purification method to collect labeled orphan ligand/target receptor complex and then characterizing the target receptor, for example, by sequencing, molecular weight, activity, and/or other technique known in the art (alternatively, the labeled complex may be isolated using other methods known in the art, such as polyacrylamide gel electrophoresis, immunoprecipitation, etc.).

[0137] The foregoing method may be more readily practiced where the orphan ligand covalently binds to its target receptor as exemplified below, but its application is not so limited, so that the method may also be applied to orphan ligands which non-covalently bind to their receptor.

[0138] 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. In certain embodiments, 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.

[0139] 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.

Administration

[0140] The compounds and compositions of the present invention may be administered in any appropriate manner. For example, 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). For example, 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 LD₅₀) or causes an undesired effect in a proportion of the treated population. Preferably, 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.

[0141] 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. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an individual, 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 are well known in the art and 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. In a preferred embodiment, when such pharmaceutical compositions are for human administration, 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.

[0142] 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. Such 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.

[0143] 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.

[0144] Pharmaceutical 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.

[0145] Solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like) 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. These 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.

[0146] 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. Besides inert diluents, 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.

[0147] Pharmaceutical 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.

[0148] 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.

[0149] Pharmaceutical 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. 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. These 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.

[0150] In some cases, in order to prolong the effect of a drug containing a modulator of the present invention, it is desirable to slow its absorption from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility.

[0151] The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug 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.

[0152] 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. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.

[0153] In certain embodiments, 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. As used herein, 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). For example, the different therapeutic compounds can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic compounds.

[0154] It is contemplated that 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).

[0155] The following examples are provided to further illustrate the methods, compounds, and compositions of the present invention. These examples are illustrative only and are not intended to limit the scope of the invention in any way.

EXAMPLES

Example 1

High-throughput assays for suppressors of mutant-huntingtin-induced apoptosis

PC 12 Neuronal Assay

[0156] We adapted a previously developed PC 12 cell model for mutant- huntingtin-induced apoptosis, created by Erik Schweitzer in Allan Tobin's laboratory (34), into a robust high-throughput, 384-well plate high-throughput screening platform (HTS) (Figure 1). In this model, PC12 cells were stably transfected with a construct containing the first exon of the human huntingtin gene, which is the location of the polyglutamine expansion in the disease, fused to enhanced green fluorescent protein (EGFP). Two constructs were used: in one, 25 glutamines (Q25) were encoded in exon 1 ; in the second, 103 glutamines (Q103) were encoded, to represent a severe form of HD. To increase the stability of the repeat, 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).

[0157] We used the viability dye Alamar Blue to measure apoptosis in high- throughput in this cell system. Alamar Blue is reduced by cellular reductases in viable cells, leading to a shift in its fluorescence spectrum (35). This shift in Alamar blue fluorescence correlates well with Trypan Blue exclusion, a more conventional assay for cell viability that measures the integrity of the plasma membrane (26, 27).

[0158] In the screen, tebufenozide was added to induce Q103 expression.

After 72 hours, Alamar blue was added and fluorescence measured. Q103- expressing cells show a decrease in viability of ~70% (Figure 1C). Moreover, a pan- caspase inhibitor such as BOC-D-FMK (FMK) can completely prevent this loss of viability, demonstrating that it is a caspase-dependent, apoptotic mechanism (Figure 1C). We optimized the cell density in the assay and used 7,500 cells per well, yielding a Z' factor of 0.8, which indicates a robust, screenable assay (Figure 1C).

[0159] We screened about 47,000 small molecules in this assay for their ability to prevent Q103-induced apoptosis. The tested compounds were derived from several libraries that we assembled from commercial suppliers. We used our TIC library of 24,000 natural products, natural product analogs and synthetic drug-like compounds, assembled from Timtec, lnterbioscreen and Chembridge (30). We also used our collection of 20,000 combinatorial-synthesis-derived compounds from Comgenex (26) and our collection of 3,000 annotated, biologically active compounds assembled from Sigma, Aldrich, Fluka, RBI and Microsource Discovery (21 , 36). We found four compounds - SUP-1 , -2, -3, and -4 - with a significant ability to prevent Q103-induced apoptosis in this assay system, and several additional compounds with weaker activity (Figure 2).

Example 2

Characterization of hit compounds

[0160] We characterized hit compounds identified from the PC 12 cell viability assay in a number of additional assays. First, we tested their ability to restore normal cell morphology, i.e. the morphology of PC12 cells lacking Q103. We found the top four compounds (Ae., SUP-1 , -2, -3, and -4) restored normal cell morphology, whereas the weaker hits did not have this activity (Figure 4 and data not shown). Second, we tested the effects of the hit compounds on Q103 expression to confirm they there were not acting simply by preventing the induction of Q103 expression. We found the top four compounds did not affect Q103 expression (data not shown). Third, we tested whether any of the hit compounds affected Q103 aggregation in cells. We found no significant effect on aggregation (Figure 4 and data not shown).

Example 3

Effect of hit compounds on caspase activation and mitochondrial release of cytochrome c

[0161] We wanted to determine at what point in the apoptosis cascade these compounds (i.e., SUP-1 , -2, -3, and -4) intervene. Ideal compounds, both as therapeutic agents and as chemical probes, would intervene fairly upstream in the

Q103-induced apoptotic cascade, which would reveal some of the most upstream aspects of Q103 toxicity. We found that an effector caspase, caspase-3, and to some extent effector caspases-6 and 7, are cleaved into their activated forms 15 hours after induction of Q103 expression (Figure 3). The four hit compounds prevent this early caspase activation, while the pan-caspase inhibitor BOC-D-fmk does not suppress the appearance of these active fragments (Figure 3, 24B-C and data not shown). In addition, the four hit compounds do not directly inhibit caspase activity in cell lysates, unlike BOC-D-FMK (Figure 3A). Thus, 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. Finally, 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). Caspase-2 assay

[0162] Arteannuin B (SUP-3) and 16F16 (SUP-2) were tested for suppression of caspase-2 activation by serum withdrawal. PC12 cells were cultured for 18 hours in media without serum but supplemented with either 2.5 μg/ml 16F16 (SUP-2) or 3.5 μg/ml arteannuin B (SUP-3). Neither compound suppressed the appearance of caspase-2 active fragments by western blot of active caspase-2 fragments (Fig. 18).

Example 4

Defining the Structure-Activity Relationship for SUP-2

[0163] One of the four hit compounds, 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_N2 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).

[0164] 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.

Application of copper-mediated Huisgen cycloaddition chemistry (HCC) to target identification

[0165] To overcome this problem, we adapted HCC₁ sometimes also called

"Click" chemistry, i.e. the copper-mediated cycloaddition of an azide and an alkyne, which has been shown to be bio-orthogonal (38), but facile in the presence of cell lysates or even intact cells (39, 40) (Figure 5). The advantage we saw in using this reaction was that we could introduce a small change in the structure, such as an alkyne, and then bind the target protein covalently through the chloromethylcarbonyl functionality (41). Finally, after such labeling of the protein, we could use a copper- mediated cycloaddition to install a biotin or fluorescent tag onto the labeled protein (42, 43). This strategy allowed us to identify the target of SUP-2, which turned out to be the same target for all four hit compounds.

