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WO1998013488A2 - Process for the determination of app secretase modulators and the use thereof as agents in the treatment of alzheimer's disease - Google Patents

Process for the determination of app secretase modulators and the use thereof as agents in the treatment of alzheimer's disease Download PDF

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Publication number
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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Prior art keywords
app
cells
pcep
secretase
expression vector
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PCT/EP1997/005149
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French (fr)
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WO1998013488A3 (en
Inventor
Thomas Dyrks
Jonathan-David Turner
Marion HÄRTEL
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Schering Aktiengesellschaft
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Priority to AU47757/97A priority Critical patent/AU4775797A/en
Publication of WO1998013488A2 publication Critical patent/WO1998013488A2/en
Publication of WO1998013488A3 publication Critical patent/WO1998013488A3/en

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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

Described are 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 βA4 release, and the modulators that can be identified and isolated by that process as pharmaceutical agents for the treatment of Alzheimer's disease. Also described are expression vectors, cells transfected with the expression vectors and the use thereof in the identification of APP secretase modulators, and a kit for determining APP secretase activity.

Description

Process for the determination of APP secretase modulators and the use thereof as agents in the treatment of Alzheimer's disease
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 βA4 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.
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 :
1. Intracellular deposits in the form of neurofibrillary tangels (NFTs), chiefly consisting of the Tau protein. 2. Extracellular amyloid plaques, with the amyloid βA4 peptide as chief component.
3. Amyloid βA4 peptide deposits in the walls of cortical blood vessels.
4. Massive neurone loss in the limbic system and in the cerebral cortex, together with gliosis and atrophy.
Amyloid βA4 peptide, which forms the chief component of extracellular deposits, is a peptide 39-43 amino acids long that is released by two proteolytic activities (β and γ- secretases) from a 695-770 amino acid-long transmembrane-bound amyloid precursor protein (APP). Depending on various cell regulation mechanisms, a third proteolytic activity (α-secretase) can prevent amyloid βA4 release by enzymatic processing within the amyloid βA4 sequence. In addition to the fact that amyloid βA4 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 βA4 and neurodegeneration.
Decisive findings for clarifying the molecular processes involved in AD come from gene technology. Approximately 3% of all AD cases have an autosomal dominant inheritance, the FAD loci having been mapped on chromosome 21 , 19, 14 and 1. Three of the FAD genes have been identified and sequenced so far. It has been possible to demonstrate that mutations in the amyloid precursor protein (APP on chromosome 21) or in the "presinilin" genes (PS1 on chromosome 14 and PS2 on chromosome 1) segregate with the disease. On the basis of those FAD mutations it has been possible to demonstrate conclusively that amyloid βA4 is causatively involved in the development of the pathological indicators of AD. All mutations found hitherto result in a qualitative or quantitative change in the amyloid βA4 peptide. Modulation (inhibition) of the formation of amyloid βA4 has therefore been one of the main therapeutic approaches. The simplest modulation of amyloid βA4 formation is effected by a direct or indirect inhibition or stimulation of the proteases involved in amyloid βA4 release (APP secretases α, β or γ). On the basis of various publications (Kojima S. and Omori M. (1992) FEBS 304, 57-60, McDermott J., Biggins J.A. and Gibson A.M. (1992) BBRC 185, 746-752, Nelson R.B., Siman R., Iqbal M.A. and Potter H. (1993) Journal of Neurochemistry 61 , 567-577, Sahasrabudhe S.R., Brown A.M., Hulmes J.D., Jacobsen J.S., Vitek M.P., Blume A.J. and Sonnenberg J.L. (1993) Journal of Biological Chemistry 268, 16699-16705, Papastoitsis G., Siman R., Scott R., and Abraham CR. (1994) Biochemistry 33, 192-199, Matsumoto A. and Fujiwara Y. (1994) Biochemistry 34, 3941-3948, Schόnlein C, Lόffler J. and Huber G. (1994) BBRC 201 , 45-53 and Dreyer R. N., Bausch K. M., Fracasso P., Hammond L J., Wunderlich D., Wirak D. O., Davis G., Brini C. M., Buckholz T. M., Konig G.,
Kamarck M. E. and Tamburini P. P. (1994) European Journal of Biochemistry, 224, 265-271) the fact that the APP secretases require a membrane-bound substrate in order to be able to cleave efficiently and specifically must be taken as a starting point.
