WO2011056222A1 - Procédés de traitement de troubles associés à une agrégation protéique - Google Patents

Procédés de traitement de troubles associés à une agrégation protéique Download PDF

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WO2011056222A1
WO2011056222A1 PCT/US2010/002898 US2010002898W WO2011056222A1 WO 2011056222 A1 WO2011056222 A1 WO 2011056222A1 US 2010002898 W US2010002898 W US 2010002898W WO 2011056222 A1 WO2011056222 A1 WO 2011056222A1
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protein
disease
atz
promoter
caenorhabditis elegans
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PCT/US2010/002898
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English (en)
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Stephen C. Pak
David Hirsch Perlmutter
Gary A. Silverman
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University Of Pittsburgh - Of The Commonwealth System Of Higher Education
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Priority claimed from US12/881,976 external-priority patent/US8809617B2/en
Application filed by University Of Pittsburgh - Of The Commonwealth System Of Higher Education filed Critical University Of Pittsburgh - Of The Commonwealth System Of Higher Education
Publication of WO2011056222A1 publication Critical patent/WO2011056222A1/fr
Priority to US13/463,638 priority Critical patent/US9072772B2/en
Priority to US14/728,619 priority patent/US9452171B2/en
Priority to US15/247,518 priority patent/US9820990B2/en

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Definitions

  • the present invention relates to methods of treating clinical disorders associated with protein aggregation comprising administering, to a subject, an effective amount of an anti- protein aggregate ("APA") compound selected from the group consisting of pimozide, fluphenazine, tamoxifen, taxol, cantharidin, cantharidic acid, salts thereof, and their structurally related compounds.
  • APA anti- protein aggregate
  • disorders are associated with the presence of protein aggregates. These disorders may alternatively be referred to as disorders of protein polymerization or of protein misfolding, but are collectively referred to herein as disorders of protein aggregation.
  • AT 1 -antitrypsin
  • the classical form of a 1 -antitrypsin ("AT") deficiency is an autosomal co-dominant disorder that affects approximately 1 in 2000 live births (25). It is caused by a point mutation that alters the folding of an abundant liver-derived plasma glycoprotein during biogenesis and also renders it prone to aggregation (43). In addition to the formation of insoluble aggregates in the ER of liver cells, there is an 85-90% reduction in circulating levels of AT, the pre-dominant physiologic inhibitor of neutrophil elastase. Individuals who are homozygous for the mutant allele are susceptible to premature development of chronic obstructive pulmonary disease. Pulmonary involvement is believed to be caused by a loss-of-function mechanism, as lack of AT in the lung permits elastase to slowly destroy the pulmonary connective tissue matrix (44).
  • AT deficiency is the most common genetic cause of liver disease in children and also causes liver disease and hepatocellular carcinoma in adults. In contrast to pulmonary involvement, liver inflammation and carcinogenesis are believed to be caused by a gain-of-toxic function mechanism. This is most clearly demonstrated by introducing the mutant human ATZ allele as transgene into genetically engineered mice (45, 1 1). Insoluble aggregates in hepatocytes, hepatic inflammation and carcinogenesis evolve even though the endogenous anti-elastases of the transgenic mouse are intact.
  • AD Alzheimer's Disease
  • AD is the most common form of age-dependent neurodegeneration. Most cases are recognized by the sporadic onset of dementia during the seventh decade of life while the less common, mutation-linked familial cases cause dementia that is recognized by the fifth decade.
  • AD is associated with the accumulation of
  • aggregation-prone peptides in the brain especially amyloid- ⁇ (" ⁇ ") peptides, but hyperphosphorylated tau proteins also contribute to the tangles and plaques that constitute the histological hallmarks of the disease.
  • ⁇ amyloid- ⁇
  • AD Alzheimer's disease
  • AD is thought to be caused by a gain-of-toxic function mechanism that is triggered by the accumulation of aggregated ⁇ and tau and worsened by aging (36).
  • Recent studies have shown that the prevalence of autophagosomes is increased in dystrophic neurons of the AD brain, a finding that is recapitulated in mouse models of the disease (37).
  • Most of the evidence suggests that autophagy plays a role in disposal of aggregated proteins that might have toxic effects on neurons (38, 39).
  • the neuropathological effects of ⁇ in a mouse model of AD were ameliorated by enhancing autophagy via overexpression of the autophagy protein beclin 1 (39).
  • Parkinson's Disease is associated with the presence of protein aggregates in the form of "Lewy Bodies", which contain a number of proteins including one or more of alpha-synuclein, ubiquitin,
  • Huntington's Chorea is associated with aggregates of huntingtin protein containing a mutation that results in long tracts of polyglutamine ("polyQ") which result in improper protein processing and aggregate formation.
  • HCS high-content screening
  • HCS using cell-based assays facilitate the elimination of compounds that are directly cytotoxic, they are unable to identify those that lose their desired therapeutic effect in vivo, or demonstrate deleterious side effects on complex developmental or physiological processes, such as cellular migration or synaptic transmission, respectively.
  • forward chemical genetic screens i.e., phenotype-directed
  • live animals that model human disease phenotypes might serve as suitable alternatives to target-directed reverse chemical screens (66).
  • Drug screens using live organisms provide several distinct advantages over molecular- or cell-based assays and include: 1) the assessment of ADMET characteristics at the earliest stages of the drug discovery process, 2) the identification of leads without detailed knowledge of specific disease-related targets or molecular pathways and 3) the avoidance of ascertainment biases associated with targeting pathways or molecules whose involvement may prove to be tangential to the disease process.
  • the assimilation of live animals into drug screening protocols presents logistical challenges. These barriers include labor- and cost- intensive development of suitable disease phenotypes; screening protocols that are low-throughput and unamenable to statistically robust HCS-like formats; and the prohibitive consumption of compound libraries.
  • C. elegans in particular, should be an ideal candidate for live animal HCS campaigns, as their tissues are transparent at all developmental stages, the use of fluorescent probes and tissue-specific fluorescent transgenic markers to study physiological processes in vivo are well established, fundamental cell biological processes are highly conserved across species, and aspects of mammalian diseases can be successfully modeled in these invertebrates (reviewed in 76). Nonetheless, experimental variables that affect high-quality HCS protocols, such as sample preparation, assay strategy, and image acquisition, have yet to be optimized for any organism (77).
  • the present invention relates to methods of treatment of clinical disorders associated with protein aggregation comprising administering, to a subject in need of such treatment, an effective amount of an anti- protein polymer ("APA") compound selected from compounds listed in TABLES 4 and 5 herein, and particularly selected from the group consisting of pimozide, fluphenazine (e.g., fluphenazine hydrochloride), tamoxifen (e.g., tamoxifen citrate), taxol, cantharidin, cantharidic acid, salts thereof and their structurally related compounds.
  • APA anti- protein polymer
  • treatment with one or more of these APA compounds may be used to ameliorate the symptoms and signs of AT deficiency as well as other disorders marked by protein aggregation, including, but not limited to, Alzheimer's Disease, Parkinson's Disease, and Huntington's Disease.
  • the present invention relates to methods and compositions for high content drug screening in C. elegans which may be used to identify compounds that treat disorders associated with protein
  • the assay system of the invention utilizes an all-liquid work-flow strategy that essentially eliminates a major bottleneck in the screening process and fully exploits the advantages of C. elegans as a platform for in vivo high-content and high-throughput pre-clinical drug discovery campaigns. According to the invention, adapting an automated system that streamlines the image acquisition and data analysis
  • FIGURE 1 A-D. Overlap-extension PCR strategy for introducing introns.
  • synthetic introns ⁇ 70 bp
  • Oligonucleotides primers flanked with synthetic intronic sequences (colored) were used to amplify small (-250 bp) fragments of the ATZ cDNA.
  • B PCR fragments were gel purified and adjacent fragments were incubated for 45 s at 95 °C and 68 °C for 10 cycles in the presence of dNTPs and Pfu DNA polymerase to promote annealing and extension of complementary strands.
  • C Agarose gel showing individual PCR fragments (lanes 1-5). Overlap extension products 1+2 (lane 6), 4+5 (lane 7), [l+2]+3 (lane 8), [l+2+3]+[4+5] (lane 9).
  • D ATZ with 4 synthetic introns (colored).
  • FIGURE. 2A-E DIC (left) and fluorescence (right) photomicrographs of transgenic animals. For orientation in each paired set of figures, white arrowheads indicate corresponding basal surfaces of intestinal cells.
  • A Adult worm harboring Pnhx-2::GFP shows diffuse intracellular GFP expression within the intestinal cells.
  • B and
  • C Pnhx-2::sGFP and Pnhx-2::sGFP::ATM transgenic animals secrete GFP into the extracellular pseudocoelomic space (asterisks). Note only background autofluorescent granules (lysosomes) in intestinal cells, but no cytoplasmic GFP at even high integration.
  • Pnhx-2::sGFP::ATZ animals accumulate ATZ "globules" within intestinal cell cytoplasm (arrowheads with dot) and fail to secrete detectable amounts of fusion protein into the pseudocoelomic space.
  • E A second larval (L2) stage, Pnhx-2::sGFP::ATZ animal showing prominent intracellular inclusions of sGFP::ATZ (red arrowheads) that are comparable to those of the adult. In some animals, a second type of granule was seen occasionally (arrowheads with "x"). The subcellular location of this granule has not yet been identified.
  • FIGURE 3A-B Immunoblot of worm lysates after SDS-PAGE.
  • Immunoblots were probed with anti-human AT (A) and anti-GFP (B) antibodies.
  • Lane 1 N2; lane 2, Pnhx-2::sGFP; lane 3, Pnhx-2::sGFP::ATM; lane 4, Pnhx- 2::sGFP::ATZ; lane 5, purified plasma AT standard.
  • FIGURE 4A-D Electron micrographs of ATZ globule-containing intestinal cells of transgenic worms. Cross and transverse sections of early larval stage worms expressing sGFP::ATM (A) or sGFP::ATZ (B) transgenes. Arrowheads point to large intracellular inclusions similar to those found in ATZ liver. A close-up of another ATZ inclusion (C). A higher magnification of the boxed area is shown in (D). Arrowheads point to ribosomes of the dilated ER. Int, intestinal lumen.