[0166] First, we synthesized the alkyne-modified SUP-2 analog (SUP-2A) and demonstrated that this analog was still able to prevent Q103-induced apoptosis (Figure 8A). Second, we optimized the labeling and reaction conditions, and eventually purified two related proteins as a 53 kDa doublet (Figure 7). Third, we performed the labeling in the presence of excess soluble compound and found that the labeled proteins were competed off, suggesting a specific interaction with the alkyne probe reagent (Figure 7). Finally, we showed that the other three hit compounds could also compete off the same doublet, but inactive analogs could not compete (Figure 7). These results demonstrated, somewhat surprisingly, that all four hit compounds interact with the same two 53 kDa target proteins.

[0167] The optimized HCC assay (direct PDI binding assay) is set forth below with reference to SUP-2A:

[0168] Cell lysate was prepared by trypsinizing PC12 cells, washing with media followed by PBS₁ 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: 43μl of cell lysate (2 million cell equivalents) was incubated with 35 μM SUP-2A (0.6μl of 2.5mg/ml SUP-2A stock in DMSO). 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 dH₂O), 0.1mM ligand (from 1.7mM in DMSO:t-BuOH 1 :4), 1mM Cu(II) sulfate (from 5OmM stock in dH₂O). 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.

Huisgen Cycloaddition Chemistry (HCC) Competition Assay

[0169] For chemical competitions to labeled proteins, 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.

Exact Mass 530 27 Molecular Weight: 583.64

(HCC Ligand) (Rhodamine azide tag)

Example 5

Protein disulfide isomerases as novel regulators of neuronal apoptosis

[0170] We sequenced the target proteins using mass spectrometry and discovered that they were two related protein disulfide isomerases (Figure 6): PDIA1 (SEQ ID NO:1) and PDIA3 (SEQ ID NO:2), which is also known as ERp57 (44). PDIs are chaperone proteins present primarily in the endoplasmic reticulum; but have also been detected in the cytosol and outer mitochondrial membrane (44, 45). We confirmed this by Western blot (Figure 7). We adapted a published assay (46) for measuring PDI enzymatic activity in vitro, and found that all four hit compounds, but not inactive analogs, inhibited bovine PDI activity (Figure 8).

In Vitro PDI Assay

[0171] 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 50μl total volume and performed on a 384 well plate. Daughter plates were prepared at 60μg/ml inhibitor compound in PBS. The enzyme assay buffer (PBS / 1.5 mM EDTA / 60 μM DTT) was prepared. For DTT, 3μl of 10OmM DTT stock in water was added to 5ml PBS. Next, enzyme was added by hand to the plate, 20μl/well at a dilution of 340μl (stock 1mg/ml) -> 3060μl assay buffer for 3.4 mis total was about 0.7μM. Then, transfer and mix inhibitor compound 20μl from PBS daughter plate for a final concentration of 20ug/ml hit compound (Biomek). Plates were incubated at 37°C for 20 minutes. Next, the substrate assay buffer (PBS / 1.5 mM EDTA) was prepared. Then, substrate was added to the plate, 10μl/well (300μM in substrate assay buffer) for 60μM final (1 :116 dilution of 35mM stock). Incubation was carried out for 1hr at 37°C. The plate was read using a diabzGSSG PE Victor³ protocol (340 nm excitation and 420 nm emission filter, 0.1 sec integration). Representative results from the in vitro PDI assay are shown in Figure 9.

[0172] In view of the foregoing, we speculated that 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.

[0173] 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). 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).

[0174] Although 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 intriguing, 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.

Example 6

PDI-induced MOMP in purified mitochondria

[0175] Given that PDI has been reported to be present in non-ER locations, including mitochondria, we speculated that mutant huntingtin might induce increased PDI levels and that PDI might activate the mitochondrial apoptotic cascade. Indeed, when we tested the effect of recombinant bovine PDI on purified mitochondria, we found that PDI induced mitochondrial outer membrane permeabilization in this cell- free system. Thus, PDI has an unexpected pro-apoptotic activity (See, e.g., Figure 16).

[0176] If 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.

[0177] We tested whether the MOMP-inducing activity of PDI was mediated by BAX, which is a major MOMP pathway in mammalian cells (Figure 16C). lmmunodepletion of BAX from these extracts had no effect on the MOMP-inducing activity of PDI. Moreover, PDI can induce a significant degree of MOMP, even in the absence of cytosolic extract, demonstrating that cytosolic BCL proteins are not needed. Adding cytosol to the assay enhances PDI-induced MOMP, while adding PDI-inhibitors suppresses MOMP (Fig. 24a).Together these data suggest that PDI- driven MOMP is independent of cytosolic BCL proteins and may be mediated by a novel pathway, although we cannot exclude the possibility of BCL protein interactions that may co-purify with the mitochondria. [0178] In summary, we have identified a potentially novel mechanism by which mis-folded proteins activate apoptosis, via PDI-driven MOMP (Figure 17). We have identified PDI inhibitors that block this apoptotic response. Thus, our data suggest that inhibition of PDI is a new means of treating, preventing, or ameliorating the effects of HD and any other neurodegenerative disease, which may be regulated by the same or similar mechanism.

Example 7

High-throughput screen for drug-like PDI inhibitors and characterization of these drug-like PDI inhibitors

[0179] 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. We screened a new library of 22,126 compounds, which we had filtered first in silico to be able to penetrate the blood-brain barrier and to be drug-like, e.g., lacking toxic or reactive functionalities.

[0180] Initially, all 22,126 compounds were screened in the PC12 Assay at one concentration of 4 μg/mL using the procedure of Example 1. Any compound that showed a %Rescue of 50% or more was retested in the PC12 Assay (Example 1) and also tested in the in vitro PDI inhibition assay according to the procedure used in Example 5. The compounds that prevented PC12 Q103-induced cell death consistently, and also inhibited PDI, were then re-tested in a thorough dilution series in three assays: PC12 Assay (Example 1), PDI in-vitro inhibition assay (Example 5), and HCC competition assay (to measure direct PDI binding) (Example 4). [0181] Out of 22,126 compounds that were screened, 78 showed a rescue of

≥50%. Upon retesting of these 78 compounds, 16 reconfirmed a rescue of >50% in the PC12 Q103 assay, and 8 compounds inhibited PDI enzymatic activity (Figure 14). We obtained these top eight hits and four related analogs, and tested them in a two-fold dilution across 10 wells. In the PC12 Q103 assay, the concentration range of compounds was from 20 μg/mL to 0.039μg/mL. Compound 141 , 16F16 (SUP-2), and cystamine were used as controls. Cystamine concentration in the assay was from 200 μg/mL to 0.391 μg/mL. In the PDI inhibition assay, the concentrations for compounds were from 160 μg/mL to 0.312μg/mL. Cystamine's concentration in the in-vitro PDI assay was from 200 μg/mL to 0.391 μg/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.

