WO1998013488A2 - Procede pour determiner les modulateurs de la secretase de l'app et utilisation de ces derniers comme agents dans le traitement de la maladie d'alzheimer - Google Patents

Procede pour determiner les modulateurs de la secretase de l'app et utilisation de ces derniers comme agents dans le traitement de la maladie d'alzheimer Download PDF

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WO1998013488A2
WO1998013488A2 PCT/EP1997/005149 EP9705149W WO9813488A2 WO 1998013488 A2 WO1998013488 A2 WO 1998013488A2 EP 9705149 W EP9705149 W EP 9705149W WO 9813488 A2 WO9813488 A2 WO 9813488A2
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app
cells
pcep
secretase
expression vector
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PCT/EP1997/005149
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WO1998013488A3 (fr
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Thomas Dyrks
Jonathan-David Turner
Marion HÄRTEL
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Schering Aktiengesellschaft
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the present invention relates to a process for determining the activity of APP secretase using cellular model systems, those cellular systems being used directly for screening for pharmacological modulators of amyloid ⁇ A4 release, and to the modulators that can be identified and isolated by that process as pharmaceutical agents for the treatment of Alzheimer's disease.
  • the invention relates also to expression vectors, to cells transfected with the expression vectors and to the use thereof in the identification of APP secretase modulators, and to a kit for determining APP secretase activity.
  • AD Alzheimer ' s disease
  • AD is the most frequently occurring dementia disease in the elderly. 20% of all people aged 80 suffer from this disease.
  • AD is a neuro- degenerative disease having the following characteristic pathological indicators :
  • NFTs neurofibrillary tangels
  • Amyloid ⁇ A4 peptide deposits in the walls of cortical blood vessels.
  • Amyloid ⁇ A4 peptide which forms the chief component of extracellular deposits, is a peptide 39-43 amino acids long that is released by two proteolytic activities ( ⁇ and ⁇ - secretases) from a 695-770 amino acid-long transmembrane-bound amyloid precursor protein (APP).
  • APP amyloid precursor protein
  • ⁇ -secretase a third proteolytic activity can prevent amyloid ⁇ A4 release by enzymatic processing within the amyloid ⁇ A4 sequence.
  • amyloid ⁇ A4 is the chief component of extracellular deposits, it has been possible in a large number of in vitro and in vivo systems to demonstrate the neurotoxic potential of the peptide and accordingly an experimental correspondence between the formation of amyloid ⁇ A4 and neurodegeneration.
  • an expression vector is prepared that comprises a DNA sequence under the control of a promoter, the DNA sequence coding for a fusion polypeptide comprising i) one or more membrane anchor domains, ii) one or more recognition sites for an APP secretase and iii) one or more polypeptide component(s) releasable by cleavage at the recognition site, b) the expression vector is introduced into a cell that is capable of expressing one or more APP secretases, c) the APP secretase activity in the cells obtained according to step (b) is analysed based on the release of the polypeptide component(s) (iii) and d) optionally the secretase activity is determined in the presence of an added test substance in order to identify modulators that stimulate or inhibit the APP secretase activity.
  • Suitable promoters include all promoters known in the literature and familiar to the person skilled in the art that are suitable for the purpose in question.
  • a process for the quantitative and differential determination of the APP secretases involved in amyloid ⁇ A4 release and for the direct isolation of corresponding modulators is described which is characterised in that a) first of all a suitable expression vector is prepared by i) recombinant fusion of the secretory form of a protein, which secretory form is fused to the recognition sites for APP ⁇ - or ⁇ -secretase, with ii) a suitable transmembrane anchor sequence and iii) expression under the control of a suitable promoter, b) eukaryotic cells are stably transfected with the expression vector so produced, c) the transfected cells are selected with a suitable marker, d) the selected stable cells are analysed immunologicaily or enzymatically by means of the secreted reporter domain using current standard methods and finally e) with the use of suitable vectors as negative and positive controls, the modulators that stimulate or inhibit the ⁇ - or ⁇ -secretase activity are determined.
  • the expression vectors that may be used in process step a) may be, preferably, the vectors pCEP/SEAP ⁇ secTM1 , pCEP/SEAP ⁇ secTM2, pCEP/SEAP ⁇ secTM1 , pCEP/SEAP ⁇ secTM2, pCEP/SEAP ⁇ secswTM1 and pCEP/SEAP ⁇ secswTM2. They may also be functionally equivalent vectors.
  • the invention relates also to the expression vectors.