  • FIGURE 5 Array ScanVTi screen shot interface. Bright field and flourescent image of sGFP::ATZ animals in one of four fields from well Dl .
  • FIGURE 6 Detection of worm boundaries and ATZ aggregates.
  • Objects blue outline (there are two bright lines at the outer boundaries of each worm; the outer one is red (e.g. marked by an arrowhead with a dot) and the inner one is blue (e.g. marked by an arrowhead with an "x")) and spots (red dots, e.g. see dots indicated by white arrowheads) selected by the ArrayScanVTi algorithms. Only these selected elements were used for subsequent data analysis (see Figs. 7 and 8).
  • FIGURE 7 Detection of fluorescent "aggregates" in N2, sGFP::ATM and sGFP::ATZ worms. Top images are merged bright field and GFP fluorescence images (top) were analyzed quantitatively for size and number of fluorescent
  • FIGURE 8A-C Comparison of aggregate number (A), total aggregate area (B) and average aggregate size (C) in N2, sGFP::ATM and sGFP::ATZ animals. Data were derived from those animals shown in FIGURE 7.
  • FIGURE 9 New improved transgenic line with mCherrry expression (marked by a white arrowhead) in the head region.
  • FIGURE 10A-E Global strategy for high-throughput screening of lead compounds that alter ATZ aggregation.
  • A Compounds, media and E. coli are dispensed into 96- well plates using a high-throughput robotic liquid handler.
  • B Worms are sorted based on size and GFP fluorescence and automatically transferred into 96-well plates.
  • C An automated high-throughput microscopic imaging system captures images and converts them into numerical data that identifies changes in aggregate number, size and intensity.
  • D Lead compounds are identified and further scrutinized to confirm positives and eliminate nuisance compounds.
  • E New compounds are synthesized to develop potential therapeutics.
  • FIGURE 1 B-score analysis of LOPAC compounds.
  • FIGURE 12A-E Animal (object) detection using the ArrayScan V TI . Thirty- five adult or mixed stage animals were dispensed into 384-well plates, imaged and analyzed using the ArrayScan V TI and SpotDetector BioApplication.
  • A A brightfield image of adult animals.
  • B SpotDetector correctly identified all the worms in the field as indicated by the blue outline (e.g. marked with white arrowheads, approximately >30 worms).
  • C A representative brightfield image of a well containing 36 animals with a predetermined percentage (0, 25, 50, 75 and 100%) of adults sorted into a 384-well plate.
  • (D) SpotDetector was optimized to identify large (L4 and adult stage) worms (blue outline, e.g. shown by white arrowheads) and exclude smaller (LI , L2 and L3 stage) worms (orange outline, e.g. shown by arrowheads with dots).
  • FIGURE 13 A-Q Automated detection and quantification of cells, tissues, subcellular protein aggregates or autophagy in individual animals.
  • A- J The SpotDetector BioApplication was used to identify and quantitate different types of transgene expression (left of panels) in adult animals.
  • the brightfield channel (left panels) was used to discriminate between complete adult animals (outlined in blue, e.g. indicated by white arrowheads) and debris or incomplete animals (outlined in orange, e.g. indicated by white arrowheads with dots), while a fluorescence channel (colored overlays in right panels) was used to detect different types of fluorescently tagged transgenes in correctly identified objects.
  • K-N Fluorescence images of well- fed (K) and starved (M) animals expressing the autophagy marker, mCherry::LGG-l .
  • mCherry::LGG-l was diffusely cytoplasmic (K).
  • induction of autophagy by starvation leads to a punctate fluorescence pattern within intestinal cells, as LGG-1 is incorporated in to autophagosomes (M).
  • L, N Higher magnification of the boxed areas in k and m, respectively.
  • O-Q The different types of transgene expression were quantified by spot count (O), spot area (P) or spot intensity (Q) per animal.
  • FIGURE 14A-N Identification of live cells or dead animals using C elegans.
  • FIGURE 15A-C Identification of animals in a mixed population using a fluorescent head- marker. Thirty-six animals expressing the pharyngeal marker, P m y 0 - 2 mRFP were sorted into wells of 384-well plate. The wells contained different percentages (0-100%) of L4/young, and the SpotDetector Bio Application was optimized to select this group and reject younger animals (LI , L2and L3 stages).
  • A A brightfield-mRFP composite image of transgenic worms at different stages expressing P myo-2 mRFP.
  • B A SpotDetector image showing the ability to differentiate adults (magenta overlay, e.g.
  • FIGURE 16A-K High-content analysis of transgenic animals expressing the wild-type (ATM) and mutant (ATZ) forms of human a 1 -antitrypsin (AT) fused to GFP. Thirty-five young adult animals were sorted into wells of a 385- well plate and imaged using the ArrayScan V TI .
  • A, D Brightfield images of sGFP::ATM and sGFP: :ATZ expressing transgenic animals, respectively.
  • B, E SpotDetector images of fluorescent red-heads for corresponding transgenic lines pictured in a and d, respectively.
  • FIGURE 17A-K LOPAC library screen.
  • A Total spot area per animal (object),
  • B z-scores and
  • C B-scores from a representative screen assaying the effects of 1280 LOPAC compounds on sGFP::ATZ accumulation in transgenic animals.
  • the x-axis represents the molecular identification (Mol ID) number of the compound.
  • Mol ID molecular identification
  • Known autofluorescent compounds were excluded from the plot. Selected compounds, based on rank-order were analyzed for dose-dependent responses.
  • Error bars represent SEM. Number of animals used was 140 for each compound concentration and 520 for the DMSO control. Significance was determined using an unpaired Student's Mest. Asterisks indicate values that differed significantly from animals treated with DMSO. *P ⁇ 0.01 and **P ⁇ 0.001.
  • FIGURE 18A-E Induction of autophagy by hit compounds. Images of transgenic animals expressing Pnhx-2mCherry::lgg-l treated with various compounds are shown. Images were acquired using a Nikon instruments TiEclipse widefield light microscope fitted with a 20x Plan Apochromat objective. Images were then deconvolved using Volocity (Perkin Elmer, v 5.3.2). Deconvolved z planes were then merged to a single plane. Well-fed animals treated with DMSO (A) show a diffuse mCherry expression throughout the intestine. In contrast, animals treated with Cantharidin (B), Fluphenazine (C) and Pimozide (D) show a markedly punctate distribution pattern indicative of increased autophagic activity. Starved (E) animals are included as a positive control for autophagy. Scale bar, 50 ⁇ .
  • FIGURE 19 Dose response effect of pimozide in HCS assay.
  • FIGURE 20 Dose response effect of fluphenazine in HCS assay.
  • FIGURE 21 Dose response effect of fluphenazine in HCS assay.
  • FIGURE 22 HTO/Z cells were incubated for 24 hrs in the absence or presence of fluphenazine.
  • Treatment agents which may be used according to the invention include the APA compounds listed in TABLES 4 and 5 herein, and particularly pimozide, fluphenazine, tamoxifen, taxol, cantharidin, cantharidic acid, their salts (where applicable) and structurally related compounds.
  • Pimozide is l-[l-[4,4-bis(4-fluorophenyl)butyl]-4-piperidyl]-l ,3- dihydrobenzoimidazol-2-one (IUPAC name) and has the structural formula:
  • Compounds structurally related to pimozide include other compounds of the diphenulbutyl piperidine class as well as the compouns clopimozide, penfluridol, piamperone and R28935.
  • Fluphenzine also known, for example, as “Prolixin®” and “Permitil®” is a drug of the piperazine subclass of phenothiazines, and specifically is 4-[3-[2-(Trifluoromethyl)phenothiazin-10-yl] propyl]- 1 -piperazineethanol, frequently available as dihydrochloride having molecular formula C 2 2H2 8 F3N 3 OS 2 HCl.
  • the molecular structure of the dihydrochloride form is:
  • fluphenazine is also commercially available as fluphenazine decanoate.
  • Complexes of fluphenazine with compounds other than hydrochloride and decanoate fall within the scope of the invention.
  • Compounds structurally related to fluphenazine include other members of the piperazine subclass of phenothiazines and include, but are not limited to, 2- (trifluoromethyl)-10-[3-(diethanolamino)-2-hydroxypropyl] phenothiazine, fluphenazine-4-chlorophenoxy-isobutyrate ester; compounds set forth in (102); 2- nitro-10-[3-[4-(2-hydroxyethyl)-l-piperazinyl]propyl] phenothiazine; 2-(2,2- dicyanoethenyl)-10-[3-[4-(2-hydroxyethyl)-l -piperazinyl [propyl] phenothiazine; 2- (2-nitro-ethenyl)-10-[3-[4-(2-hydroxyethyl)-l -piperazinyl [propyl] phenothiazine and 10-[3-[4-(2-hydroxye
  • Tamoxifen e.g., tamoxifen citrate, sold as Nolvadex® and Istubal®
  • Tamoxifen citrate sold as Nolvadex® and Istubal®
  • tamoxifen citrate sold as Nolvadex® and Istubal®
  • tamoxifen citrate sold as Nolvadex® and Istubal®
  • Its molecular formula is C 2 H 29 NO (and if citrate is present, add C 6 H 8 0 7 ) and the structural formula of tamoxifen is:
  • Tamoxifen may optionally be administered in the form of a citrate salt or in combination with a compound other than citrate.
  • tamoxifen Compounds structurally related to tamoxifen include, but are not limited to, tamoxifen's metabolite, 4-hydroxy tamoxifen; (Z)-4-hydroxy-N- desmethyltamoxifen (endoxifen); a nido-carborane analog of tamoxifen, boroxifen; and compounds as described in (104).
  • tamoxifen's metabolite 4-hydroxy tamoxifen
  • Z -4-hydroxy-N- desmethyltamoxifen
  • endoxifen a nido-carborane analog of tamoxifen, boroxifen
  • Taxol also known by the generic term paclitaxel and the trade names of formulations Taxol® and Onxal®, is 5 ⁇ ,20- ⁇ -1 ,2 ⁇ ,4,7 ⁇ ,10 ⁇ ,13 ⁇ - hexahydroxytax- 1 1 -en-9-one 4,10-diacetate 2-benzoate 13-ester with (2R,3S)-N- benzoyl-3-phenylisoserine.