[0182] We found 3 compounds that came up as a hit in all three assays: rescued PC12 Q103 cells, inhibited PDI-substrate binding, and competed off the propargyl analog binding to PDI (see Figures 10-14 for the assay data). Four additional compounds rescued htt cell toxicity with Alamar Blue, and inhibited PDI in one of the two in-vitro assays.

[0183] In sum, we now have several PDI inhibitors that may be tested in other

HD models and for their suitability for in vivo studies.

[0184] We tested two of the compounds C13 (BBC7M13) and compound D13

(BBC7E8) to determine their effect on caspase activation and mitochondrial release of cytochrome c (Example 3). Using purified mitochondria, we found that PDI is capable of inducing MOMP in the absence of cytosolic proteins. Adding cytosol to the assay enhances PDI-induced MOMP, while adding the two PDI-inhibitors suppresses MOMP (Fig. 24A). Parallel experiments using intact cells also showed that inhibiting PDI in Q103-expressing cells suppresses mitochondrial release of cytochrome c and activation of effector caspases-3, -6, and -7 (Fig. 24B-C).

Example 8

Identification of Compound 141 as Securinine and Characterization of

Securinine

Identification of Compound 141 as Securinine

[0185] To elucidate the structure of Compound 141 , we performed thorough spectroscopic and analytical characterization. The compound was subject to liquid chromatography-mass spectrometry (LC-MS), thin-layer chromatography (TLC), nuclear magnetic resonance (¹H-NMR, ¹³C-NMR, and ¹³C-DEPT quantitative NMR), Infrared spectroscopy, UV spectroscopy, elemental analysis, and high-resolution mass spectrometry (HRMS). In addition, we performed several chemical tests to narrow down possible functional groups on the molecule. Analytical data for Compound 141 are summarized below.

[0186] Compound 141 : LC-MS(m/z): [LC-MS(m/z): [M]⁺ found 218.1 ; TLC

(CHCI₃: MeOH, 98:2 v/v): R_F=0.29, f?_F=0.12; ¹H-NMR (400 MHz, CDCI₃): δ 6.60 (d, J= 9.1 Hz, 1 H), 6.41 (dd, J= 5.3Hz, J=9.2Hz, 1 H), 5.55 (s, 1 H), 3.82 (t, J= 4.7Hz, 1 H), 2.97 (dt, J= 3.8Hz, J=10.5Hz, 1 H), 2.51 (dd, J= 4.1 Hz₁ J=9.3Hz, 1 H), 2.42 (m, 1 H), 2.10 (dd, J= 2.4Hz, J=11.3Hz, 1H), 1.88 (m, 1 H), 1.78 (d, J=9.2Hz, 1 H), 1.59 (m, 6H), 1.23 (m, 2H); ¹³C-NMR (75MHz, CDCI₃): δ 173.83, 170.24, 140.34, 121.58, 105.27, 89.67, 63.18, 58.96, 48.93, 42.46, 27.43, 26.07, 24.68; ¹³C-DEPT NMR (75MHz, CDCI₃) CH: δ 140.64, 121.89, 105.54, 63.43, 59.22 CH₂: 6 49.20, 42.71 , 27.66, 26.29, 24.92 CH₃ δ^~: none; IR (Attenuated Total Reflection): 1738.21 cm^'1; UV/vis: λ_max 272nm; elemental analysis (% calculated, % found for Ci₃Hi₅O₂N): C (71.87, 71.91), H (6.96, 7.05), N (6.45, 6.52), B (0, 0.24), F (0, <0.1), P (0, <0.05), S (0, 0.40), Cl (0, <0.1); HRMS(m/z): [M]⁺ calculated for C₁₃H₁₅O₂N, 217.11028; found, 217.110279; ninhydrin test (tests for presence of amines): positive; bromocresol green test (test for presence of functional groups with pKa<5): negative; Schiff s test (tests for presence of aldehydes): negative.

[0187] From these data, we determined the three possible structures of

Compound 141 that give specific ¹³C-NMR shifts in the 85 to 175 p. p.m. region. Searching SciFinder for all possible compounds with the molecular formula of C₁₃H₁₅NO₂ and having one of these structures, resulted in one match — securinine (structure shown in Fig. 20B). We purchased securinine from TimTec (cat.# ST057165) and tested it alongside Compound 141 in the PC12 HttQ103 viability assay with Alamar Blue. We also characterized securinine by NMR (Figure 19). Both ¹H-NMR and ¹³C-NMR spectra of securinine were identical to those of Compound 141 (compare the NMR data below with that of Compound 141 above; see also Figure 19).

[0188] Securinine: 1 H-NMR (400 MHz, CDCI3): δ 6.61 (d, J= 9.1 Hz, 1H), 6.42

(m, 1 H), 5.55 (s, 1 H), 3.82 (s, 1 H), 2.97 (d, J= 10.6Hz, 1H), 2.46 (m, 2H), 2.10 (m, 12H), 1.88 (d, J= 13.4Hz, 1 H), 1.78 (d, J= 9.3Hz, 1 H), 1.58 (m, 4H), 1.24 (s, 1 H); 13C-NMR (75MHz, CDCI3): δ 173.76, 170.16, 140.24, 121.46, 105.09, 89.54, 63.04, 58.79, 48.80, 42.34, 27.29, 25.92, 24.56;

[0189] From these data, we concluded that Compound 141 is securinine. Securinine binds and inhibits PDI

[0190] Previously, we showed that one of the other hit small molecules from the screen, compound 16F16, binds and inhibits protein disulfide isomerase. Although securinine and 16F16 share little structural similarity, we tested the possibility that securinine also binds and inhibits PDI. First, we performed a PDI binding competition experiment. We added to cell lysates securinine at 20-fold excess compared to a 16F16-derived binding probe, named 16F16A. The dark band at ~55kDa (the molecular weight of PDI) was present when the 16F16A probe was incubated with vehicle only, and disappeared when it was incubated with securinine. Hence, securinine was able to compete for binding to PDI with the 16F16A probe.

[0191] This PDI-binding activity was consistent with the results of an in vitro

PDI reductase assay. Briefly, di (o-aminobenzoyl) glutathione disulfide (diabz- GSSG) contains a disulfide bond that can be reduced by PDI; two fluorphores self- quench when in close proximity to each other. When diabz-GSSG was incubated with bovine PDI, an increase of fluorescence was observed. When 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.

In Vitro PDI Reductase Assay

[0192] Daughter plates were prepared with Biomek FX by transferring 2μl_ of compound to be tested from a stock DMSO plate at 20 mg/mL into a daughter plate with 98μL of 0.1 M sodium phosphate buffer pH 7. Bovine PDI (Sigma-Aldrich, cat. # P3818) was diluted 1 :10 in enzyme assay buffer (1.5mM EDTA, 60μM DTT, in sodium phosphate buffer, pH 7) and added at 20μL/well to a 384-well assay plate (Corning Inc., cat. # 3712). 20μL/well of compound were transferred from the daughter plate to an assay plate with Biomek FX and the assay plate was incubated at 37°C, 9.5% CO₂ for 15 minutes. Following incubation, 10μL/well of diabz-GSSG solution (1.5mM EDTA, 300μM diabz-GSSG in sodium phosphate buffer pH 7) were added to the assay plate and incubated for 1h at 37°C, 9.5% CO₂. Plates were read on a fluorescence plate reader (PerkinElmer Victor3V) with 340 nm excitation filter and 420 nm emission filter.