  • the secretory protein that may be used in process step a) may be urokinase (Langer G., Toschi L, Dieckmann J., Schleuning W.-D. (1995) Gene 161 , 287-292) or preferably the secretory form of alkaline phosphatase.
  • the membrane or transmembrane anchor sequence that may be used in process step a) may be the transmembrane domain of any protein or, preferably, the sequences of human APLP2.
  • the promoter that may be used in process step a) ii) may be any eukaryotic or viral promoter, preferably the Cytomegalovirus promoter.
  • the cells used in process step b) are eukaryotic cells, preferably human neuroblastoma cells, especially Sy5y cells.
  • the present invention relates also to cells transfected with the expression vectors, such as eukaryotic cells, preferably human neuroblastoma cells, especially Sy5y cells.
  • the present invention relates also to the use of the transfected cells for the identification of APP secretase modulators.
  • the markers used in process step c) may in principle be any customary eukaryotic selection marker known to the person skilled in the art.
  • the selection marker preferably used in process step c) is hygromycin.
  • Current standard methods according to process step d) are, for example, Western blots, immunoprecipitation, enzymatic detection reactions etc..
  • the vectors suitable for use in process step e) are preferably, for the negative control, the vector pCEP/SEAPTM and, for the positive control, the vector pCEP/SEAPsec, or functionally equivalent vectors.
  • the invention relates also to the modulators obtainable and obtained by the process according to the invention.
  • the modulators stimulate or inhibit the ⁇ - and ⁇ -secretase activity.
  • Such modulators are, for example, compounds such as 1 ,1 ,2,2-cyclopropane tetracarbonitrile and 9,10-dihydro-12-methylene-ethane anthracen-11-one, which may be used as pharmaceutical compounds in the treatment of Alzheimer's disease.
  • the invention relates also to pharmaceutical agents or compositions that comprise at least one modulator, optionally together with formulation substances and additives customary in pharmacy.
  • the present invention relates also to pharmaceutical agents or compositions in the form of tablets, tablets with controlled release of the active ingredient, dragees, pills, capsules, film-coated tablets and film-coated tablets with controlled release of the active ingredient.
  • the pharmaceutical agents or compositions of the invention are prepared in a manner known perse , in a suitable dose, with the customary solid or liquid carriers or diluents and the customary pharmaceutical and commercial excipients according to the desired mode of administration.
  • the preferred preparations are in a form suitable for oral, enteral or parenteral administration.
  • Such forms of administration are, for example, tablets, film-coated tablets, dragees, capsules, pills, powders or depot forms and also suppositories.
  • Suitable tablets can be obtained, for example, by mixing the active ingredient with known excipients, for example inert diluents such as dextrose, sugars, sorbitol, mannitol, polyvinylpyrrolidone, disintegrators, such as maize starch or alginic acid, binders, such as starch or gelatin, glidants, such as magnesium stearate or talc, and/or agents for obtaining a depot effect, such as carboxypolymethylene, carboxylmethyl cellulose, cellulose acetate phthalate or polyvinyl acetate.
  • the tablets may also consist of several layers.
  • dragees can be produced by coating cores, prepared analogously to tablets, with agents conventionally used in dragee coatings, for example polyvinyl- pyrrolidone or shellac, gum arabic, talc, titanium dioxide or sugars.
  • Dragee casings may also consist of several layers, in which the excipients mentioned above for tablets may be used.
  • Active ingredient-containing capsules may be produced, for example, by mixing the active ingredient with an inert carrier, such as lactose or sorbitol, and encapsulating the mixture in gelatin capsules.
  • an inert carrier such as lactose or sorbitol
  • the modulators according to the invention may also be used, in suitable solutions, such as, for example, physiological saline, as infusion or injection solutions.
  • oily solutions such as, for example, solutions in sesame oil, castor oil and cottonseed oil.
  • Solubilisers such as, for example, benzyl benzoate or benzyl alcohol, may be added to increase solubility.
  • the modulators obtainable and obtained by the process according to the invention may be used in the treatment of Alzheimer's disease.
  • the present invention relates also to the use of the modulators obtainable by the process in the manufacture of an agent for the treatment of Alzheimer's disease and to the use of the modulators obtained by the process in the manufacture of an agent for the treatment of Alzheimer's disease.