  • the chemical formula of taxol is C 47 H 5
  • Taxol Compounds structurally related to taxol include, but are not limited to, 2-debenzoyl-2-(m-azidobenzoyl) paclitaxel; 2-debenzoyl-2-(phenoxyacetyl) paclitaxel; 7- benzoyl paclitaxel; N-debenzoyl-N-(phenoxyacetyl) paclitaxel (see 106); 2'-deoxypaclitaxel; 2'-methoxypaclitaxel and paclitaxel 2' acetate (see 107).
  • Cantharidin is 2,6-Dimethyl-4,10-dioxatricyclo-[5.2.1.02,6]decane-
  • Cantharidic acid is 5'-3'2,3-dicarboxy-2,3-dimethyl- l ,4- epoxycyclohexane. Its chemcial formula is Ci 0 H
  • Compounds structurally related to cantharidin and cantharidic acid include, but are not limited to, norcanthiridin and analogs described in (108-1 1 1 ).
  • disorders of protein aggregation also sometimes referred to in the art as disorders of protein aggregation or accumulation
  • disorders of protein aggregation or accumulation include, but are not limited to, a 1 -antitrypsin deficiency, Alzheimer's Disease, Parkinson's Disease, Pick's Disease, corticobasal atrophy, multiple system atrophy, Lewy Body Disease, familial encephalopathy with neuroserpin inclusion bodies (FENIB), Huntington's Disease, amyloidosis (e.g., primary, secondary, familial, senile), prion-associated diseases (e.g.
  • Creuzfeld- Jacob disease mad cow's disease
  • protein aggregation resulting from ischemic or traumatic brain injury for example dementia pugilistica (chronic traumatic encephalopathy)), progressive supranuclear palsy, Lytico-Bodig disease (Parkinson dementia complex of Guam), ganglioma, subsacute sclerosing panencephalitis, certain forms of congenital diabetes, certain forms of retinitis pigmentosa, certain forms of long QT syndrome, hereditary hypofibrinogenemia, certain forms of osteogenesis imperfecta, certain forms of hereditary angioedema, Charcot-Marie-Tooth disease and Pelizaeus- Merzbacher leukodystrophy.
  • ischemic or traumatic brain injury for example dementia pugilistica (chronic traumatic encephalopathy)
  • Lytico-Bodig disease Parkinson dementia complex of Guam
  • ganglioma subsacute sclerosing panencephalitis
  • the present invention relates to methods of treating clinical disorders associated with protein aggregation comprising administering, to a subject in need of such treatment, an effective amount of one or more APA compound.
  • Suitable APA compounds are described above and are listed in TABLES 4 and 5 herein.
  • a subject in need of such treatment may be a human or a non-human subject, and may be suffering from a disorder associated with protein aggregation or be at risk of developing such a disorder due to age, family history, or exposure to a toxic agent.
  • An effective amount is an amount that (i) reduces one or more sign and/or symptom of the disorder; and/or (ii) inhibits progression of the disorder; and/or (iii) prolongs survival of the subject. It is this reduction in a sign and/or symptom, inhibition of progression, or prolongation of survival which constitutes treatment of the disorder.
  • Signs and symptoms of a disorder associated with protein aggregation depend upon the particular disorder and are known to the person skilled in the art.
  • one sign that may be "reduced” may be the accumulation of aggregated protein, in which either the rate of accumulation may be slowed or (but not necessarily) the amount of aggregated protein accumulated may stabilize or decrease.
  • signs or symptoms that may be reduced or otherwise ameliorated according to the invention include hepatitis, hepatic enlargement, hepatic fibrosis, hepatocarcinoma, impaired liver function, abdominal distension from ascites, jaundice, edema, enlarged spleen, hypersplenism, gastrointestinal bleeding, encephalopathy, renal failure, prolonged bleeding from injuries, shortness of breath, wheezing, cough, decreased serum oxygen, increased serum carbon dioxide, increased total lung capacity, decreased FEV1/FVC ratio, increased incidence of pulmonary infection, pulmonary infection, weight loss and fatigue.
  • signs or symptoms that may be reduced or otherwise ameliorated according to the invention include impairment of short term memory, impairment of abstract thinking, impairment of judgment, impairment of language skills, and mood changes.
  • the disorder is Parkinson's
  • tremor tremor
  • bradykinesia rigidity
  • impaired speech tremor
  • dementia tremor
  • signs or symptoms that may be reduced r otherwise ameliorated according to the invention include dementia and choreoform movements.
  • signs or symptoms that may be reduced or otherwise ameliorated according to the invention include thickening of the skin, rash, cardiomyopathy, congestive heart failure, cardiac arrhythmias and/or conduction defects, shortness of breath, fatigue, impaired renal function, hyothyroidism, anemia, bone damage/fracture, impaired liver function, impaired immunity, and glossitis.
  • signs or symptoms that may be reduced or otherwise ameliorated include dementia and choreoform movements.
  • the present invention provides for a method of decreasing the amount of aggregated protein in a cell comprising exposing the cell to an effective amount of one of more APA compound.
  • the cell may be a cell affected by a disorder of protein aggregation, as set forth above, for example, but not by way of limitation, a liver cell or a lung cell from a subject suffering from AT deficiency, a neuron from a subject suffering from Alzheimer's Disease, Parkinson's Disease, Huntington's disease, a prion disease, or a cell from a subject suffering from any of the other aforelisted disorders associated with protein aggregation.
  • the APA compound may be administered by any route of administration, including oral, intravenous, intramuscular, subcutaneous, intrathecal, intraperitorneal, intrahepatic, by inhalation, e.g., pulmonary inhalation, etc..
  • the present invention provides for a method of treating clinical disorders associated with protein aggregation comprising administering, to a subject in need of such treatment, an effective amount of pimozide or a structurally related compound.
  • pimozide may be administered at a total dose of between about 0.5 and 10 mg/day, or between about 0.5 and 1 mg/day, or between about 1 -2 mg/day, or between about 2 and 5 mg/day, or between about 2 and 8 mg/day, or less than 10 mg/day.
  • a compound that is structurally related to pimozide may be administered at an adjusted version of the foregoing doses, where the adjustment compensates for any difference in potency between pimozide and the structurally related compound.
  • the dose of pimozide or a structurally related compound administered produces a serum concentration or cerebrospinal fluid concentration of at least about 5 micromolar, or between about 5 and about 10 micromolar, or between about 10 and about 20 micromolar, or between about 20 and about 30 micromolar, or at least about 50 micromolar.
  • the dose of pimozide or a structurally related compound may be administered daily, about every other day, about twice a week, or about once a week.
  • the present invention provides for a method of treating clinical disorders associated with protein aggregation comprising administering, to a subject in need of such treatment, an effective amount of fluphenazine (e.g., fluphenazine hydrochloride or fluphenazine decanoate).
  • an effective amount of fluphenazine e.g., fluphenazine hydrochloride or fluphenazine decanoate.
  • fluphenazine e.g., fluphenazine hydrochloride or fluphenazine decanoate
  • fluphenazine hydrochloride or fluphenazine decanoate may be administered at a total dose of between about 0.5 and 50 mg/day, or between about 0.5 and 2 mg/day, or between about 0.5 and 5 mg/day, or between about 2 and 10 mg/day, or between about 5 and 20 mg/day, or between 10 and less than about 40 mg/day, any of which doses may optionally be administered as a divided dose.
  • a compound that is structurally related to fluphenazine may be administered at an adjusted version of the foregoing doses, where the adjustment compensates for any difference in potency between fluphenazine and the structurally related compound.
  • the dose of fluphenazine e.g., fluphenazine hydrochloride or fluphenazine decanoate
  • a structurally related compound administered produces a serum concentration or cerebrospinal fluid concentration of at least about 5 micromolar, or between about 5 and about 10 micromolar, or between about 10 and about 20 micromolar, or between about 20 and about 30 micromolar, or at least about 50 micromolar.
  • the dose of fluphenazine e.g., fluphenazine hydrochloride or fluphenazine decanoate
  • a structurally related compound may be administered daily, about every other day, about twice a week, or about once a week.
  • the present invention provides for a method of treating clinical disorders associated with protein aggregation comprising administering, to a subject in need of such treatment, an effective amount of tamoxifen (e.g., tamoxifen citrate) or a structurally related compound.
  • an effective amount of tamoxifen e.g., tamoxifen citrate
  • a structurally related compound e.g., tamoxifen citrate
  • tamoxifen e.g. , tamoxifen citrate
  • tamoxifen citrate may be administered at a total dose of between about 0.5 and about 50 mg/day or between about 0.5 and about 20 mg/day or between about 5 and about 15 mg/day or between about 10 and about 20 mg/day, any of which doses may optionally be administered as a divided dose.
  • a compound that is structurally related to tamoxifen may be administered at an adjusted version of the foregoing doses, where the adjustment compensates for any difference in potency between tamoxifen and the structurally related compound.
  • the dose of tamoxifen e.g. , tamoxifen citrate
  • a structurally related compound administered produces a serum concentration or cerebrospinal fluid concentration of at least about 5 micromolar, or between about 5 and about 10 micromolar, or between about 10 and about 20 micromolar, or between about 20 and about 30 micromolar, or at least about 50 micromolar.
  • the dose of tamoxifen (e.g., tamoxifen citrate) or a structurally related compound may be administered daily, about every other day, about twice a week, or about once a week.
  • the present invention provides for a method of treating clinical disorders associated with protein aggregation comprising administering, to a subject in need of such treatment, an effective amount of taxol or a structurally related compound.
  • taxol may be administered at a total dose of between about 25 and about 175 mg/m /day, or between about 25 and about 50 mg/m 2 /day, or between about 25 and about 100 mg/m 2 /day, or between about 25 and about 150 mg/m 2 /day, any of which doses may optionally be administered as a divided dose.
  • a compound that is structurally related to taxol may be administered at an adjusted version of the foregoing doses, where the adjustment compensates for any difference in potency between taxol and the structurally related compound.