Example 9

PDI Localizes to Mitochondria

[0193] In humans, 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). 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).

[0194] When we analyzed the localization of PDI in Q 103 cells by immunofluorescence confocal microscopy, we observed a prominent punctate pattern that resembled mitochondrial staining with Mitotraker (data not shown). This finding was intriguing because it suggested that there might be a connection between PDI and mitochondrial factors that activate the intrinsic apoptosis cascade.

[0195] We then examined the localization of PDI over time in subcellular fractions and found that following induction, Q103 cells differentially accumulate PDI on mitochondria (2.8-fold over Q25 at 24 hours). Inhibiting PDI activity with 16F16 further increased mitochondrial accumulation of PDI in Q103 cells (5.3-fold over Q25 at 24 hours; Fig 23a-c). The latter result suggested that by inhibiting the enzymatic activity of PDI, we were allowing PDI levels to increase on mitochondria, surpassing a point that would otherwise induce apoptosis.

[0196] An additional consideration regarding the subcellular localization of PDI is that the ER contacts mitochondria through mitochondrial-associated-membranes (MAM) (78-80). To more clearly decipher the location of PDI associated with mitochondria, as either lumenal ER-associated or mitochondrial membrane- associated, protease-shaving experiments on purified ER and mitochondrial fractions were performed as follows.

[0197] Briefly, 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 MgCI₂, 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 x10⁵ cell eq. for mitochondria and ~5 x10⁵ 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. For protease shaving experiments, mitochondria and ER/microsomal fractions (5 x10⁵ cell eq) were treated with trypsin (300 μg/ml, 30 min, 25⁰C) followed by quenching with SBTI (1 mg/ml).

[0198] These experiments showed that mitochondrial PDI was protease sensitive, whereas lumenal ER PDI was protease protected. These results support previously published data showing a pool of PDI being localized to the outer mitochondrial membrane (55) (Fig. 23 d-g). Example 10

Rat Brain Slice HD assay

[0199] In this model, 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. Degeneration in medium spiny neurons (MSNs), a group of striatal neurons most affected in HD (81) 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.

[0200] Degeneration of MSNs in brain-slice explants was induced by biolistic transfection of htt constructs based on previously published approaches (82). At postnatal day 10, brains were dissected from CD Sprague Dawley rats (Charles River) after euthanasia and sliced into 250 micrometer coronal sections containing striatum using a tissue microtome (Vibratome). All animal experiments were done in accordance with the Institutional Animal Care and Use Committee and Duke University Medical Center Animal Guidelines.

[0201] Brain slices were plated onto serum-supplemented culture medium and maintained at 32⁰C under 5% CO₂ 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) were coated onto 1.6 micron elemental gold particles and delivered to the brain-slice explants using a biolistic device (Helios Gene Gun, Bio- Rad). 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

within the striatum and on their characteristic morphology using fluorescent

stereomicroscopes (Leica). Those MSNs that expressed bright, even YFP fluorescence and showed two or more dendrites with continuous YFP labeling at least two cell body diameters in length were scored as healthy. All experiments were performed and data analyzed in a blinded study.

[0202] The results of the experiments show that BBC7E8 and 16F16 were able to rescue the toxicity of Htt-Q73-CFP in a dose-dependent manner. (Fig. 26).

Example 11

Knock down PDI using siRNAs in PC12 cells and test for rescue of Q103- induced apoptosis.

[0203] We will test whether RNAi-mediated reduction in PDI mRNA protects from Q103-induced apoptosis. First, we will determine the extent to which our PC12 cell clones can be transfected effectively with siRNA reagents. Towards this end, we will compare several lipid transfection reagents (Oligofectamine, Lipofectamine, Fugene etc.) and nucleofection (Amaxa) by measuring knockdown of PDI by western blot and qPCR. One concern with measuring PDI knockdown by western blot is that PDI is a fairly stable protein; thus, knockdown of PDI mRNA will not have an immediate effect on PDI protein level. We may need to re-transfect several times and allow the cells to divide multiple times before we see depletion of PDI protein.

However, this disadvantage may turn out to be an advantage — if complete

elimination of PDI is a cell-lethal or cell-stressing event, then partial knockdown caused by virtue of the long PDI protein half-life may be protective without causing cell death. In any case, this issue will be examined by varying the time course of knockdown and testing the effect on PDI depletion.

[0204] Second, if the transfection efficiency is low, we will be unlikely to observe rescue from Q103-induced apoptosis because, while 100% of cells will have Q103, only a small fraction will receive the siRNA. Thus, we may need to measure the effect of the siRNA on viability only in the transfected cells. To do this, we will co- transfect a luciferase reporter plasmid with a constitutive promoter. Such a strategy has been used recently to measure the effect of transfected cDNAs on cell viability (57).

[0205] Third, we will control for off-target effects of the siRNAs that may yield a false-positive result. To control for this possibility, we will create a mutant PDI cDNA that cannot be knocked down by our siRNA, and test whether this prevents the rescue by the siRNA. In addition, we will test the effect of the siRNA on several other PDI family members, and we will use more than one siRNA, with the assumption that off-target effects of each siRNA are unlikely to be the same.

[0206] Fourth, we will control for the effect of transfection itself. To do this, we will use a scrambled siRNA that does not target PDI and confirm that at the same concentration this siRNA does not affect PDI protein or mRNA levels.

[0207] In our original pull down assay, we identified two different PDI isoforms

(PDIA1 and PDIA3), demonstrating that both of these isoforms were purified by our affinity reagent. We confirmed the presence of a PDI isoform in the purified material using an-anti-PDI antibody in a western blot; however, the isoform specificity of this antibody is not known. When we purchased bovine PDI and observed its MOMP- inducing activity, we were not able to determine the isoform of PDI provided by the manufacturer. We will, however, be able to resolve this particular point by sequencing this commercial bovine PDI by MALDI-TOF mass spectrometry, which is underway. Currently it is unclear whether the relevant PDI isoform is PDIA1 or PDIA3, or whether inhibition of both isoforms is needed. We will resolve this issue by testing the effects of siRNAs specific to PDIA1 and PDIA3, alone and in combination.

[0208] In summary, we will use a variety of methods to test whether knockdown of PDI isoforms by siRNAs in PC 12 protects from Q103-induced apoptosis. Using these methods, we expect to confirm that PDI is a relevant target of our PDI inhibitors, at least in the PC12 model.

Example 12

Creation of a lentiviral shRNA construct for knocking down PDI in PC12 cells and rat brain slice model.