Hitherto described approaches for identifying protease inhibitors that prevent amyloid βA4 release use synthetic peptide substrates to find corresponding protease inhibitors (Sisodia S.S., Koo E.H., Beyreuther K., Unterbeck A. and Price D.L. (1990) Science 248, 492-495 and LeBlanc A.C. and Gambetti P. (1994) BBRC 204, 1371-1380). The cellular activity and specificity of the inhibitors found in that way is, however, questionable.
It would therefore be desirable to have a process that is suitable for the determination of the activity of APP secretases in intact cells and for the quantitative and differential determination of the APP secretase modulators involved in amyloid βA4 release, and that, in spite of the complexity of the intact cell system, renders possible a quantitative determination of the proteolytic activity being examined, it being possible for the modulators identifiable and obtainable by the process also to be used as pharmaceutical agents in the treatment of Alzheimer's disease. A process for the determination of the activity of APP secretase in intact cells and for the identification of modulators of APP secretase activity has now been found 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 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.
There is to be understood by the polypeptide components mentioned in a) iii), for example, reporter domains.
In particular, a process for the quantitative and differential determination of the APP secretases involved in amyloid βA4 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 α- or β-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 α- or β-secretase activity are determined. The expression vectors that may be used in process step a) are prepared by
i) recombinant fusion of the secretory form of a protein (reporter), which secretory form is fused to the recognition sites for APP α- or β- secretase, with ii) a suitable transmembrane anchor sequence and ϋi) expression of the protein under the control of a suitable promoter,
The expression vectors that may be used in process step a) may be, preferably, the vectors pCEP/SEAPαsecTM1 , pCEP/SEAPαsecTM2, pCEP/SEAPβsecTM1 , pCEP/SEAPβsecTM2, pCEP/SEAPβsecswTM1 and pCEP/SEAPβsecswTM2. 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 α- and β-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. Correspondingly, 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. The modulators according to the invention may also be used, in suitable solutions, such as, for example, physiological saline, as infusion or injection solutions.
There are suitable for parenteral administration especially 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.
It is also possible to incorporate the modulators obtainable and obtained by the process according to the invention into a transdermal system and thus administer them transdermally.
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 βA4 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 α- or β-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 α- or β-secretase activity are determined.
The eukaryotic cells that may be used in b) are preferably human neuroblastoma cells, especially Sy5y cells.
Description of the drawings
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. The proteins were transferred onto nitrocellulose. Detection was carried out with the antibody APP-CT, which is directed against the C-terminal 20 residues of APP CT = the C-terminal APP fragment after α-secretase activity.
Fig. 2 shows the analysis of the secreted reporter fragments after secretase activity. The stably transfected cells were labelled for 2 hours with 35S-methionιne. The conditioned medium was analysed by means of immunoprecipitation with anti- SEAP (SEAP track) or R1736 (α 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-methιonιne 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 μl of the conditioned medium were used for quantitative analysis with SEAP measurement by chemiluminescence Phospha-Light™, Serva) (A) or SEΞAP measurement by absorption (B).
To provide a better understanding of the embodiment examples forming the basis of this invention, a list has first of all been drawn up of all the procedures and methods known perse necessary for these experiments.
1. Plasmids and vectors used
pCEP (in vitro gen), APP cDNA (Kang et al, (1987), Nature 325, 733-736), pCMV/SEAP (Berger er a/., (1988), Gene, 66, 1-10), pSP65-PreA4 (Dyrks er a/., (1988), EMBO Journal 7, 949-957)
10
2. Oiigonucieotide primers used
The 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).