  • the present invention relates also to a kit for the determination of the activity of APP secretase in intact cells and for the identification of modulators of APP secretase activity, which is characterised in that a) an expression vector is prepared that comprises a DNA sequence under the control of a promoter, the DNA sequence coding for a fusion polypeptide comprising i) one or more membrane anchor domains, ii) one or more recognition sites for an APP secretase and iii) one or more polypeptide components releasable by cleavage at the recognition site, b) the expression vector is introduced into a cell that is capable of expressing one or more APP secretases, c) the APP secretase activity in the cells obtained according to step (b) is analysed based on the release of the polypeptide component(s) (iii) and d) optionally the secretase activity is determined in the presence of an added test substance in order to identify modulators that stimulate or inhibit the APP secretase activity.
  • the present invention relates especially to a kit for the quantitative and differential determination of the APP secretases involved in amyloid ⁇ A4 release and for the direct isolation of corresponding modulators, which is characterised in that a) first of all a suitable expression vector is prepared by i) recombinant fusion of the secretory form of a protein, which secretory form is fused to the recognition sites for APP ⁇ - or ⁇ -secretase, with ii) a suitable transmembrane anchor sequence and iii) expression under the control of a suitable promoter, b) eukaryotic cells are stably transfected with the expression vector so produced, c) the transfected cells are selected with a suitable marker, d) the selected stable cells are analysed immunologically or enzymatically by means of the secreted reporter domain using current standard methods and finally e) with the use of suitable vectors as negative and positive controls, the modulators that stimulate or inhibit the ⁇ - or ⁇ -secretase activity are
  • the eukaryotic cells that may be used in b) are preferably human neuroblastoma cells, especially Sy5y cells.
  • Fig. 1 shows the analysis of the C-terminal fragments after secretase activity.
  • the cells were lysed as described below.
  • the cell extracts were concentrated by precipitation with acetone and separated by SDS-PAGE.
  • Fig. 2 shows the analysis of the secreted reporter fragments after secretase activity.
  • the stably transfected cells were labelled for 2 hours with 35 S-methion ⁇ ne.
  • the conditioned medium was analysed by means of immunoprecipitation with anti- SEAP (SEAP track) or R1736 ( ⁇ track) and subsequent SDS-PAGE.
  • Fig. 3 shows the analysis of the secreted reporter fragments after secretase activity in the presence of ammonium chloride.
  • the stably transfected cells were pre- incubated for 40 minutes, with or without ammonium chloride, in methiomne- free medium and then metabolically labelled for 2 hours, with or without ammonium chloride, with 35s-meth ⁇ on ⁇ ne
  • the conditioned medium was analysed by means of immunoprecipitation with anti-SEAP (SEAP track) and subsequent SDS-PAGE.
  • Fig. 4 shows stably transfected cells that have been sown in microtitre plates (75 000 per well) and the next day incubated for 90 minutes, with or without ammonium chloride, in cell culture medium 30 ⁇ l of the conditioned medium were used for quantitative analysis with SEAP measurement by chemiluminescence Phospha-LightTM, Serva) (A) or SE ⁇ AP measurement by absorption (B).
  • sequences were derived from SEAP (Berger et al. (1988), Gene, 66, 1-10), APP (Kang et al. (1987), Nature, 325, 733-736) and APLP2 (Wasco er al. (1993), Nature 15 Genetics, 5, 95-99).
  • the cells (c. 1 x 10 ⁇ ) are washed once with PBS buffer and lysed directly in the cell culture dish by the addition of 2 ml of homo-buffer (4M guanidine thiocyanate, 25 mM sodium citrate pH 7; 100 mM ⁇ -mercaptoethanol, 0.5% lauroylsarcosine (v/v); 0.1% antifoam A (v/v)).
  • homo-buffer 4M guanidine thiocyanate, 25 mM sodium citrate pH 7; 100 mM ⁇ -mercaptoethanol, 0.5% lauroylsarcosine (v/v); 0.1% antifoam A (v/v)
  • the lysed cells After the lysed cells have been transferred into a 15 ml Sarstedt tube, the cells are mixed for 10 seconds and 0.2 ml of sodium acetate (2M, pH 4), 2 ml of phenol (pH 4) and 0.4 ml of chloroform are added.
  • RNA is then precipitated by adding 2 ml of isospropanol and incubating for at least one hour at -20°C.
  • the RNA is subsequently precipitated (20 minutes, 4°C, 10000 g), and the RNA precipitate is dried, dissolved in 0.5 ml of homo-buffer and transferred into a 1.5 ml Eppendorf vessel. After repeated precipitation with isopropanol (0.5 ml isopropanol), the dried RNA is dissolved in 50-100 ⁇ l of DEPC/water.