[0209] We will confirm the results of the siRNA knockdown in PC 12 cells using a lentiviral shRNA system. In the past, we have successfully used the pLKO.1 lentiviral shRNA system, which was the basis for the 150,000 shRNAs created by the RNAi Consortium co-founded by Dr. Brent Stockwell, a co-inventor of the present invention, based at the Broad Institute (28). We have knocked down KRAS, BRAF, NRAS, VDAC1, VDAC2, VDAC3 and many other genes using this system. The pLKO.1 lentiviral vector is effective for targeting both mouse and human shRNAs. Thus, although it has not been tested yet in rat cells, it is likely to be effective in this context. [0210] Accordingly, we will use the standard protocol created by the RNAi

Consortium to design 57-mer oligonucleotides targeting rat PDIA1 and PDIA3, which we will have synthesized by IDT, the supplier of oligonucleotides to the Consortium. We will then use the standard Consortium protocol, which we have implemented successfully to clone these 57-mers into the pLKO.1 vector, and confirm the resulting shRNA plasmids by sequencing and restriction digest (which is used as a quality control measure for overall plasmid seize and integrity). Thus, we will create pLKO.1 shRNA constructs targeting rat PDIA1 and PDIA3. We will synthesize five different constructs targeting each PDI isoform, generate virus, and use to infect PC 12 cells and test the effectiveness of each plasmid by qPCR and by western blot.

[0211] We will then test the effectiveness of each shRNA at suppressing

Q103-induced apoptosis in the PC12 HD model, using the controls and strategies described above for the siRNAs. In this way, we will define a lentiviral shRNA system for knocking down rat PDI, which will serve to both confirm the siRNA and small- molecule results, and to create a system transferable to Dr. Donald Lo's rat brain slice HD model.

[0212] Subsequently, we will test the effectiveness of these lentiviral shRNA constructs in the brain slice HD model. We will confirm that knockdown of PDI can be achieved by western blot and by immunofluorescence in this model, and test the effect on striatal cell loss induced by mutant huntingtin. The small molecule PDI inhibitors we have discovered are being tested in the rat brain slice HD model. (See, e.g., Example 10 and Figures 25A-C and 26) Together, we expect that the shRNA results, like the small molecule results, will confirm that inhibiting PDI can be protective in the brain slice model. Example 13

Test mutants of PDI that impair its enzymatic activity for their effects on the

MOMP-inducing activity of PDI

[0213] We will determine the extent to which the MOMP-inducing activity of

PDI can be dissociated from the reductase, oxidase and isomerase activity of PDI for

two reasons. First, this would confirm that one could identify compounds that block the MOMP-inducing activity of PDI without affecting the enzymatic activities. It is possible that potently inhibiting the enzymatic activities of PDI may induce an ER stress response and lead to cell dysfunction or cell death. Thus, an ideal compound would inhibit only the MOMP-inducing activity of PDI. Second, determining the dissociability of the MOMP and enzymatic activities would further define the mechanism by which PDI induces MOMP. If the enzymatic activities of PDI are required for its MOMP-inducing activity, then it is likely that PDI acts enzymatically on substrates on the outer mitochondrial membrane, reducing or isomerizing disulfides. On the other hand, if these two activities can be dissociated, then it is likely that PDI docks on the outer membrane through a protein-protein interaction, and induces MOMP through altered protein-protein interactions. These two scenarios would suggest different approaches to identifying the mechanism of PDI- induced MOMP. Specifically, in one case it would be necessary to identify PDI substrates (using expression cloning and sib selection, for example), whereas in the

other case, it would be necessary to identify PDI-interacting proteins (using two- hybrid or affinity purification methods, for example).

[0214] We will create a series of PDI enzymatic active site mutants that have already been described (44, 58-64) and test their ability to induce MOMP. Each mutant will be generated in an Invitrogen Gateway donor vector, so that it can subsequently be transferred into a bacterial expression vector, a mammalian expression vector or a lentiviral vector with equal facility using recombination. The proteins will be expressed in E. coli, using methods similar to those we have used in the past, for example using a His tag and Ni²⁺ chromatography. The proteins will be characterized by PAGE, MALDI-TOF and reactivity by western blot. Then the mutants will be tested in the MOMP assay. The mutants will also be cloned into a mammalian expression vector and tested for their ability to exacerbate or induce apoptosis in response to Q103 expression. By carrying out these experiments, we expect to dissociate the distinct activities of PDI.

Example 14

Measure the level and localization of PDI in mouse HD models

[0215] We will measure the level, activity and subcellular localization of PDI in discrete brain regions in the R6/2 and YAC128 HD mouse models. Our central hypothesis is that PDI plays a role in HD neurodegeneration. We will use two different mouse models of HD to confirm this. The R6/2 model is a widely used and convenient HD model that is commercially available (65, 66). R6/2 mice harbor exon 1 of huntingtin with an expanded CAG region (65, 66). Thus, this model is quite similar to the PC12 cell model we are using, which also contains exon 1 of huntingtin. In addition, pan-caspase inhibitors and cystamine have a modest protective effect in this model (67), although little overt apoptosis is observed.

[0216] We will also use the YAC HD model, which contains the full-length huntingtin transgene with 72Q (68). These mice show inclusions and cell loss in the striatum (68). To confirm that PDI is involved in these mouse models of HD, we will measure the level, activity and localization of PDI in striatum, cortex, hippocampus, cerebellum and total brain in these mice. To measure the level of protein, we will use a quantitative, two-color western blotting system used extensively in our lab (the LICOR Odyssey system). In this system, we use infrared dyes conjugated to two different secondary antibodies, e.g. one color for PDI and one color for a control protein (such as actin, alpha-tubulin or GAPDH). Because the signal intensity is linearly related to the amount of protein over a large dynamic range, we can obtain reliable quantification of protein levels using this assay. We will use this format to determine PDI levels in each brain region. It may be, for example, that over time expanded polyglutamine causes increased PDI expression, contributing to increased neuronal apoptosis. We have seen evidence of this in the PC 12 model (data not shown).

[0217] It is likely that PDI will be localized to mitochondria in brain regions that subsequently degenerate in response to mutant huntingtin expression. Thus, we will fractionate cytosol and mitochondria and measure the relative abundance of PDI in each subcellular localization, along with several control proteins to confirm the effectiveness and reliability of our separation. This will allow us to determine if PDI and ERp57 are re-localized to mitochondria in response to mutant huntingtin expression in specific brain regions, as we have seen in PC12 cells. In all of these experiments using HD mice, we will perform the experiment in parallel in wild-type mice to determine the dependence of any finding on mutant huntingtin expression. Example 15

Defining the molecular mechanism of small molecule inhibition of PDIs

[0218] In order to understand how the compounds of the present invention inhibit PDIs, and to confirm PDIs as the relevant protein targets, we will identify the binding site on PDI and ERp57 using mass spectrometry. We will make mutations in each protein that disrupts the interaction with the inhibitors and confirm that these inhibitor-resistant mutants prevent the ability of the inhibitors to suppress Q 103- induced apoptosis. We will generate a co-crystal structure of one or more of the inhibitors in complex with PDI and ERp57. We expect that these studies will define the mechanism of inhibition of PDI/ERp57 and validate PDI and/or ERp57 as the relevant target protein.