AP5/868
CGACTCACTA TAGGGAGACC (Seq. ID No. 1)
20 AP3Cla
CCATCGATCC CCGGGTGCGC GGCGTCGG (Seq. ID No. 2)
bAAP/LP-2
TTCCGACATA GTAGCAGTGC TCTCATTGGC (Seq. ID No. 3)
~> 5 bA P-2/AP
ACTGCTACTA TGTCGGAATT CTGCATCCAT (Seq. ID No. 4)
bCAP/LP-2
30 AAATTGGTGC CACTGCGGGA GGACTTCAGT (Seq. ID No. 5)
bCLP-2/AP
CCGCAGTGGC ACCAA I I I I I GATGATGAAC (Seq. ID No. 6)
35 APPCIal62
CTCCTTATCG ATAATGGAGA GTTC (Seq. ID No. 7)
KALPCIal
CTGAGTATCG ATGCTCTCAT TGGCCTG (Seq. ID No.8) FAPLP2Xhol
CCGCTCGAGC TAAATCTGCA TCTGCTCCAG (Seq. ID No. 9)
APPCIal CGAATCGATT CAGGATATGA AGTTCAT (Seq. ID No. 10)
aAAP/LP-2
GTGGGTTCAA GCAGTGCTCT CATTGGCCTG (Seq. ID No. 11)
aALP-2/AP
AGCACTGCTT GAACCCACAT CTTCTGAAAA (Seq. ID No. 12)
AB LP-2/AP
CTCCCGCAGG AACACCAATT TTTGATGATG (Seq. ID No. 13)
AB AP/LP-2 .
TTGGTGTTCC TGCGGGAGGA CTTCAGTCTG (Seq. ID No. 14)
TM5 GGACTAGTGG CCCACTGCGG GAGGACTTC (Seq. ID No.15)
T 3
CCGCTCGAGC CTAAATCTGC ATCTGCTCCA GG (Seq. ID No. 16)
Cloning process
Preparation of the plasmid DNA, restriction enzyme digestion, DNA electrophoresis, DNA ligation and bacterial transformation were carried out according to methods generally known to the person skilled in the art (Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, N.Y.).
4. RNA preparation
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 β-mercaptoethanol, 0.5% lauroylsarcosine (v/v); 0.1% antifoam A (v/v)). 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. The whole is then mixed for a further 10 seconds and centrifuged for 20 minutes (4°C, 10000 g), and the supernatant is transferred into a new Sarstedt tube. The 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 μl of DEPC/water.
5. RT-PCR
18 μl of commercially available Rtase mix are added to 2 μl of RNA (1 μg). Reverse transcription is then carried out: 10 min. 21°C, then 15 min. 42°C, then 5 min. 99°C and cooling to 4°C. For amplification of the cDNA, 80 μl of commercial PCR mix are added per reaction and the appropriate PCR programme is started: 5 min. 95°C: addition of 0.5 μl of Ampli-Taq-polymerase per reaction after 3.5 min. = "hot start"; followed by 30 cycles with: 1 min. 20 sec. 94°C; 1 min. 20 sec. 55°C; 1 min. 20 sec. 72°C; and a cycle for 10 min. at 72°C, after which the samples are cooled to 4°C.
5.1 PCR
The PCR reactions were carried out as follows:
Figure imgf000013_0001
Cycles: 25
denaturation 1 min. 95°C annealing 1 min. 55°C extension 2 min. 72°C final extension 5 min. 72°C
6. Cell culture and transfection:
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 μg/ml) and 10 % (v/v) foetal calf serum (FCS).
For stable transfection, 5 times 105 cells were sown on 60 mm dishes and incubated overnight at 37°C. Once the cells had achieved approximately 80 % confluence, they were transfected by means of lipofectin. For that purpose the cells were washed three times with Opti-MEM I before the DNA/lipofectin solution was added. The DNA/lipofectin solution was prepared beforehand by mixing diluted DNA (10-20 μg of DNA in 1.5 ml of Opti-MEM) and diluted lipofectin (30-50 μg of lipofectin in 1.5 ml of Opti-MEM) and incubated for 30 minutes at room temperature. After an incubation of 8 hours at 37°C, 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 μg/ml the first week, then 300 μg/ml).