  • the sub-clone of the neuroblastoma cell line SH-Sy5y was cultured in equal amounts of minimal essential medium (MEM, with Earle's salts and L-glutamine) and Ham's F-12, supplemented with non-essential amino acids (Eagle's formulation), penicillin (50 U/ml), streptomycin (40 ⁇ g/ml) and 10 % (v/v) foetal calf serum (FCS).
  • MEM minimal essential medium
  • FCS 10 % (v/v) foetal calf serum
  • the DNA/lipofectin solution was prepared beforehand by mixing diluted DNA (10-20 ⁇ g of DNA in 1.5 ml of Opti-MEM) and diluted lipofectin (30-50 ⁇ g of lipofectin in 1.5 ml of Opti-MEM) and incubated for 30 minutes at room temperature.
  • the cells were cultured in culture medium for a further 24-48 hours and stable lines were selected by the addition of hygromycin B (400 ⁇ g/ml the first week, then 300 ⁇ g/ml).
  • the cells were preincubated for 40 minutes with 2 ml of MEM (6 cm dish) without methionine.
  • the labelling of the cells was carried out with 300 ⁇ Ci of [ 35 S] methionine in 1.5 ml of MEM without methionine for 2-3 hours.
  • the medium (conditioned medium) was then collected, and the cells were washed with PBS at 4°C and scraped off the dish.
  • the centrifuged-off cells were resuspended in 600 ⁇ l of sol-buffer (50 mM tris-HCI [pH7.5], 150 mM NaCI, 2 mM
  • the immunoprecipitation was carried out according to the method of Anderson er al. (Methods Enzymol. 96, 110-120).
  • the conditioned medium was standardised on 50 mM tris-HCI [pH7.5], 150 mM NaCI, 2 mM EDTA, 1% Triton X-100 and commercial protease-inhibitor cocktail.
  • the conditioned medium and the cell lysate were incubated for 30 minutes with 5 ⁇ l of pre-immunoserum and 50 ⁇ l (5 mg) of protein A/Sepharose.
  • the samples were briefly centrifuged and the supernatants incubated overnight at 4°C with antibody (10 ⁇ l of SEAP, 5 ⁇ l of A4CT) and 50 ⁇ l (5mg) of protein A/Sepharose.
  • the antibody-protein A complexes were washed three times with washing buffer A (10 mM tris-HCI [pH 7.5], 150 mM NaCI, 0.2 % Triton X- 100, 2 mM EDTA), twice with washing buffer B (10 mM tris-HCI [pH 7.5], 500 mM NaCI, 0.2 % Triton X-100, 2 mM EDTA) and once with washing buffer C (10 mM tris- HCI [pH7.5]) and then incubated twice in Laemmli test buffer for 5 minutes at 100°C.
  • the analysis of the labelled proteins was carried out by means of tris-tricine SDS- PAGE and phosphoimaging.
  • the gel electrophoresis was carried out according to Schagger and Jagow ((1987) Analytical Biochemistry 166, 368-379).
  • 200 ⁇ l of the cell lysate are incubated with 1 ml of acetone for 30 minutes at -40°C and then centrifuged off for 10 minutes at 13000 rpm; the supernatant is discarded and the pellet is dried at 37°C.
  • the precipitated protein is dissolved in 40 ⁇ l of 8M urea for 15 minutes at 56°C, 40 ⁇ l of Laemmli test buffer are added twice and the whole is incubated for a further 15 minutes at 56°C for the purpose of denaturation.
  • the gel is washed for 30 minutes in transfer buffer (25 mM tris, 192 mM glycine, 0.05 % SDS, 20% methanol).
  • transfer buffer 25 mM tris, 192 mM glycine, 0.05 % SDS, 20% methanol.
  • the proteins are for 2.5 hours electrophoretically transferred (60 V, 280 mA, 4°C) in transfer buffer onto 0.45 ⁇ m of nitrocellulose.
  • the filter After saturation of the nitrocellulose filter in TBST/powdered milk (20 mM tris/pH 7.6, 127 mM NaCI, 0.5% Tween 20, 1% powdered milk) for at least 1 hour at room temperature (RT), the filter is washed twice with TBST (20 mM tris/pH 7.6, 127 mM NaCI, 0.5% Tween 20®) and incubated for a further hour (1-16 hours depending on antibody) at RT with the primary antibody in TBST/powdered milk.