[0219] In order to gain a clearer picture of how the inhibitors of the present invention bind to and affect PDI and ERp57, we will incubate bovine PDI and ERp57 with SUP-2 to covalently label each protein. We will digest with several different proteases and submit the resulting sample for ES/LC-MS-MS analysis to identify peptide residues modified by SUP-2. We will thus use mass spectrometry to determine the site at which PDI and ERp57 are covalently linked to SUP-2. We know the other inhibitors (i.e., SUP-1 , -3, and -4) bind in an overlapping way because they all compete for binding of the alkyne analog of SUP-2. We will test for the competability of this binding with the other inhibitors to show that it is a specific binding site.

[0220] Once we identify the binding site, we will use the published x-ray structure of PDI to select mutations in the binding site that are likely to prevent binding of the inhibitors, but not affect PDI enzymatic activity. We will make each mutant in the rat PDI cDNA using overlap extension PCR, clone the mutant cDNA into a Gateway donor vector, and verify the clone by DNA sequencing. We will transfer the clones into a Gateway destination mammalian expression vector using recombination, reconfirm the sequence, and overexpress each mutant by transfection in Q 103 PC12 cells.

[0221] We will test whether SUP-1 , -2, -3, and -4, cystamine, and other PDI inhibitors identified according to the methods of the present invention can prevent Q103-induced apoptosis in the presence of each mutant. A mutant that cannot bind one or more of these inhibitors, but that can still induce apoptosis, should overcome the suppressor activity of the inhibitors. We will perform these experiments on PDI and ERp57 mutants individually and in combination, to test the notion that both PDI and ERp57 are needed for inducing apoptosis.

[0222] For these experiments, it will be important to have a high transfection efficiency, so that most of the cells are able to undergo apoptosis, even in the presence of inhibitors (i.e. most of the cells must express the binding-defective mutant). If we cannot achieve high transfection efficiency in these cells, we will transfer the mutant cDNA into a Gateway lentiviral vector (which we have used in the past) using recombination, and again confirm the identity of the clone by sequencing. We will then generate lentivirus, as we have done in the past, and infect the cells, rather than transfecting them. However, if again we do not succeed in getting a high infection efficiency, we will turn to use of a reporter vector for measuring viability. In this strategy, we will co-transfect or co-infect PC12 Q103 cells with a CMV-luciferase reporter plasmid and measure luciferase activity to determine viability only of the transfected cells. This strategy was recently used in a high-throughput genetic suppressor screen (57), and overcomes the problem of low transfection efficiency.

[0223] Once we identify one or more mutations in PDI/ERp57 that prevent cell death rescue with inhibitors, we will overexpress and purify each of these effective

mutants using a published protocol (69). In brief, we will clone the rat PDI/ERp57

cDNA (using PCR, restriction digests and sequencing to verify each clone) into the E. co// expression vector pET-15b (Novagen). Amino-terminally hexahistidine-tagged

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, HPLC₁ CD and PDI enzymatic activity.

Example 16

Defining the role of PDI in polyglutamine-induced apoptosis, and the effect of

Q103 on PDI.

[0224] We will test the effect of PDI and ERp57 on Bcl-2 family proteins and mitochondrial function in intact cells, in isolated mitochondria and in vitro. We will also measure the effect of Q103 expression on PDI and ERp57 function and localization in cells. These studies will define how PDI and ERp57 are affected by Q103 expression and the effects of these proteins on activation of the intrinsic apoptotic pathway.

[0225] PDI and ERp57 are related ER chaperone proteins that have not been implicated in apoptosis directly. Thus, 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. We expect that 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.

[0226] Not wishing to be bound by a particular theory, it is believed that Q103 aggregates cause the translocation of PDI and/or ERp57 into mitochondria, where they induce cytochrome c release. Our data to date demonstrate that PDI does indeed become localized to mitochondria upon Q103 expression. We will test whether PDI and ERp57 bind directly to Q103 aggregates by purifying aggregates on a filter trap, incubating with PDI or ERp57, washing and blotting for PDI/ERp57. We will also look for co-localization of PDI or ERp57 and Q103 aggregates in cells, at different time points after induction of Q103, and after treatment with inhibitors. We expect that these experiments will confirm that direct binding of PDI or ERp57 to Q103 aggregates activates PDI and/or ERp57 for migration into mitochondria.

[0227] Alternatively, it is possible that some other stimulus (either Q 103- aggregate-dependent or independent) activates PDI/ERp57 translocation into mitochondria. In this case, there may be a post-translational modification of PDI and/or ERp57 that causes one or both to migrate into mitochondria. To test this hypothesis, we will purify PDI and ERp57 from Q 103 and Q25 (as a control) cells using several methods (immunoprecipitation of native PDI/ERp57, overexpression of epitope-tagged PDI/ERp57, and purification with the SUP-2A affinity reagent) and compare the post-translational modifications of PDI and ERp57 in these different cell lines, with or without induction of Q103 or Q25. To detect post-translational modifications, we will use the nanospray ESI LC-MS/MS instrument.

[0228] We will also test the effect of the PDI inhibitors of the present invention on cytochrome c and SMAC release from mitochondria. Our data to date demonstrate that Q103 induction in PC12 cells causes both SMAC and cytochrome c release; therefore, we will confirm that these PDI inhibitor compounds act upstream of this mitochondrial permeability transition.

[0229] Not wishing to be bound by a particular theory, we believe that PDI and/or ERp57 localize to mitochondria and induce BCL independent MOMP (Figure 16C). To confirm this, we will test the ability of PDI and ERp57 to interact with Bax, Bak, bid, Bcl-2 and Bcl-xL using co-immunoprecipitation from Q103-induced cells and in purified mitochondria. In addition, we will test the ability of PDI and ERp57 to interact with other proteins localized to mitochondria that may participate in PDI driven MOMP. If we identify interactions between PDI or ERp57 and BcI family proteins or other mitochondrial proteins, we will map the interaction by making deletion and point mutations to define the region of PDI/ERp57 that interacts. These mutants will also confirm that a binding-defective PDI/ERp57 is not able to induce apoptosis in response to Q103 expression. This experiment will be performed in cells in which we have knocked down the endogenous PDI and/or ERp57.

[0230] Thus, we will determine how Q103 expression causes PDI and/or

ERp57 translocation to mitochondria, and what the effects of PDI and ERp57 are in mitochondria.

[0231] In summary, these results will make a case for a causal, mechanistic role for PDI and ERp57 in HD-associated neuronal loss and the resulting defects in motor and memory functions. This will define a novel pathway for inducing apoptosis in neuronal cells.

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[0233] Although illustrative embodiments of the present invention have been described herein, it should be understood that the invention is not limited to those described, and that various other changes or modifications may be made by one skilled in the art without departing from the scope or spirit of the invention.

Claims

WHAT IS CLAIMED IS:

1. A method for modulating neurodegeneration comprising administering to a patient in need thereof an effective amount of a compound that modulates protein disulfide isomerase (PDI)-induced cellular toxicity.

2. The method according to claim 1, wherein the compound is a PDI inhibitor.

3. The method according to claim 1, wherein the neurodegeneration is caused by a condition selected from the group consisting of a polyglutamine disease, a prion disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, and a disease characterized by misfolded protein-induced cell death.

4. The method according to claim 3, wherein the polyglutamine disease is selected from the group consisting of Huntington's Disease (HD), spinal and bulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA), and spino-cerebellar ataxia (SCA) types 1 , 2, 3, 6, 7, and 17.