  • the dose of taxol or a structurally related compound administered produces a serum concentration or cerebrospinal fluid concentration of at least about 5 micromolar, or between about 5 and about 10 micromolar, or between about 10 and about 20 micromolar, or between about 20 and about 30 micromolar, or at least about 50 micromolar.
  • the dose of taxol or a structurally related compound may be administered daily, about every other day, about twice a week, about once a week, about once every two weeks, about once every three weeks, about once a month, about once every six weeks, about once every eight weeks, about once every ten weeks, about once every twelve weeks, about once every sixteen weeks, about once every 20 weeks, or about once every 24 weeks.
  • taxol is preferably administered intravenously. To avoid a hypersensitivity reaction, it may be desirable to premedicate a subject about to receive taxol or a structurally related compound with one or more medication, for example
  • dexamethasone e.g. 20 mg 12 and 6 hours prior to treatment
  • diphenhydramine e.g. 50 mg IV 30-60 minutes prior to treatment
  • cimetidine e.g. 300 mg 30-60 minutes prior to treatment
  • ranitidine e.g., 50 mg 30-60 min prior to treatment
  • the present invention provides for a method of treating clinical disorders associated with protein aggregation comprising administering, to a subject in need of such treatment, an effective amount of cantharidin, cantharidic acid, or a structurally related compound, where said effective amount does not have substantial toxic effects on the subject (both cantharidin and cantharidic acid are known to have toxic effects at relatively low concentrations, so that substantially non-toxic doses would need to be determined using standard pharmaceutical techniques which consider that an anti-protein aggregate effect was achieved at a concentration of 100 micromolar in the C. elegans assay).
  • the substantially non-toxic dose of cantharidin, cantharidic acid, or a structurally related compound may be administered daily, about every other day, about twice a week, or about once a week.
  • Treatment according to any of the foregoing methods may be administered continuously or for intervals interrupted by breaks.
  • the present invention provides for at least two types of assay constructs: first, an aggregation-prone protein construct which encodes a protein with a tendency to aggregate (which may also be referred to as a " aggregatable protein”), the expression of which results in the generation of aggregated protein in C. elegans and second, a marker construct which comprises a marker gene that encodes a marker protein, the expression of which assists in the characterization of animals in the assay, either by facilitating counting, developmental staging, organ localization, or some other characterization of the worm.
  • Said constructs may comprise a promoter active in C.
  • elegans which may optionally confer tissue specific, location-specific, and/or developmental stage-specific expression, operably linked to a nucleic acid encoding a protein with a tendency to aggregate or a marker protein (or, in non-limiting embodiments, both).
  • Said constructs may optionally be comprised in vectors known in the art ⁇ e.g., a plasmid, phage or virus) or may be introduced directly into C.
  • constructs may further comprise additional elements known in the art, for example, but not limited to, one or more selection marker, a translation
  • C. elegans has been extensively characterized, and lists of cell-type and location specific promoters are known in the art (see, for example, C. elegans II, second edition, Cold Spring Harbor Monograph Series, Vol 33, Cold Spring Harbor Press, Cold Spring Harbor, NY (1997), and www.wormbase.org. For example, and not by way of limitation:
  • neuron-specific promoters include, but are not limited to, ace-1, acr-5, aex-3, apl-1, alt-J, cat-1, cat-2, cch-1, cdh-3, ceh-2. ceh-2, ceh-6, ceh-10, ceh-14, ceh-17, ceh-23, ceh-28, ceh-36, che-1, che-3, cfi-1, cgk-1, cha-1, cnd-1, cod-5, daf-1, daf-4, daf-7, daf-19, dbl-1, des-2, deg-1, deg-3, del-1, eat-4, eat-16, ehs-1, egl-10, egl-17, egl-19, egl-2, egl-36, egl-5, egl-8, fax-1, flp-1, flp-1 , flp-1 , flp-3, f
  • pharynx specific promoters include the ceh-22, hlh-6 and myo-2 promoters;
  • gut-specific promoters include the nhx-2, vit-2, cpr-1, ges-1, mtl-1, mtl-2, pho-1, spl- 1, vha-6 and elo-6 promoters.
  • Non-limiting examples of proteins with a tendency to aggregate include, but are not limited to, the human proteins: AT mutants ATZ, Siiyama and Mmalton;
  • the protein with a tendency to aggregate is comprised in a fusion protein with a second, detectable protein, for example, but not limited to, a fluorescent protein such as green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, yellow
  • a portion of the native protein with a tendency to aggregate may optionally be omitted in the encoded fusion protein, and/or an additional protein or peptide may be comprised in the fusion protein, for example to link the protein with a tendency to aggregate with the detectable protein, provided that the tendency of the protein to aggregate is not substantially impaired (in specific non- limiting examples, at least about 90 percent or at least about 95 percent or at least about 98 percent of the native protein with a tendency to aggregate (examples of a "aggregatable portion") is present in the fusion protein).
  • Non-limiting examples of marker proteins are the various fluorescent proteins that are fluorescent in vivo known in the art, including, but not limited to, green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc..
  • expression of the marker construct results in a single detectable region (or image) per worm.
  • a single detectable region means that using a detection means appropriate for the assay, expression of the marker results in a pattern of detectable signal which can be perceived as a single region (for example, the pharyngeal region, the head region, the worm surface, or the entire worm) so that perception of that region allows for the discrimination of one worm from another and therefore facilitates accurate counting of worms.
  • the marker gene is operably linked to a pharynx-specific (e.g. myo-2) C. elegans promoter.
  • expression of the marker construct results in a defined number of detectable regions (or images) per worm.
  • a defined number of detectable regions means that using a detection means appropriate for the assay, expression of the marker results in a pattern of detectable signal which can be consistently perceived as a defined number of regions (for example, 2 or 3 or 4, etc. regions), so that perception of that region allows for the discrimination of one worm from another and therefore facilitates accurate counting of worms (where the number of images counted reflects the number of worms presented multiplied by a factor which is the defined number of detectable regions per worm).
  • one or more marker gene is operably linked to one or more promoter so that the marker is expressed two detectable regions per animal, so that the number of worms present may be determined by counting the number of detectable regions and dividing by two.
  • An analogous approach could be used to produce worms having three detectable regions which could be determined by counting the regions and dividing by three, etc.
  • the invention provides for other methods of defining worms and worm boundaries.
  • the present invention provides for a transgenic C. elegans worm expressing myo-3::mCherry which is specifically expressed in the muscles and effectively creates an outline of the worm.
  • Such worms may be further engineered to express ATZ::GFP in any tissue and quantify ATZ aggregation/polymerization within the myo-3::mCherry boundary.
  • the present invention further provides for worms carrying at least three transgenes.
  • a transgenic C. elegans may be engineered to express the fusion proteins ATZ::YFP, myo-2::CFP and LGG- 1 ::mCherry. These worms may be used to identify/study the effect of a particular compound on ATZ disposition and autophagy, simultaneously.
  • UTR unfolded protein response
  • elegans may be engineered to express myo-2::mCherry (red-head marker), nhx-2::sGFP::ATZ (intestine) and lgg-1 ::CFP (blue marker for autophagy).
  • myo-2::mCherry red-head marker
  • nhx-2::sGFP::ATZ intestine
  • lgg-1 ::CFP blue marker for autophagy
  • the red head (mCherry) region may be used to determine the number of worms in the well
  • the green (GFP) regions may be measured to determine the extent of ATZ
  • aggregation/polymerization and the blue (CFP) region may be used to measure the level of autophagy.
  • non-limiting embodiments of the invention in order to facilitate expression in C. elegans, which is believed to be more efficient in the presence of introns, where a nucleic acid encoding either a protein with a tendency to aggregate (or aggregatable portion thereof) or a marker protein lacks introns, one or more ⁇ e.g., 1 , 2, 3 or 4, etc.) "synthetic" intron may be introduced (either by engineering blunt-end restriction sites into the protein-encoding DNA ⁇ e.g., cDNA) by site-directed mutagenesis or by overlap-extension PCR (see below)). Synthetic introns are between 48 - 51 bp in length and include consensus splice acceptor (AGGUAAGU) and splice donor (CAGG) sequences at the 5' and 3' ends, respectively.
  • AGGUAAGU consensus splice acceptor
  • CAGG splice donor
  • the present invention provides for an aggregated protein construct comprising a nucleic acid encoding a protein with a tendency to aggregate (for example, but not limited to, a human protein such as ATZ, huntingtin, synuclein, amyloid beta, neuroserpin, ubiquitin, neurofilament protein, alpha B crystallin and tau, or a non-human equivalent thereof, or an aggregatable portion thereof) optionally comprised in a fusion protein with a detectable protein such as a fluorescent protein, operably linked o a C. elegans promoter, where said promoter may, for example, be a neuron-specific promoter, a gut (e.g. intestinal) specific promoter, a muscle specific promoter, a pharynx-specific promoter, or a tail specific promoter, and where said nucleic acid encoding a protein with a tendency to aggregate optionally comprises one or more synthetic intron.
  • a human protein such as ATZ, huntingtin, synuclein, amy
  • an aggregated protein construct according to the invention which is expressed in intestinal cells of C.
  • Pnhx-2sGFP::ATM may be generated by inserting a 4 kb nhx-2 promoter fragment into Hindlll/Xbal restriction sites of the expression vector, pPD95.85. Then a Kasl restriction site may be introduced by site-directed mutagenesis into the GFP translational stop codon. A 1.4 kb fragment containing the ATM cDNA and 3 synthetic introns may be cloned into the Kasl site.
  • Pnhx-2sGFP::ATZ may be generated by site-directed mutagenesis of Pnhx-2sGFP::ATM, thereby generating the E342K (Z) mutation.
  • synthetic introns resembling those found in C. elegans may be introduced into the ATZ cDNA using overlap-extension PCR (FIGURE 1 A-B).
  • Large oligonucleotides consisting of ⁇ 50 nucleotides of synthetic intron (carefully designed to contain appropriate 5' and 3' donor /acceptor sequences) and ⁇ 22 nt sequence complementary to the ATZ coding region may be synthesized and used as primers to amplify small regions of the ATZ cDNA (see FIGURE 1 A).