  • TBST room temperature
  • SEAP Assay SEAP measurement by chemiluminiscence
  • 100 ⁇ l of the heat-treated medium have 100 ⁇ l of assay buffer (2M dietha ⁇ olamine, pH 7.8, 1 mM MgCl2, 20 mM L-homoarginine) added, and the whole is incubated at room temperature for 5 minutes.
  • 100 ⁇ l of reaction buffer (0.1M diethanolamine, CSPD) are added to the incubate and, after 20 minutes, the chemiluminescence is measured for 5 seconds in a luminometer.
  • SEAP Assay SEAP measurement by absorption
  • All steps are carried out in 96-well microtitre plates.
  • 75 000 cells are sown per 96 wells.
  • the analysis is carried out in 200 ⁇ l of culture medium with and without test substance for from 1 to 20 hours at 37°C. Subsequently, 100 ⁇ l of the medium are incubated at 65°C for 60 minutes. After 2 minutes on ice, 100 ⁇ l of the heat-treated medium have 100 ⁇ l of assay buffer (2M diethanolamine, pH 7.8, 1 mM MgCI 2l 20 mM L-homoarginine) and 20 ⁇ l of p- nitrophenol phosphate (120 mM in assay buffer) added.
  • assay buffer 2M diethanolamine, pH 7.8, 1 mM MgCI 2l 20 mM L-homoarginine
  • the N-terminal 511 amino acids of the secretory form of human placenta! alkaline phosphatase (SEAP; human from the placenta, Berger et al. (1988), Gene, 66, 1-10) were amplified by means of PCR from the pCMV/SEAP expression vector (Tropix, Serva, Cat.No. AV10C) as template and the oligonucleotide primers AP5/868 and AP3Cla, and cloned by way of Hind 3 / Cla I into pBluescript SK. The vector pBC/SEAP is obtained.
  • the APP fragment amino acids 604 to 622, referred to here as ⁇ sec (numbering according to APP695), was amplified by means of PCR from APP cDNA (Kang er a/., (1987), Nature 325, 733-736) as template and the oligonucleotide primers APPCIal and aALP-2/AP. Then, the C-terminal 72 amino acids of APLP2 (Wasco et al., (1993), Nature genetics 5, 95-99), referred to here as TM1 , were amplified by means of PCR from vector SP65/APLP2-CT as template and the oligonucleotide primers aAAP/LP-2 and FAPLP2Xhol.
  • the APP fragment and the APLP2 fragment were then fused by means of PCR with the oligonucleotide primers APPCIal and FAPLP2Xhol.
  • the resulting APP/APLP2 hybrid molecule was cloned by way of Clal / Xho I into pBC/SEAP, vector pBCSEAP ⁇ secTM being obtained.
  • the resulting fusion fragment SEAP ⁇ secTM was cloned by way of Hind3/Xhol into the expression vector pCEP4.
  • the APP fragment, amino acids 604 to 615, referred to here as ⁇ sec was amplified by means of PCR from APP cDNA (Kang et al., (1987), Nature, 325, 733-736) as template and the oligonucleotide primers APPCIal and aBLP-2/AP.
  • TM2 the C-terminal 80 amino acids of APLP2 (Wasco er al., (1993), Nature genetics 5, 95-99), referred to here as TM2, were amplified by means of PCR from the vector SP65/APLP2-CT as template and the oligonucleotide primers aBAP/LP-2 and FAPLP2Xhol.
  • the APP fragment and the APLP2 fragment were then fused with the oligonucleotide primers APPCIal and FAPLP2Xhol by means of PCR.
  • the resulting APP/APLP2 hybrid molecule was cloned by way of Clal / Xho I into the vector pBC/SEAP resulting in the vector pBCSEAP ⁇ secTM2.
  • the resulting fusion fragment SEAP ⁇ secTM was cloned by way of Hind3/Xhol into the expression vector pCEP4.
  • TM2 the C-terminal 72 amino acids of APLP2 (Wasco et al., (1993), Nature genetics 5, 95-99), referred to here as TM2, were amplified by means of PCR from the vector SP65/APLP2-CT as template and the oligonucleotide primers bAAP/LP-2 and FAPLP2Xhol.
  • the APP fragment and the APLP2 fragment were then fused with the oligonucleotide primers APPClai62 and FAPLP2Xhoi by means of PCR.
  • the resulting APP/APLP2 hybrid molecule was cloned by way of Clal / Xho I into pBC/SEAP, resulting in the vector pBCSEAP ⁇ secTMl .