5. The method according to claim 1 , wherein the neurodegeneration is caused by Huntington's disease (HD).

6. The method according to claim 3, wherein the prion disease is selected from the group consisting of Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt- Jakob Disease (vCJD), Gerstmann-Straussler-Scheinker Syndrome, Fatal Familial Insomnia, Bovine Spongiform Encephalopathy (BSE), Chronic Wasting Disease (CWD), Scrapie, Transmissible mink encephalopathy, Feline spongiform encephalopathy, and Ungulate spongiform encephalopathy.

7. The method according to claim 2, wherein a target of the PDI inhibitor is at least one PDI.

8. The method according to claim 7, wherein the at least one PDI is selected from the group consisting of PDIA1 , PDIA3 (ERp57), and combinations thereof.

9. The method according to claim 2, wherein the PDI-inhibitor is selected from the group consisting of SUP-1 , SUP-2, SUP-3, SUP-4, cystamine, analogs, enantiomers, optical isomers, diastereomers, N-oxides, crystalline forms, hydrates, pharmaceutically acceptable salts, and combinations thereof.

10. The method according to claim 2, wherein the PDI-inhibitor is selected from the group consisting of compound C13 (BBC7M13), compound D13 (BBC7E8), compound J15, compound F14, securinine, analogs, enantiomers, optical isomers, diastereomers, N-oxides, crystalline forms, hydrates, pharmaceutically acceptable salts, and combinations thereof.

11. The method according to any one of claims 1 or 2, wherein the compound is administered as part of a pharmaceutical composition.

12. A method of modulating neuronal apoptosis associated with a polyglutamine disease comprising administering to a patient in need thereof an effective amount of a compound that modulates protein disulfide isomerase (PDI)- induced cellular toxicity.

13. The method according to claim 12, wherein the compound is a PDI inhibitor.

14. The method according to claim 12, wherein the polyglutamine disease is selected from the group consisting of 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.

15. The method according to claim 14, wherein the polyglutamine disease is HD.

16. The method according to claim 13, wherein a target of the PDI inhibitor is at least one PDI.

17. The method according to claim 13, wherein a target of the PDI inhibitor is selected from the group consisting of PDIA1 , PDIA3 (ERp57), and combinations thereof.

18. The method according to claim 13, wherein the PDI-inhibitor is selected from the group consisting of SUP-1 , SUP-2, SUP-3, SUP-4, cystamine, analogs, enantiomers, optical isomers, diastereomers, N-oxides, crystalline forms, hydrates, pharmaceutically acceptable salts, and combinations thereof.

19. The method according to claim 13, wherein the PDI-inhibitor is selected from the group consisting of compound C13 (BBC7M13), compound D13 (BBC7E8), compound J15, compound F14, securinine, analogs, enantiomers, optical isomers, diastereomers, N-oxides, crystalline forms, hydrates, pharmaceutically acceptable salts, and combinations thereof.

20. The method according to any one of claims 12 or 13, wherein the compound is administered as part of a pharmaceutical composition.

21. The method according to claim 12, wherein the modulation is a decrease in neuronal apoptosis.

22. A method for modulating mutant-huntingtin-induced neuronal apoptosis comprising administering to a patient in need thereof an effective amount of a compound that modulates protein disulfide isomerase (PDI)-induced cellular toxicity.

23. The method according to claim 22, wherein the compound is a PDI inhibitor.

24. The method according to claim 23, wherein a target of the PDI inhibitor is at least one PDI.

25. The method according to claim 23, wherein a target of the PDI inhibitor is selected from the group consisting of PDIA1 , PDIA3 (ERp57), and combinations thereof.

26. The method according to claim 23, wherein the PDI-inhibitor is selected from the group consisting of SUP-1 , SUP-2, SUP-3, SUP-4, cystamine, analogs, enantiomers, optical isomers, diastereomers, N-oxides, crystalline forms, hydrates, pharmaceutically acceptable salts, and combinations thereof.

27. The method according to claim 23, wherein the PDI-inhibitor is selected from the group consisting of compound C13 (BBC7M13), compound D13 (BBC7E8), compound J15, compound F14, securinine, analogs, enantiomers, optical isomers, diastereomers, N-oxides, crystalline forms, hydrates, pharmaceutically acceptable salts, and combinations thereof.

28. The method according to any one of claims 22 or 23, wherein the compound is administered as part of a pharmaceutical composition.

29. A method for treating, preventing, or ameliorating the effects of Huntington's disease (HD) in a patient comprising administering to a patient in need thereof an amount of a compound that modulates protein disulfide isomerase (PDI)- induced cellular toxicity.

30. The method according to claim 29, wherein the compound is a PDI inhibitor.

31. The method according to claim 30, wherein the PDI-inhibitor is selected from the group consisting of SUP-1 , SUP-2, SUP-3, SUP-4, cystamine, analogs, enantiomers, optical isomers, diastereomers, N-oxides, crystalline forms, hydrates, pharmaceutically acceptable salts, and combinations thereof.

32. The method according to claim 30, wherein the PDI-inhibitor is selected from the group consisting of compound C13 (BBC7M13), compound D13 (BBC7E8), compound J15, compound F14, securinine, analogs, enantiomers, optical isomers, diastereomers, N-oxides, crystalline forms, hydrates, pharmaceutically acceptable salts, and combinations thereof.

33. The method according to any one of claims 29 or 30, wherein the compound is administered as part of a pharmaceutical composition.

34. A method for reducing or suppressing misfolded protein-induced cytotoxicity, which is associated with a neurodegenerative disease, the method comprising administering to a patient in need thereof an amount of a compound that is sufficient to reduce or suppress the misfolded protein-induced cytotoxicity.

35. The method according to claim 34, wherein the compound is a PDI inhibitor.

36. The method according to claim 34, wherein the neurodegenerative disease is selected from the group consisting of a polyglutamine disease, a prion disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, and Alzheimer's disease.

37. The method according to claim 36, wherein the polyglutamine disease is selected from the group consisting of Huntington's Disease (HD), spinal and bulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA), and spino-cerebellar ataxia (SCA) types 1 , 2, 3, 6, 7, and 17.

38. The method according to claim 34, wherein the neurodegenerative disease is Huntington's disease (HD).

39. The method according to claim 36, wherein the prion disease is selected from the group consisting of Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease (vCJD), Gerstmann-Straussler-Scheinker Syndrome, Fatal Familial Insomnia, Bovine Spongiform Encephalopathy (BSE), Chronic Wasting Disease (CWD), Scrapie, Transmissible mink encephalopathy, Feline spongiform encephalopathy, and Ungulate spongiform encephalopathy.

40. The method according to claim 31 , wherein the PDI-inhibitor is selected from the group consisting of SUP-1 , SUP-2, SUP-3, SUP-4, cystamine, analogs, enantiomers, optical isomers, diastereomers, N-oxides, crystalline forms, hydrates, pharmaceutically acceptable salts, and combinations thereof.