7. Metabolic labelling of cell cultures and preparation of cell lysates
After removal of the culture medium, 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 μCi of [35S] 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. For lysis, the centrifuged-off cells were resuspended in 600 μl of sol-buffer (50 mM tris-HCI [pH7.5], 150 mM NaCI, 2 mM
EDTA, 1% Triton X-100 and commercial protease-inhibitor cocktail ) and incubated for 30 minutes on ice. The cell nuclei were centrifuged off at 10000 x g for 5 minutes. The supernatant (cell lysate) was stored at -20°C for further analysis. 8. Immunoprecipitation
The immunoprecipitation was carried out according to the method of Anderson er al. (Methods Enzymol. 96, 110-120). For the immunoprecipitation, 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. For the preincubation, the conditioned medium and the cell lysate were incubated for 30 minutes with 5 μl of pre-immunoserum and 50 μl (5 mg) of protein A/Sepharose. The samples were briefly centrifuged and the supernatants incubated overnight at 4°C with antibody (10 μl of SEAP, 5 μl of A4CT) and 50 μl (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.
9. Tris-tricine-SDS-polyacrylamide gel electrophoresis
The gel electrophoresis was carried out according to Schagger and Jagow ((1987) Analytical Biochemistry 166, 368-379).
10. Precipitation with acetone
200 μl 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 μl of 8M urea for 15 minutes at 56°C, 40 μl 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.
11. Western Blot Analysis
When the gel electrophoresis is complete, the gel is washed for 30 minutes in 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 μm of nitrocellulose. 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. After washing three times with TBST, incubation is carried out for at least 30 minutes at RT with secondary antibody (1 :7500), coupled to peroxidase, in TBST/powdered milk, followed by washing a further three times with TBST and development with a commercial ECL kit.
12. SEAP Assay :SEAP measurement by chemiluminiscence
All steps are carried out in 96-well microtitre plates. One day before the analysis, 75 000 cells are sown per 96 wells. The analysis is carried out in 200 μl of culture medium plus/minus test substance for from 1 to 20 hours at 37°C. Subsequently, 90 μ I of dilution buffer (0.05M tris-HCI, pH 7.4; 0.15M NaCI) are added to 30 μl of the medium and the whole is incubated for 60 minutes at 65°C. After 2 minutes on ice, 100 μl of the heat-treated medium have 100 μl of assay buffer (2M diethaπolamine, pH 7.8, 1 mM MgCl2, 20 mM L-homoarginine) added, and the whole is incubated at room temperature for 5 minutes. 100 μl 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.
13. SEAP Assay: SEAP measurement by absorption
All steps are carried out in 96-well microtitre plates. One day before the analysis, 75 000 cells are sown per 96 wells. The analysis is carried out in 200 μl of culture medium with and without test substance for from 1 to 20 hours at 37°C. Subsequently, 100 μl of the medium are incubated at 65°C for 60 minutes. After 2 minutes on ice, 100 μl of the heat-treated medium have 100 μl of assay buffer (2M diethanolamine, pH 7.8, 1 mM MgCI2l 20 mM L-homoarginine) and 20 μl of p- nitrophenol phosphate (120 mM in assay buffer) added. The evaluation is carried out in an ELISA reader at OD 405 as terminal point determination or as kinetics, the amount of enzyme being calculated as follows: 1 mU SEAP = 0.04 A405 increase/min. The following embodiment Examples illustrate the invention without the invention being limited thereto :
Example 1
Preparation of the cloning and expression vectors
1.1 Vector pBC/SEAP
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.
1.2 Vector SP65/APLP2-CT
The C-terminal 82 amino acids of human APLP2 (Wasco et al., (1993), Nature genetics 5, 95-99) were amplified by means of the above-described RT-PCR and subsequent PCR with the specific oligonucleotide primers TM5 and TM3 and cloned by way of Spel/Xhol into Sp65/preA4, the vector SP65/APLP2-CT being obtained.
1.3 Vector pCEP/SEAPαsecTM1
The APP fragment , amino acids 604 to 622, referred to here as αsec (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 pBCSEAPαsecTM being obtained. For cellular expression, the resulting fusion fragment SEAPαsecTM was cloned by way of Hind3/Xhol into the expression vector pCEP4.