  • the resulting fusion fragment For cellular expression, the resulting fusion fragment
  • SEAP ⁇ secTMl was cloned by way of Hind3/Xhol into the expression vector pCEP4. 17
  • Vector pCEP/SEAP ⁇ secswTM1 was generated analogously to pCEP/SEAP ⁇ secTM1 using APP751sw (Urmoneit et al. 1995, Journal of Molecular Neuroscience, Vol. 6, 23-32) instead of APP cDNA as template.
  • the APP fragment and the APLP2 fragment were then fused with the oligonucleotide primers APPCIal62 and FAPLP2Xhol by means of PCR and the resulting APP/APLP2 hybrid molecule was cloned by way of Clal / Xho I into pBC/SEAP.
  • the vector pBCSEAP ⁇ secTM2 was obtained.
  • the resulting fusion fragment SEAP ⁇ secTM2 was cloned by way of Hind3/Xhol into the expression vector pCEP4.
  • Vector pCEP/SEAP ⁇ secswTM2 was generated analogously to pCEP/SEAP ⁇ secTM2 using APP751sw (Urmoneit et al. 1995, Journal of Molecular Neuroscience, Vol. 6, 23-32) instead of APP cDNA as template.
  • TM The C-terminal 70 amino acids of APLP2 (Wasco et al., (1993), Nature genetics 5, 95- 99), referred to here as TM, were amplified by means of PCR from SP65/APLP2-CT as template and the oligonucleotide primers KALPCIal and FAPLP2Xhol and cloned by way of Clal / Xho I into pBC/SEAP, resulting in the vector pBCSEAPTM.
  • the resulting fusion fragment SEAPTM was cloned by way of Hind3/Xhol into the expression vector pCEP4. 1.8 Vector pCEP/SEAPsec
  • the Hind III / Xba I fragment from the pCMV/SEAP expression vector (Tropix, Serva, Cat. No. AV10C), coding for the secretory form of human placenta! alkaline phosphatase (SEAP; human from the placenta, Berger et al. (1988), Gene, 66, 1-10) was intermediately cloned into pBluescript SK and then cloned by way of Hind3/Notl into the expression vector pCEP4.
  • Sy5y cells are transfected with the above-described expression vectors.
  • a stable expression is achieved not by dilution and cloning but simply by selection with hygromycin.
  • Analysis of the stable cells is effected both immunologically (immunoprecipitation with SEAP and APP antibodies) and by means of enzymatic detection of the secreted reporter (SEAP assay).
  • SEAP secreted reporter
  • the stably transfected cells release the reporter (SEAP) into the medium only after ⁇ or ⁇ -secretase activity.
  • the amount of secreted reporter reflects the level of the respective proteolytic activity.
  • the amount of released reporter can be measured by current standard methods, such as, for example, chemiluminescence, down to sub- nanomolar concentrations.
  • the sensitivity achieved thereby allows a quantitative determination of the ⁇ or ⁇ -secretase activity in the "high capacity screen"-compatible 96-well format.
  • Published ⁇ and ⁇ -secretase modulators such as, for example, phorbol ester and NH4CI, exhibit the expected effects in the system described herein.
  • the expression of the reporter fusion constructs should, after the respective secretase activity, result in a secreted N-terminal fragment (SE ⁇ AP) and the corresponding intracellular, membrane-bound C-terminal fragment.
  • Fig. 1 shows a Western blot analysis, developed with an antibody to the C-terminus of APP/APLP2 (anti-APP-CT (Dr.
  • SEAPsec ⁇ TM constructs The antibody R1736 (Dr. Selkoe/Dr. Haass, Brigham and Women's Hospital, 221 Long Wood Ave, Boston, MA 02119) recognises the C- terminal amino acid of APP, which is released after ⁇ -secretase activity. Immuno- reactivity with R1736 thus indicates sequence-specific ⁇ -secretase activity. As can be seen in Fig. 2, track ⁇ , only the SEAPsec ⁇ TM 1 and SEAPsec ⁇ TM2 cells secrete significant amounts of SEAP with the R1736 epitope.
  • ⁇ -secretase activity can be inhibited by compounds that render alkaline endosomal/lysosomal compartments (NH4CI, chloroquine, methylamine etc.), whilst the ⁇ -secretase activity is not affected by those conditions.