41. The method according to claim 35, wherein the PDI-inhibitor is selected from the group consisting of compound C13 (BBC7M13), compound D13 (BBC7E8), compound J15, compound F14, securinine, analogs, enantiomers, optical isomers, diastereomers, N-oxides, crystalline forms, hydrates, pharmaceutically acceptable salts, and combinations thereof.

42. The method according to any one of claims 34 or 35, wherein the compound is administered as part of a pharmaceutical composition.

43. A method of modulating caspase activation in a cell comprising contacting the cell with a caspase-modulating amount of a compound that modulates protein disulfide isomerase (PDI)-induced cellular toxicity.

44. The method according to claim 43, wherein the compound is a PDI inhibitor.

45. The method according to claim 43, wherein the cell is a neuron.

46. The method according to claim 44, wherein the PDI-inhibitor is selected from the group consisting of SUP-1 , SUP-2, SUP-3, SUP-4, cystamine, analogs, enantiomers, optical isomers, diastereomers, N-oxides, crystalline forms, hydrates, pharmaceutically acceptable salts, and combinations thereof.

47. The method according to claim 44, wherein the PDI-inhibitor is selected from the group consisting of compound C13 (BBC7M13), compound D13 (BBC7E8), compound J15, compound F14, securinine, analogs, enantiomers, optical isomers, diastereomers, N-oxides, crystalline forms, hydrates, pharmaceutically acceptable salts, and combinations thereof.

48. The method according to claim 43, wherein the modulation is a decrease in caspase activation.

49. The method according to claim 43, wherein the caspase is selected from the group consisting of caspase-3, caspase-6, caspase-7, caspase-9, and combinations thereof.

50. A method of modulating mitochondrial outer membrane permeabilization (MOMP) in a cell comprising contacting the cell with a MOMP- modulating amount of a compound that modulates protein disulfide isomerase (PDI)- induced cellular toxicity.

51. The method according to claim 50, wherein the compound is a PDI inhibitor.

52. The method according to claim 50, wherein the cell is a neuron.

53. The method according to claim 50, wherein 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.

54. The method according to claim 50, wherein the MOMP-modulating amount of the compound is sufficient to prevent or reduce caspase activation.

55. The method according to claim 51 , wherein the PDI-inhibitor is selected from the group consisting of SUP-1 , SUP-2, SUP-3, SUP-4, cystamine, analogs, enantiomers, optical isomers, diastereomers, N-oxides, crystalline forms, hydrates, pharmaceutically acceptable salts, and combinations thereof.

56. The method according to claim 51 , wherein the PDI-inhibitor is selected from the group consisting of compound C13 (BBC7M13), compound D13 (BBC7E8), compound J15, compound F14, securinine, analogs, enantiomers, optical isomers, diastereomers, N-oxides, crystalline forms, hydrates, pharmaceutically acceptable salts, and combinations thereof.

57. The method according to claim 50, wherein the modulation is a decrease in MOMP.

58. A method for identifying a target of a candidate compound identified in an assay as modulating a cellular phenotype of interest comprising:

a. derivatizing a candidate compound to make it Huisgen cycloaddition chemistry (HCC) compatible;

b. 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;

c. carrying out HCC to covalently attach a detectable label to the derivatized candidate compound; and

d. determining whether the labeled derivatized candidate compound is bound to the target.

59. The method according to claim 58, wherein the derivatizing step comprises introducing 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.

60. The method according to claim 58, wherein the derivatizing step comprises introducing 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.

61. The method according to claim 59, wherein 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.

62. The method according to claim 60, wherein 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.

63. The method according to claim 58 further comprising identifying the target, if it is present, to which the derivatized candidate compound bound.

64. The method according to claim 63, wherein the target modulates protein disulfide isomerase (PDI)-induced cellular toxicity.

65. The method according to claim 64, wherein the candidate compound is a PDI inhibitor.

66. The method according to claim 65, wherein the candidate compound is selected from the group consisting of SUP-1, SUP-2, SUP-3, SUP-4, cystamine, analogs, enantiomers, optical isomers, diastereomers, N-oxides, crystalline forms, hydrates, pharmaceutically acceptable salts, and combinations thereof.

67. The method according to claim 65, wherein the candidate compound is selected from the group consisting of compound C13 (BBC7M13), compound D13 (BBC7E8), compound J15, compound F14, securinine, analogs, enantiomers, optical isomers, diastereomers, N-oxides, crystalline forms, hydrates, pharmaceutically acceptable salts, and combinations thereof.

68. The method according to claim 58, wherein the detectable label is selected from the group consisting of radioactive labels, fluorescent labels, chromogenic labels, enzymatic labels, and a target antigen for a labeled antibody.

69. The method according to claim 58, wherein steps b. and c. are carried out in vivo.

70. The method according to claim 58, wherein steps b. and c. are carried out in vitro.

71. The method according to claim 58, wherein the target is bound to a solid substrate.

72. The method according to claim 58, which is a high-throughput screening assay.

73. The method according to claim 58, wherein the modulated cellular phenotype is neuronal apoptosis.

74. The method according to claim 73, wherein the neuronal apoptosis is induced by a neurodegenerative disease selected from the group consisting of a polyglutamine disease, a prion disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, and a disease characterized by misfolded protein-induced cell death.

75. The method according to claim 74, wherein the polyglutamine disease is selected from the group consisting of 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.

76. The method according to claim 75, wherein the polyglutamine disease is Huntington's disease (HD).

77. The method according to claim 74, wherein the prion disease is selected from the group consisting of Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease (vCJD), Gerstmann-Straussler-Scheinker Syndrome, Fatal Familial Insomnia, Bovine Spongiform Encephalopathy (BSE), Chronic Wasting Disease (CWD), Scrapie, Transmissible mink encephalopathy, Feline spongiform encephalopathy, and Ungulate spongiform encephalopathy.

78. The method according to claim 58, wherein the sample comprises a cell or a cellular extract.

79. A compound for treating, preventing, or ameliorating the effects of a neurodegenerative disease identified according to the method of claim 58.

80. A pharmaceutical composition comprising a compound according to claim 79.

I l l

81. A method of identifying a compound useful for the treatment of a neurodegenerative disease comprising 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.

82. A method of identifying a compound useful for the treatment of a

neurodegenerative disease comprising 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.

83. A method according to any one of claims 81 or 82, wherein the determining step comprises carrying out virtual high throughput screening to identify compounds that bind to or inhibit at least one PDI.

84. A method for identifying candidate compounds for use in treating a neurodegenerative disease comprising:

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%, wherein

n . _. (Average Induced Value) - (Average Background Value) %Rescue = -^ - '-÷- ≡ Ξ > _x 100;

(Average Uninduced Value) -(Average Background Value)

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, and

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% in the cell viability assay as candidate compounds for use in treating a neurodegenerative disorder.

85. The method according to claim 84, wherein 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.

86. The method according to claim 84, wherein the cell viability assay is a PC12 neuronal assay.

87. The method according to claim 84 further comprising carrying out, on the candidate compounds selected from step d., (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.

88. The method according to claim 87, wherein the direct PDI binding assay comprises:

a. derivatizing a candidate compound selected from step d. to make it 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.

89. The method according to any one of claims 84, 86, or 87, wherein at least one of the assays is a high throughput screen.