  • the amplified fragments may be joined pairwise using overlap-extension PCR to generate larger fragments containing intronic regions (see FIGURES IB and 1 C).
  • the complete ATZ fragment containing all of the synthetic introns may be amplified using primers flanked with Kas I recognition sites (see FIGURE ID) and cloned into the expression vector pPD95.85 to generate Pnhx-2::sGFP::ATZ.
  • constructs encoding neuroserpin which are expressed in intestinal or neuronal cells of C. elegans are shown in TABLE 3 below.
  • a marker construct according to the invention that is expressed selectively in the C. elegans pharynx, a
  • transcriptional Pmyo-2mRFP fusion (where RFP is "Red Fluorescent Protein") construct may be constructed by subcloning the myo-2 promoter and the mRFP cDNA into the Sphl/Xbal and Nhel/EcoRV sites of the canonical expression vector, pPD49.26, respectively.
  • RFP Red Fluorescent Protein
  • a "model system” comprises a C. elegans adapted to serve as a model of a disorder of protein aggregation.
  • a model system may be a Caenorhabditis elegans carrying a transgene comprising an aggregated protein construct comprising a nucleic acid encoding a protein with a tendency to aggregate (for example, but not limited to, a human protein such as ATZ, huntingtin, synuclein, amyloid beta, neuroserpin, ubiquitin, neurofilament protein, alpha B crystallin and tau, or a non-human equivalent thereof, or an aggregatable portion thereof), optionally comprised in a fusion protein with a detectable protein such as a fluorescent protein, operably linked o a C.
  • a detectable protein such as a fluorescent protein
  • said promoter may, for example, be a neuron-specific promoter, a gut (e.g. intestinal) specific promoter, a muscle specific promoter, a pharynx-specific promoter, or a tail specific promoter, and where said nucleic acid encoding a protein with a tendency to aggregate optionally comprises one or more synthetic intron, as described in the section above.
  • said C. elegans may further comprise an additional transgene comprising a marker construct comprising a marker gene operably linked to a C. elegans promoter and encoding a marker protein, as described in the section above.
  • the protein with a tendency to aggregate is comprised in a fusion protein with a first fluorescent protein and where the marker construct encodes a second fluorescent protein
  • the first and second fluorescent protein are not the same (for example, so that their fluorescent emissions are distinguishable (for example, they may have a different wavelength)).
  • expression of the marker construct results in a single or otherwise consistently countable detectable region (or image) per worm.
  • the present invention provides for a model system for a 1 -antitrypsin deficiency comprising a C. elegans carrying a transgene comprising an aggregated protein construct comprising a nucleic acid encoding ATZ optionally comprised in a fusion protein with a detectable protein such as a fluorescent protein, operably linked o a C. elegans promoter, where said promoter is a gut specific promoter, and where said nucleic acid encoding ATZ optionally comprises one or more synthetic intron.
  • said C. elegans may further comprise an additional transgene comprising a marker construct comprising a marker gene operably linked to a C. elegans promoter and encoding a marker protein.
  • the present invention provides for a model system for a disorder associated with protein aggregatation in neurons comprising a Caenorhabditis elegans carrying a transgene comprising an aggregated protein construct comprising a nucleic acid encoding a protein with a tendency to aggregate (for example, but not limited to, a human protein such as huntingtin, synuclein, amyloid beta, neuroserpin, ubiquitin, neurofilament protein, alpha B crystallin and tau, or a non-human equivalent thereof, or an aggregatable portion thereof), optionally comprised in a fusion protein with a detectable protein such as a fluorescent protein, operably linked to a C.
  • a human protein such as huntingtin, synuclein, amyloid beta, neuroserpin, ubiquitin, neurofilament protein, alpha B crystallin and tau, or a non-human equivalent thereof, or an aggregatable portion thereof
  • a detectable protein such as a fluorescent protein
  • said promoter may, for example, be a neuron-specific promoter, a gut ⁇ e.g. intestinal) specific promoter, a muscle specific promoter, a pharynx-specific promoter, or a tail specific promoter, and where said nucleic acid encoding a protein with a tendency to aggregate optionally comprises one or more synthetic intron, as described in the section above.
  • said C. elegans may further comprise an additional transgene comprising a marker construct comprising a marker gene operably linked to a C. elegans promoter and encoding a marker protein.
  • the protein with a tendency to aggregate in a model system for AD may be human amyloid beta, or an aggregatable portion thereof; in a model system for Parkinson's disease the protein with a tendency to aggregate may be human synuclein, or an aggregatable portion thereof; in a model system for Huntington's disease the protein with a tendency to aggregate may be human huntingtin, or an aggregatable portion thereof; in a model system for chronic traumatic brain injury the protein with a tendency to aggregate may be human tau protein, or an aggregatable portion thereof; and so forth.
  • Transgenic C. elegans may be prepared by methods known in the art, including, but not limited to, microinjection or microparticle bombardment.
  • an aggregated protein construct and/or a marker construct may be introduced by injection into the gonad of a young adult hermaphrodite worm, for example, at a concentration of about 80 ng/ ⁇ .
  • the present invention provides for a method of determining whether a test compound has activity in treating a disorder of protein aggregation (and/or activity in reducing the amount of protein polymer), comprising:
  • transgenic C. elegans carrying (a) a first transgene comprising a nucleic acid encoding a human protein with a tendency to aggregate, or an aggregatable portion thereof, operably linked o a C. elegans promoter, where the expression of the human protein results in a detectable accumulation of human protein in the C. elegans (which may be detectable, for example, because the human protein with a tendency to aggregate, or an aggregatable portion thereof, may be comprised in a fusion protein with a first fluorescent protein) and (b) a second transgene comprising a marker construct comprising a marker gene encoding a marker protein ⁇ e.g. , a second fluorescent protein) operably linked to a C. elegans promoter;
  • the marker protein uses the marker protein, determining the number of C. elegans in said plurality (for example, where the marker protein generates a single or a definite number of images per worm, counting those images as a direct method of determining the number of worms, and where the marker protein may be a second fluorescent protein having a fluorescent distinguishable from that of the first fluorescent protein fused to the aggregatable protein);
  • test compound uses the results of (ii) and (iii), determining the change in the amount of human protein per worm resulting from the administration of test compound; , wherein, if administration of the test compound results in a significant decrease in the amount of human protein per worm, then the test compound is indicated to be therapeutically effective in a disorder of protein aggregation.
  • test compounds are preferably practiced in a high-throughput format where at least 96 or at least 384 (or more) test compounds may be tested in parallel.
  • a 96 well-based assay may be performed as follows. Fifty-five young adult stage worms may be dispensed into 96-well plates using the COPAS BioSort (worm sorter). Worms may then be immobilized by the addition of 0.1 M sodium azide to facilitate image capture. The plates may be placed into a computerized high throughput plate reader, ArrayScanVTi (Thermofisher Cellomics Products). ArrayScanVTi may be set up to rapidly scan wells and capture multiple images using brightfield and fluorescence parameters. Algorithms, as discussed below, may be used to identify and quantify defined spots (fluorescent granules) and objects (individual animals).
  • FIGURE 5 A typical screen shot of the ArrayScanVTi interface is shown in FIGURE 5.
  • brightfield and GFP fluorescence images were taken from 4 different fields within each well using a 5x Carl Zeiss objective.
  • a single field showing a brightfield and GFP fluorescence overlay illustrates GFP aggregates throughout the length of the intestine (FIGURE 5, well Dl).
  • FIGURE 6 shows exactly the elements from FIGURE 5 that were selected for data analysis.
  • ArrayScanVTi correctly identified all adult worms in the field of view (blue outline) and excluded all eggs and other debris that would alter the analysis.
  • the algorithm identified all the ATZ aggregates (FIGURE 6, red spots, e.g. as indicated by white arrowhead) within the set boundary (FIGURE 6, red outline, the outer of two line boundaries at the perimeter of each worm).
  • a transgenic C. elegans that expresses an ATZ/GFP fusion protein and carries a second transgene encoding a red fluorescent protein (mCherry) as marker protein which is expressed only in the head (pharynx) region of the worm.
  • mCherry red fluorescent protein
  • the expression of a second fluorescent marker that has a distinct expression pattern than GFP::ATZ (intestine) has several major advantages. First, the bright expression of the mCherry protein significantly improves focus time and efficiency.
  • red heads can be easily counted to obtain accurate worm number per well.
  • GFP::ATZ aggregates can be more efficiently and accurately measured. By simply dividing the total GFP fluorescence in the well by the total number red heads, the average GFP fluorescence per worm can be determined, a capability that was problematic to achieve using the brightfield object identification algorithms.
  • a 384- well assay may be performed as follows.
  • a 384- well-based assay has several advantages over the 96-well format. First, one can screen more compounds using the same number of worms needed for a 96-well plate. Second, images can be captured using the 2.5x objective. This reduces the number of fields needed to capture the well from 16 to 1.
  • the Arrayscan VTi may be fitted with a 0.63x coupler. The 0.63x coupler allows the capture of 100% of the well (as opposed to -90% using the lx coupler) allowing one to account for all the worms on in the well, leading to a considerably smaller variance between replicate wells.
  • Two ⁇ of stock (lOmM) compounds may be diluted with 98 ⁇ of S-medium to a final concentration of 200 ⁇ drug in 2% DMSO and S-medium. Fifteen ⁇ of the diluted compounds may then be transferred to 384-well plates using a robotic liquid handler (EP3). Prior to the experiment, fifteen ⁇ of 4x OP50/antibiotic solution may be added to each well. Using the COPAS Biosort, 35 L4-young adult stage worms may be deposited into each well and allowed to incubate for 24 or 48 h at 22°C.
  • worms may be immobilized by the addition of sodium azide or levamisole to a final concentration of 12.5 mM and 4 mM, respectively.
  • the worms may then be imaged using the high speed, automated imaging device, ArrayScan VTi. Image capture and data analysis may be performed using the Spot Detector BioApplication with algorithms optimized for worms. B-score statistical analyses may be performed to identify compounds that had a significant (> 2 SDs away from the mean) effect on ATZ aggregation.
  • FIGURE 1 A summary of a typical LOPAC screen is shown in FIGURE 1 1.