  • Fig. 3 shows an immunoprecipitation of the medium with SEAP-antibody after metabolic labelling in the presence or absence of 20 mM NH4CI. It can be seen clearly that the processing of SEAPsec ⁇ TM2 but not of SEAPsec ⁇ TMl is inhibited under those conditions. In addition to the immunological analysis of the specific regulation of the reporter fusion constructs, the modulation of the reporter secretion was determined quantitatively by means of the SEAP assay.
  • phorbol ester In contrast to NH4CI, phorbol ester (PMA) stimulates ⁇ -secretase activity without having an effect on ⁇ -secretase activity. It was also possible for that secretase- specific regulation to be reproduced with the reporter fusion constructs.
  • Fig. 4 shows an SEAP assay in which the secreted SEAP was measured as a function of phorbol ester (Fig. 4A) and NH4CI (Fig. 4B).
  • the secretion of SEAP in the case of the SEAP ⁇ TM cells can be stimulated by PMA by approximately 400%, which agrees very well with the hitherto published data.
  • BafilomycinAI 0.2 ⁇ M inhibits SEAP secretion of SEAP ⁇ sec and SEAP ⁇ secsw by -35 %
  • SEAP ⁇ sec corresponds to SEAP ⁇ secTM 1 and SEAPsec ⁇ TM2
  • SEAPased corresponds to SEAPsecaTMI
  • SEAPasec2 corresponds to SEAPsecaTM2
  • SEAP ⁇ sec corresponds to SEAP ⁇ secTMl and SEAP ⁇ secTM2
  • SEAP ⁇ sed corresponds to SEAPsec ⁇ TMl
  • SEAP ⁇ sec2. corresponds to SEAPsec ⁇ TM2
  • SEAP ⁇ secsw corresponds to SEAP ⁇ secswTMl and SEAP ⁇ secswTM2
  • SEAP ⁇ secswl corresponds to SEAPsec ⁇ swTMl
  • SEAP ⁇ secsw2. corresponds to SEAPsec ⁇ swTM2 Example 6
  • the process according to the invention is used as described above for screening for pharmacological modulators of amyloid ⁇ A4 release.

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Abstract

L'invention concerne un procédé pour déterminer l'activité de la secrétase de l'APP (protéine précurseur de l'amyloïde) à l'aide de systèmes de modèles cellulaires. Ces systèmes cellulaires sont directement utilisés pour analyser les modulateurs pharmacologiques de la libération de l'amyloïde βA4. L'invention traite aussi des modulateurs qui peuvent être identifiés et isolés par ce procédé comme agents pharmaceutiques pour traiter la maladie d'Alzheimer. L'invention a également pour objet des vecteurs d'expression, des cellules transfectées par les vecteurs d'expression, et l'utilisation de ces derniers pour identifier les modulateurs de la secrétase de l'APP, et un ensemble pour déterminer l'activité de cette enzyme.
PCT/EP1997/005149 1996-09-24 1997-09-22 Procede pour determiner les modulateurs de la secretase de l'app et utilisation de ces derniers comme agents dans le traitement de la maladie d'alzheimer WO1998013488A2 (fr)

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AU47757/97A AU4775797A (en) 1996-09-24 1997-09-22 Process for the determination of app secretase modulators and the use thereof as agents in the treatment of alzheimer's disease

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DE19641180A DE19641180A1 (de) 1996-09-24 1996-09-24 Verfahren zur Darstellung von APP-Sekretase Modulation und deren Verwendung als Mittel zur Behandlung der Alzheimer'schen Erkrankung
DE19641180.7 1996-09-24

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WO1998013488A2 true WO1998013488A2 (fr) 1998-04-02
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DE19920514A1 (de) * 1999-05-05 2000-11-16 Boehringer Ingelheim Pharma Methoden zur Auffindung von Proteasen, die spezifisch membrangebundene Substrate spalten
US6245884B1 (en) 1998-10-16 2001-06-12 Vivian Y. H. Hook Secretases related to alzheimer's dementia
WO2001075088A1 (fr) * 2000-04-05 2001-10-11 Esbatech Ag Procede d'identification de polypeptides ayant une activite protease
US6313268B1 (en) 1998-10-16 2001-11-06 Vivian Y. H. Hook Secretases related to Alzheimer's dementia
WO2002010354A2 (fr) * 2000-08-01 2002-02-07 Institut De Recherche Cliniques De Montreal (Ircm) Secretase/sheddase avec activite d'asp-ase sur l'enzyme de clivage app du site beta (bace asp2, memepsine 2)
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US6627739B1 (en) 1999-02-10 2003-09-30 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