  • compound tracking and data analysis for the primary HCS assay may be performed using ActivityBaseTM (IDBS, Guildford, UK), CytoMiner (UPDDI) software and visualized using SpotfireTM DecisionSite® (TIBCO Software Inc., Somerville, MA, USA) software, as described in 60-63.
  • Custom calculators were written to process the HCS data and perform the z-score and B-score statistical analysis (64,65).
  • the Z'-factor 30 may be used.
  • the Z'-factor may be calculated from the mean and the standard deviation of the negative and positive control populations as follows:
  • ⁇ is the standard deviation
  • ⁇ is the mean
  • p and n are positive and negative controls, respectively.
  • Z'-factors between 0.5 and 1.0 indicate the separation band (signal window) between the positive and negative controls is wide and the assay is of excellent quality and suitable for HTS/HCS.
  • Z'-factors between 0 and 0.5 indicate a good quality screen, whereas a score ⁇ 0 indicates the assay is of poor quality and unsuitable for HTS/HCS.
  • the B-score may be calculated from all of the sample measurements on an assay plate and used an iterative mathematical model to eliminate systematic row and column artifacts on a plate.
  • the mathematical model of the B-score may be described as:
  • Y ijp is the compound measurement at z t h row and y ' t h column of the /3 ⁇ 4i plate
  • ⁇ ⁇ is the 'true' activity value
  • s ijp is the random error of the assay on the plate
  • ⁇ ⁇ and YC jp represent the row and column artifacts on the pth plate, respectively.
  • a two-way median polish statistic method may be applied to estimate the B-score of a HCS assay.
  • the random error estimate, s jjp of the measurement at z ' t h row and t h column of the /1 ⁇ 4 plate may be calculated by fitting a two-way median polish as: where Y ijp is the fitted compound value, ⁇ is the estimated average of the plate, and R ip and C jp are the estimated systematic artifacts for the t
  • the median absolute deviation (MAD) of the random error estimate on p th plate may be computed as:
  • the B score may be calculated as:
  • the compounds may be ranked according to ascending B-score values. Rank-scores may be calculated by taking the average of compound rankings from two independent drug screens. Compounds with rank-scores ⁇ 1 10 may be considered to significantly decrease the accumulation of sGFP::ATZ inclusions. Conversely, compounds with rank-scores >1225 may be considered to significantly increase the accumulation of sGFP::ATZ inclusions. Selected compounds may be chosen for further analysis, for example in a human cell culture and/or animal model assay.
  • AT deficiency Several characteristics of AT deficiency make it an attractive target for chemoprophylaxis strategies involving high-throughput screening of small molecule libraries.
  • the disease predominantly involves an ER translocation defect, wherein mutant ATZ protein retains some of its anti-elastase activity (i.e., ATZ can inhibit neutrophil elastase albeit not as efficiently as AT).
  • ATZ anti-elastase activity
  • small molecules that increase ATZ secretion could theoretically prevent tissue damage in both lung and liver.
  • the severity of tissue injury caused by ATZ is thought to be influenced by genetic and environmental modifiers that regulate endogenous quality control mechanisms for disposal of misfolded proteins. Compounds that enhance these degradative processes could therefore be used in patients to prevent liver damage in combination with strategies specifically designed to prevent lung damage.
  • a tractable genetic model of this disease would greatly enhance the
  • the conformational disease of AT deficiency can be modeled in C.
  • Animals expressing wild-type AT (ATM) secrete the protein.
  • animals expressing ATZ develop intracellular inclusions and show a slow growth and larval arrest/lethality phenotype.
  • an assay using this model has been adapted to allow for automated high-throughput screening.
  • ATM wild-type M allele also referred to as AT in many publications
  • ATM-Saar 32 amino acid C-terminal truncation mutant that is non-polymerogenic and accumulates in the
  • ATZ-Saar 32 amino acid C-terminal truncation mutant that also has the point mutation of ATZ, but is non- polymerogenic and accumulates in the ER; referred to as AT saar Z in our previous publications.
  • ATZ was fused to the green fluorescent protein (GFP).
  • GFP-fusions have been used extensively in cell culture and other systems, their usefulness in studying ATZ aggregation has not been tested in C. elegans. Since ATZ
  • the C. elegans intestine is the center of metabolic activity and the organ that most closely resembles the human liver (where ATM is normally synthesized). In addition, the intestine would be the predominant site of absorption for compounds added to the media.
  • the synthetic signal peptide from the C. elegans expression vector, pPD95.85 was used (Fire Lab C. elegans Vector Kit, 1995).
  • introns are introduced by engineering unique blunt end restriction enzyme sites in the cDNA by site-directed mutagenesis. This approach is labor intensive, inefficient and dictated by the presence of favorable sequences.
  • a strategy was devised for intron insertion using overlap-extension PCR (FIGURE 1 A-B). Large oligonucleotides consisting of -50 nt of synthetic intron (carefully designed to contain appropriate 5' and 3' donor /acceptor sequences) and ⁇ 22 nt sequence complementary to the ATZ coding region were synthesized and used as primers to amplify small regions of the ATZ cDNA (FIGURE 1 A).
  • the amplified fragments were joined pairwise using overlap-extension PCR to generate larger fragments containing intronic regions (FIGURES 1 B and 1 C). Once the 5 pieces were joined together, the complete ATZ fragment containing all of the synthetic introns was amplified using primers flanked with Kas I recognition sites (FIGURE ID) and cloned into the expression vector pPD95.85 to generate Pnhx-2::sGFP::ATZ. A control construct designed to express ATM, Pnhx-2::sGFP::ATM, was also generated. Prior to the insertion of the ATZ fragment, several modifications were made to pPD95.85 vector to facilitate cloning and to ensure proper transgene fusion.
  • FENIB Familial encephalopathy with neuroserpin inclusion bodies
  • GFP : ATZ
  • Transgenic animals expressing AT were generated by injecting the plasmids in TABLE 2, into the gonads of young adult hermaphrodites at a concentration of 80 ng/ ⁇ .
  • GFP-positive progeny were selected and propagated to confirm germline transmission.
  • Injected DNA typically forms extrachromosomal arrays that are transmitted (at rates ranging from 0- 95%) to subsequent generations in a non-Mendelian manner.
  • Stable, integrated lines, with 100% transmission rates, can be generated by exposing animals to high doses of gamma radiation.
  • worms with integrated arrays display more stable and consistent transgene expression.
  • integrated lines are particularly advantageous for genetic screens and for experiments requiring analysis of large populations. For these reasons, stable, integrated lines were generated by exposing transgenic animals to 35Gy (3500 Rads) of gamma radiation. After stable integrants were identified, they were outcrossed 6 times to remove spurious mutations that may have been acquired as a result of the radiation treatment.
  • total worm lysates were analyzed by immunoblotting. Mixed staged worms were lysed by sonication. Following centrifugation to remove cell debris, worm lysates were boiled in sample buffer for 5 min and fractionated by SDS-PAGE. Gels were transferred to nitrocellulose membranes and AT and GFP protein bands were visualized by probing with anti-AT and anti-GFP antibodies, respectively (FIGURE 3).
  • the anti-human AT antibody did not react with any proteins in the lysates of the parental N2 (lane 1 ) or control sGFP (lane 2) expressing worms (FIGURE 3A).
  • worms were fixed in glutaraldehyde, stained with osmium tetroxide and examined under an electron microscope (FIGURE 4).
  • No abnormal structures were observed in the intestinal cells of worms expressing sGFP::ATM (FIGURE 4A).
  • large intracellular globules were present in sections of worms expressing sGFP::ATZ (FIGURE 4B and C, arrowheads). These globules appeared to be surrounded by ribosomes (FIGURE 4D), suggesting retention of the ATZ proteins within the ER.
  • C. elegans is a powerful genetic organism useful for the study of human diseases. Recently, there have been growing interest in using C. elegans as a tool in drug discovery. Efforts have been hampered by one major bottleneck - automated image capture and data analysis.
  • the present invention now provides a fully automated, whole organism-based high-content screen (HCS) for drugs and RNAis modulating the disease phenotype. This method can be easily adapted to other C. elegans models and provides a stepping-stone for future C. elegans-based drug and RNAi screens.
  • HCS high-content screen
  • ArrayScanVTi Thermofisher Cellomics Products.
  • ArrayScanVTi was set up to rapidly scan wells and capture multiple images using bright field and fluorescence parameters. Complex algorithms were set up to identify and quantify defined objects (fluorescent granules) and events (individual animals).
  • FIGURE 5 A typical screen shot of the ArrayScanVTi interface is shown in FIGURE 5.
  • bright field and GFP fluorescence images were taken from 4 different fields within each well using a 5x Carl Zeiss objective.
  • a single field showing a bright field and GFP fluorescence overlay illustrates GFP aggregates throughout the length of the intestine (FIGURE 5, well Dl).
  • FIGURE 6 shows exactly the elements from FIGURE 5 that were selected for data analysis.
  • ArrayScanVTi correctly identified all adult worms in the field of view (blue outline) and excluded all eggs and other debris that would alter the analysis.
  • the algorithm identified all the ATZ aggregates (FIGURE 6, red spots) within the set boundary (FIGURE 6, red outline).
  • red heads can be easily counted to obtain accurate worm number per well.
  • GFP::ATZ aggregates can be more efficiently and accurately measured. By simply dividing the total GFP fluorescence in the well by the total number red heads, the average GFP fluorescence per worm can be determined, a capability that was problematic to achieve using the brightfield object identification algorithms.
  • a 384-well-based assay has several advantages over the 96-well format. First, one can screen more compounds using the same number of worms needed for a 96-well plate. Second, images can be captured using the 2.5x objective. This reduces the number of fields needed to capture the well from 16 to 1. In addition, the
  • Arrayscan VTi was fitted with a 0.63x coupler.
  • the 0.63x coupler allows the capture of 100% of the well (as opposed to -90% using the lx coupler) allowing one to account for all the worms on in the well. This has lead to a considerably smaller variance between replicate wells.
  • a small molecule screen of the LOPAC library identifies drugs potentially useful for the treatment of AT-deficiency and other protein aggregation disorders.
  • a pilot screen of the LOPAC (library of pharmacologically active compounds) library was performed. The global strategy for high-throughput screen is shown in FIG. 10A-E.
  • FIGURE 1 A summary of a typical LOPAC screen is shown in FIGURE 1 1. Using the above approach, three screens of the LOPAC library were performed which identified a number of hit compounds that have potential therapeutic value (TABLE 4).
  • PKA protein kinase
  • Pimozide Ca2+ channel antagonist antipsychotic; D2 dopamine receptor antagonist
  • Pmyo-2mRFP fusion construct was constructed by subcloning the myo-2 promoter and the mRFP cDNA into the Sphl/Xbal and Nhel/EcoRV sites of the canonical expression vector, pPD49.26 (a kind gift from Dr. Andrew Fire, Stanford University School of Medicine), respectively.
  • pPD49.26 a kind gift from Dr. Andrew Fire, Stanford University School of Medicine
  • To generate the Pnhx-2mCherry:: lgg-1 construct a 3.5 kb genomic fragment containing the lgg-1 promoter, coding region and 3'-UTR was amplified and cloned into pCR®-Blunt II-TOPO® vector (Invitrogen, Carlsbad, CA, USA).
  • Pnhx-2sGFP::ATZ was generated by site-directed mutagenesis of Pnhx-2sGFP::ATM, thereby generating the E342K (Z) mutation.
  • Worm strains VK413 (Pnhx-
  • VK1093 Pnhx-2mCherry:: lgg-1
  • VK821 Pmyo-2mRFP
  • Strains VK689 Pnhx-2sGFP::ATM
  • VK694 Pnhx-2sGFP::ATZ
  • the worm strain expressing Pclh-4GFP (pFL6IIclh-4) were a gift from Keith Nehrke 40.
  • N2 and GF66 (Pvha-4Q82::YFP, 21) were obtained from Caenorhabditis Genetics Center (CGC), http://www.cbs.umn.edu/CGC/).
  • Worms were routinely cultured at 22 ° C on nematode growth medium (NGM) plates seeded with E. coli strain, OP50, unless otherwise specified.
  • Imaging of transgenic animals using ArrayScan VTI Twenty N2 or transgenic L4-adult stage worms were transferred to 384-well plates containing 60 ⁇ of PBS and anesthetized with 30 ⁇ of 0.02 M NaAz prior to image capture.
  • Fluorescent transgene expression within valid objects, was quantified in the T ITC or GFP channels.
  • SpotDetector BioApplication was optimized to identify transgene expression as spots. Parameters were optimized such that spots of varying shape, size and intensity could be identified. For this paper, spot count, spot total area and spot total intensity per object were used to compare transgene expression in different animals.
  • This method generated a population of ⁇ 200,000 age- synchronized animals for small molecule screening.
  • mice were washed off plates and transferred into 50 ml conical tubes and allowed to settle by gravity for 5 minutes. After discarding the supernatant, animals were washed again with 50 ml of PBS to remove excess bacteria and other debris that could interfere with worm sorting .
  • MatriMinistoreTM (Spokane, WA, USA) automated compound storage and retrieval system.
  • daughter plates were withdrawn from the -20 °C freezer, thawed at ambient temperature and centrifuged 1-2 min at 50 x g.
  • the plate seals were removed and 98 ⁇ of S-medium were added to the wells using the Flex Drop dispenser (Perkin Elmer, Waltham, MA, USA).
  • This intermediate stock of library compounds was at a concentration of 200 ⁇ in 2% DMSO.
  • the diluted compounds were mixed by repeated aspiration and dispensation using a 384-well P30 dispensing head on the Evolution-P3 (EP3) liquid handling platform (Perkin Elmer), and then 15 ⁇ of each compound were transferred to the wells of assay plates. In the primary screen, compounds were screened individually at a final concentration of 50 ⁇ .
  • EP3 Evolution-P3
  • Assay plate preparation for drug screen On the day of the screen, assay plates containing 15 ⁇ of each compound were thawed and centrifuged at 214 x g for 60 s. Fifteen microliters of 4x assay medium, which was prepared by mixing 4.0 ml OP50, 25.4 ml S-medium, 0.6 ml lOOx antibiotic-antimycotic stock solution (stock contained 10,000 units penicillin, 10 mg streptomycin and 25 ⁇ g amphotericin B/ml, Sigma) and 24.0 ⁇ 1 M FUDR, were added to each well. Animals were then sorted into the wells using the COPASTM BIOSORT worm sorter.
  • Animal sorting using the COPASTM BIOSORT To reduce assay variability, a tightly-synchronized population of worms was selected based on size (i.e., stage of development) and fluorescence intensity (i.e., transgene expression) using the COPASTM BIOSORT. L4 to young adult-stage worms were initially selected using empirically-determined time-of-flight (TOF) and coefficient of extinction (EXT) values. Animals were also gated based on GFP fluorescence intensity.
  • TOF time-of-flight
  • EXT coefficient of extinction
  • Imaging of animals using the ArrayScan VTI Prior to imaging, worms were anesthetized by adding 30 ⁇ of 0.02 M NaN3 in PBS to each well.
  • HCS data analysis Compound tracking and data analysis for the primary HCS assay were performed using ActivityBaseTM (IDBS, Guildford, UK), CytoMiner (UPDDI) software and visualized using SpotfireTM DecisionSite®
  • Custom calculators were written to process the HCS data and perform the z-score and B-score statistical analysis (100, 101). As a measure of assay quality and robustness, the Z' -factor 30 was used. The Z'-factor was calculated from the mean and the standard deviation of the negative and positive control populations as follows:
  • ⁇ is the standard deviation
  • ⁇ is the mean
  • p and n are positive and negative controls, respectively.
  • Z'-factors between 0.5 and 1 .0 indicate the separation band (signal window) between the positive and negative controls is wide and the assay is of excellent quality and suitable for HTS/HCS.
  • Z'-factors between 0 and 0.5 indicate a good quality screen, whereas a score ⁇ 0 indicates the assay is of poor quality and unsuitable for HTS/HCS.
  • the z-score (Xj - ⁇ )/ ⁇ , where Xj was the raw measurement on the ith compound, and X and ⁇ were the mean and standard deviation of all the sample measurements on a plate.
  • the B-score was calculated from all of the sample measurements on an assay plate and used an iterative mathematical model to eliminate systematic row and column artifacts on a plate.
  • the mathematical model of the B-score was described as:
  • 3 ⁇ 4 ⁇ + 3 ⁇ 4 + rc J , + 3 ⁇ 4 ,
  • Y jjp was the compound measurement at i t h row column of the plate
  • ⁇ ⁇ was the 'true' activity value
  • s ijp was the random error of the assay on the /? t h plate
  • ⁇ ⁇ and YC jp represented the row and column artifacts on the pth plate, respectively.
  • a two-way median polish statistic method was applied to estimate the B-score of a HCS assay. The implemented procedures are described below.
  • the random error estimate, i iJp of the measurement at t h row column of the plate was calculated by fitting a two-way median polish as:
  • Rank-scores were calculated by taking the average of compound rankings from two independent drug screens. Compounds with rank-scores ⁇ 1 10 significantly decreased the accumulation of sGFP::ATZ inclusions. Conversely, compounds with rank-scores >1225 significantly increased the accumulation of sGFP::ATZ inclusions. Selected compounds (based on cost and availability) from both groups were chosen for further analysis.
  • Hit compound characterization Compounds that were identified as potential hits were purchased (if available) and retested for verification. Compounds that failed to produce a dose-dependent response were not analyzed further.
  • Compounds that produced a response in a dose-dependent manner were further tested for a time-dependent response.
  • Compound dose-response curves were performed by dispensing 15 ⁇ of a 4X stock solution into 384-well plates containing 15 ⁇ of assay medium (see above). Thirty-five animals were sorted into each well bringing the volume to -60 ⁇ . The final compound concentrations in each well varied from 0-100 ⁇ . Assay plates were incubated in a 22 °C incubator for 24 or 48 hours. Each compound was tested in quadruplicate in at least 2 independent experiments.
  • ArrayScan VTI consists of an inverted light microscope (Axiovert 200M, Carl Zeiss) configured with a motorized objective turret with Plan-Neofluar objectives, a motorized 5-position filter cube turret, a mechanized stage, a 12-bit cooled CCD camera and controller software. Samples are illuminated for brightfield imaging using a broad white-light source and for florescence imaging in up to 4 different spectra using a mercury-based light source.
  • the program can be configured to detect animals at, for example, the L1-L2 stage and exclude those at the L3-L4 adult stages.
  • the ArrayScan VTI can detect fluorescent "spots" in up to 4 different channels within each object and the SpotDetector Bio Application can display the data as a total fluorescent spot number, spot area or spot intensity per object. It was next determined whether this application was sensitive enough to ' indentify different cell types (pharyngeal cells, excretory cell, intestinal cells), pathologic protein deposition (polyQ aggregates) or a physiological process
  • Fluorescent images were obtained for C. elegans strains carrying transgenes with tissue-specific promoters driving fluorescent protein expression in the pharynx (Pmyo-2RFP), the excretory gland cell (Pclh-4GFP) or intestinal cells (Pvha-6Q82::YFP, Pnhx-2GFP or Pnhx- 2mCherry::lgg-l). Except for the polyQ82-containing construct, which generates cytosolic aggregates (78), the others yielded a diffuse cytoplasmic fluorescence pattern under baseline conditions (FIGURES 13A-13 J).
  • FIGURES 130-13Q In comparison to the minimal background fluorescence of wild-type (N2) animals, that of the transgenic animals was markedly increased using the SpotDetector BioApplication to measure either the total spot number, area or fluorescence intensity per animal (FIGURES 130-13Q). Depending on the nature of the transgene expression pattern, certain comparisons were more meaningful. For example, total spot area or total spot intensity per animal, rather than total spot count, were better at discriminating pharyngeal or intestinal expression in comparison to background (FIGURES 13C-13F, 13P, 13Q). In contrast, total spot count per animal, was the more sensitive parameter to follow when assessing the presence of the secretory cell and the degree of protein aggregation in the animals expressing polyQ82 (FIGURES 13G-13J, 130).