WO2003087842A1 (fr) * 2002-04-18 2003-10-23 Esbatech Ag Procede pour identifier des modulateurs d'une activite de secretase
EP1366061A2 (fr) * 2001-02-05 2003-12-03 Andrx Corporation Methode de traitement de troubles de la maturation du precurseur de la proteine beta amyloide
WO2003102177A1 (fr) * 2002-05-31 2003-12-11 Otsuka Pharmaceutical Co., Ltd. Procede de criblage de compose modifiant la production de la proteine amyloide $g(b)
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US7045311B2 (en) 2001-10-25 2006-05-16 Monogram Biosciences, Inc. Whole cell assay systems for cell surface proteases
US7115410B1 (en) 1999-02-10 2006-10-03 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
DE10259834B4 (de) * 2001-12-20 2007-11-29 F. Hoffmann-La Roche Ag Test und Screeningverfahren zur Identifizierung von Inhibitoren von Beta-Sekretasen
US7335632B2 (en) 2001-10-23 2008-02-26 Comentis, Inc. Beta-secretase inhibitors and methods of use thereof
US7456007B1 (en) 1998-12-31 2008-11-25 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
US7514408B1 (en) 1999-12-02 2009-04-07 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
US8679534B2 (en) 1997-12-12 2014-03-25 Andrx Labs, Llc HMG-CoA reductase inhibitor extended release formulation

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DE19941039A1 (de) * 1999-08-28 2001-03-01 Boehringer Ingelheim Pharma gamma-Sekretase in vitro Testsystem
AU3173301A (en) * 2000-02-11 2001-08-20 European Molecular Biology Laboratory Methods and compositions for treatment of alzheimer's disease by enhancing plasmin or plasmin-like activity

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US6245884B1 (en) 1998-10-16 2001-06-12 Vivian Y. H. Hook Secretases related to alzheimer's dementia
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US7456007B1 (en) 1998-12-31 2008-11-25 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
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US7276349B2 (en) 1999-02-10 2007-10-02 Elan Pharmaceuticals, Inc. Beta-secretase enzyme compositions and methods
DE19920514A1 (de) * 1999-05-05 2000-11-16 Boehringer Ingelheim Pharma Methoden zur Auffindung von Proteasen, die spezifisch membrangebundene Substrate spalten
US7244708B2 (en) 1999-06-28 2007-07-17 Oklahoma Medical Research Foundation Inhibitors of memapsin 2 and use thereof
US6545127B1 (en) 1999-06-28 2003-04-08 Oklahoma Medical Research Foundation Catalytically active recombinant memapsin and methods of use thereof
US7514408B1 (en) 1999-12-02 2009-04-07 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
WO2001075088A1 (fr) * 2000-04-05 2001-10-11 Esbatech Ag Procede d'identification de polypeptides ayant une activite protease
WO2002010354A3 (fr) * 2000-08-01 2007-10-18 Montreal Inst Rech Cliniques Secretase/sheddase avec activite d'asp-ase sur l'enzyme de clivage app du site beta (bace asp2, memepsine 2)
WO2002010354A2 (fr) * 2000-08-01 2002-02-07 Institut De Recherche Cliniques De Montreal (Ircm) Secretase/sheddase avec activite d'asp-ase sur l'enzyme de clivage app du site beta (bace asp2, memepsine 2)
EP1366061A4 (fr) * 2001-02-05 2006-04-26 Andrx Corp Methode de traitement de troubles de la maturation du precurseur de la proteine beta amyloide
EP1366061A2 (fr) * 2001-02-05 2003-12-03 Andrx Corporation Methode de traitement de troubles de la maturation du precurseur de la proteine beta amyloide
US7335632B2 (en) 2001-10-23 2008-02-26 Comentis, Inc. Beta-secretase inhibitors and methods of use thereof
US7045311B2 (en) 2001-10-25 2006-05-16 Monogram Biosciences, Inc. Whole cell assay systems for cell surface proteases
DE10259834B4 (de) * 2001-12-20 2007-11-29 F. Hoffmann-La Roche Ag Test und Screeningverfahren zur Identifizierung von Inhibitoren von Beta-Sekretasen
WO2003087842A1 (fr) * 2002-04-18 2003-10-23 Esbatech Ag Procede pour identifier des modulateurs d'une activite de secretase
WO2003102177A1 (fr) * 2002-05-31 2003-12-11 Otsuka Pharmaceutical Co., Ltd. Procede de criblage de compose modifiant la production de la proteine amyloide $g(b)

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