  • Macroautophagy is a cellular process in which a double membrane envelops cytosolic components or organelles (autophagosome) and delivers this material to a lysosome (autophagolysosome) for degradation and recycling (reviewed in 79).
  • LGG-l/LC3/Atg8 is a diffuse cytosolic protein that participates in
  • LGG-1 fused to mCherry changes its cytoplasmic distribution pattern from diffuse (lower fluorescence intensity) to punctate (higher fluorescence intensity) (80).
  • a strain expressing a Pnhx-2mCherry::lgg- l transgene was examined after starvation, a potent inducer of intestinal autophagosome formation (81).
  • the diffuse cytoplasmic fluorescence in the intestinal cells was well above that of the N2 background (FIGURES 13K- 13L, 130- 13Q).
  • Detection of live cells and dead animals The nematode has served as an informative system to study the genetics of different modes of cell death. It was determined whether this imaging system could distinguish between live or dead cells using either the loss or gain of a fluorescent marker, respectively, mec-4, a member of the DEG/ENaC membrane cation channel superfamily, is expressed exclusively in the 6 mechanosensory neurons of C. elegans (82).
  • a reporter strain containing an integrated transgene, ZB164 bzIs8[Pmec-4GFP]; mec-4(+), diving GFP expression in the mechanosensory neurons exhibits ⁇ 4-5 fluorescent cell bodies per L4/young adult animal (83).
  • the mec-4(+) and mec- 4(d) strains averaged ⁇ 6 and ⁇ 2 cells/animal, respectively (FIGURE 14M)(45).
  • the system was capable of discriminating between wild-type and mutant animals based on the differential viability of just six mechanosensory neurons.
  • the SpotDetector BioApplication was programmed to select (pseudocolored red heads) or exclude (pseudocolored white heads) fluorescent spots above or below, respectively, a pre-determined threshold value based on a combination of fluorescent spot area and intensity (FIGURE 15B).
  • the algorithm correctly identified all 9 young adult animals and excluded ⁇ 24 of the larval forms (FIGURE 15B). Since, the area of the red-heads was proportionally smaller than that of adult animals, the total count was rarely confounded by overlapping pharyngeal "spots". Thus, there was excellent correlation between the number of adult animals detected by the spot count and the actual number of animals in the wells (FIGURE 4C). It was concluded that the number of adult animals accurately detected in a well of a 384 well plate increased from ⁇ 10 to at least 35, when fluorescence imaging of the red-head marker, rather than the brightfield imaging of individual animals was used to obtain a valid animal count.
  • Pnhx2sGFP : ATZ, contains a human ATZ minigene fused C-terminal to GFP with N- terminal signal peptide (sGFP).
  • An intestinal-specific promoter, nhx-2 drove fusion gene expression (85).
  • this common Z mutation induces protein misfolding and accumulation within the endoplasmic reticulum of hepatocytes resulting in cellular injury and cirrhosis (reviewed in 86).
  • sGFP::ATZ aggregated within the endoplasmic reticulum of intestinal cells.
  • an integrated transgenic line, expressing the wild-type fusion protein, sGFP::ATM. was generated.
  • This protein was efficiently secreted into the intestinal lumen and pseudocoelomic space and was detectable microscopically only after a relatively long integration time.
  • both strains were co-injected with the Pmyo-2mCherry transgene.
  • Approximately 35 animals expressing sGFP::ATZ or sGFP::ATM were sorted into 384-well plates. To minimize variability, only
  • Pnhx2sGFP::ATZ animals within a tight fluorescence window were sorted into the wells. Nearly the entire well was imaged in channel 1 using brightfield illumination (FIGURES 16A, 16D). These images, which were not used to identify individual animals, simply confirmed that that comparable numbers of young adult animals of both lines were sorted into the wells. Using channel 2 and 3, respectively,
  • FIGURE 16F expression in the two different transgenic lines.
  • the red-heads detected in channel 2 were used to determine a "head count” and to show that the actual number of animals sorted into each well were nearly identical (FIGURE 16G).
  • the images obtained in channel 3 were used to measure three different parameters in each of the wells containing sGFP::ATM or sGFP::ATZ expressing animals (FIGURES 16H- 16J) .
  • Compound screen To test the HCS protocol, a pilot drug screen was performed using the library of pharmacologically active compounds (LOPAC1280TM, 1280 compounds). Tight gating parameters for total fluorescence were used to sort 35 young adult Pnhx2sGFP::ATZ animals into wells of 384-well plates containing 50 ⁇ of a LOPAC compound and 0.5 % DMSO. Pnhx2sGFP::ATZ animals incubated with 0.5% DMSO served as untreated controls and were placed in the first-two and last- two columns of each plate. In a representative experiment, plate 1 of the LOPAC library was set-up for screening on day 1 , and 3 other plates were set-up on the next day.
  • LOPAC1280TM pharmacologically active compounds
  • the ArrayScan VTI reads microtiter plates by rows, alternating from left-to-right, and then right-to-left. For some assays, we noted that the control fluorescence values would drift upwards slightly in rows towards the bottom of the plate. This drift appeared to correlate with an increase in chamber temperature during the scanning period and was minimized by cooling the chamber with a fan or shortening the read times by using 2.5 x objective with a 0.6 coupler. Nonetheless, intra-plate variation was controlled for by presenting the data as a B-score (FIGURE 17C). Under the same conditions, the entire screen was repeated on a single day.
  • aRank-scores were calculated by averaging compound rankings based on ascending B-scores from two independent drug screens. Compounds with rank-scores ⁇ 1 10 or > 1225 significantly decreased or increased the accumulation of sGFP::ATZ inclusions, respectively. b Overall rank-order, based on relative rank-scores, for compounds that decreased or increased sGFP::ATZ accumulation. °Compound demonstrated a dose-dependent response. d Compound failed to demonstrate a dose-dependent response. 7.3 DISCUSSION
  • C. elegans Maintenance Medium (a chemically defined, bacteria free medium) appeared to be an ideal growth medium for animals cultivated in microtiter plates, but intense autofluorescence precluded further use (88).
  • S Medium a chemically defined, bacteria free medium
  • HCS format of the invention could capture 5-channel multiparametric images of each well of microtiter plate using an automated inverted fluorescence microscope and image analysis software.
  • the scanning time of a 384-well plate was reduced to ⁇ 30 minutes.
  • -6,000 compounds could be screened in a typical workday, or ⁇ 18,000 compounds per day if the ArrayScan VTI were configured with an automated plate loader. Screening using of compound effects on a discrete variable, such as a live-dead screen, would be even faster.

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Abstract

La présente invention concerne des procédés de traitement de troubles cliniques associés à une agrégation protéique comprenant l'administration à un sujet d'une quantité efficace d'un composé anti-agrégats protéiques (« APA ») choisi dans le groupe constitué par le pimozide, la fluphénazine (par exemple, le chlorhydrate de fluphénazine), le tamoxifène (par exemple, le citrate de tamoxifène), le taxol, la cantharidine, l'acide cantharidique, leurs sels et leurs composés structurellement apparentés. L'invention est basée au moins en partie sur la découverte selon laquelle chacun des composés mentionnés ci-dessus est capable de favoriser la dégradation de protéines ATZ agrégées dans un système modèle de Caenorhabditis elegans. Selon l'invention, un traitement par un ou plusieurs de ces composés APA peut être utilisé pour améliorer les symptômes et les signes d'une déficience en AT ainsi que d'autres troubles marqués par une agrégation de protéines, comprenant, entre autres, la maladie d'Alzheimer, la maladie de Parkinson et la maladie d'Huntington.
PCT/US2010/002898 2009-11-05 2010-11-04 Procédés de traitement de troubles associés à une agrégation protéique WO2011056222A1 (fr)

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US15/247,518 US9820990B2 (en) 2009-11-05 2016-08-25 Methods of treating disorders associated with protein aggregation

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CN108653305A (zh) * 2018-05-18 2018-10-16 南京中医药大学 以胞内渗透压为靶点治疗脑水肿的药物组合
GB2571978A (en) * 2018-03-15 2019-09-18 Andre Fisahn Uses, compositions and methods
WO2020081257A1 (fr) * 2018-10-05 2020-04-23 Vertex Pharmaceuticals Incorporated Modulateurs de l'alpha -1 antitrypsine
WO2021116707A1 (fr) * 2019-12-13 2021-06-17 Z Factor Limited 4-((2-OXOPYRIDIN-1 (2H)-YL)MÉTHYL)BENZAMIDES POUR LE TRAITEMENT D'UNE DÉFICIENCE EN α1-ANTITRYPSINE
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2571978A (en) * 2018-03-15 2019-09-18 Andre Fisahn Uses, compositions and methods
CN108653305A (zh) * 2018-05-18 2018-10-16 南京中医药大学 以胞内渗透压为靶点治疗脑水肿的药物组合
CN108653305B (zh) * 2018-05-18 2020-01-07 南京中医药大学 以胞内渗透压为靶点治疗脑水肿的药物组合
WO2020081257A1 (fr) * 2018-10-05 2020-04-23 Vertex Pharmaceuticals Incorporated Modulateurs de l'alpha -1 antitrypsine
US11623924B2 (en) 2018-10-05 2023-04-11 Vertex Pharmaceuticals Incorporated Modulators of alpha-1 antitrypsin
US11884672B2 (en) 2019-05-14 2024-01-30 Vertex Pharmaceuticals Incorporated Modulators of alpha-1 antitrypsin
WO2021116707A1 (fr) * 2019-12-13 2021-06-17 Z Factor Limited 4-((2-OXOPYRIDIN-1 (2H)-YL)MÉTHYL)BENZAMIDES POUR LE TRAITEMENT D'UNE DÉFICIENCE EN α1-ANTITRYPSINE
CN115023416A (zh) * 2019-12-13 2022-09-06 Z因子有限公司 用于治疗α1-抗胰蛋白酶缺乏症的4-((2-氧代吡啶-1(2H)-基)甲基)苯甲酰胺
CN115023416B (zh) * 2019-12-13 2024-04-02 森特萨制药(英国)有限公司 用于治疗α1-抗胰蛋白酶缺乏症的4-((2-氧代吡啶-1(2H)-基)甲基)苯甲酰胺

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