1.4 Vector pCEP/SEAPαsecTM2
The APP fragment, amino acids 604 to 615, referred to here as αsec (numbering according to APP695) 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.
Subsequently, 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 pBCSEAPαsecTM2. For cellular expression, the resulting fusion fragment SEAPαsecTM was cloned by way of Hind3/Xhol into the expression vector pCEP4.
1.5 Vector pCEP/SEAPβsecTM1
The APP fragment with the amino acids 535 to 604, referred to here as βsec,
(numbering according to APP695), was amplified by means of PCR from APP cDNA (Kang et a/., (1987), Nature, 325, 733-736) as template and the oligonucleotide primers APPCIal62 and bALP-2/AP. Then, 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 pBCSEAPβsecTMl . For cellular expression, the resulting fusion fragment
SEAPβsecTMl was cloned by way of Hind3/Xhol into the expression vector pCEP4. 17
1.5a Vector pCEP/SEAPβsecswTM1
Vector pCEP/SEAPβsecswTM1 was generated analogously to pCEP/SEAPβsecTM1 using APP751sw (Urmoneit et al. 1995, Journal of Molecular Neuroscience, Vol. 6, 23-32) instead of APP cDNA as template.
1.6 Vector pCEP/SEAPβsecTM2
The APP fragment, amino acids 535 to 614, referred to here as βsec, (numbering according to APP695), was amplified by means of PCR from APP cDNA (Kang er al., (1987), Nature, 325, 733-736) as template and the oligonucleotide primers APPCIal62 and bCLP-2/AP. Then, the C-terminal 81 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 fragment SP65/APLP2-CT as template and the oligonucleotide primers bCAP/LP-2 and FAPLP2Xhol. 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 pBCSEAPβsecTM2 was obtained. For cellular expression, the resulting fusion fragment SEAPβsecTM2 was cloned by way of Hind3/Xhol into the expression vector pCEP4.
1.6a Vector pCEP/SEAPβsecswTM2
Vector pCEP/SEAPβsecswTM2 was generated analogously to pCEP/SEAPβsecTM2 using APP751sw (Urmoneit et al. 1995, Journal of Molecular Neuroscience, Vol. 6, 23-32) instead of APP cDNA as template.
1.7 Vector pCEP/SEAPTM
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. For cellular expression, 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.
Example 2
Transfection of human neuroblastoma cells Sy5y
Sy5y cells are transfected with the above-described expression vectors. On account of the episomal character of the 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). The stably transfected cells release the reporter (SEAP) into the medium only after α or β-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 α or β-secretase activity in the "high capacity screen"-compatible 96-well format. Published α and β-secretase modulators, such as, for example, phorbol ester and NH4CI, exhibit the expected effects in the system described herein. The above-mentioned positive control (pCEP/SEAPsec) and negative control (pCEP/SEAPTM) confer on the system an additional specificity since, as a result, in the course of an HCS falsely positive compounds can easily be identified by secondary screening (inhibition of the secretion caused by a generally toxic influence and inhibition of the alkaline phosphatase is identified by cells transfected with pCEP/SEAPsec; stimulation of the secretion caused by membrane instability is identified by cells transfected with pCEP/SEAPTM). Example 3
Verification of the processing of the reporter fusion constructs by the corresponding secretase activities
The secretase activities generate, starting from physiological membrane-bound APP, various extracellularly secreted N-terminal fragments (APPsecα, APPsecβ and APPsecγ) and the corresponding intracellular membrane-bound C-terminal fragments (APPsecα => CT; APPsecβ => A4CT; APPsecγ => CT-A4). Provided processing is correct, the expression of the reporter fusion constructs should, after the respective secretase activity, result in a secreted N-terminal fragment (SEΞAP) 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. Mδnning Schering AG )). Compared with the control CEP (pCEP transfected cells) both the SEAPsecαTM2 and the SEAPsecβTMl cells exhibit an additionally generated C-terminal fragment which, on account of its size, runs just below the CT fragment of the endogenous APP (CT in the CEP tracks). The corresponding secreted N-terminal fragment can be seen in Fig. 2 after immunoprecipitation with an antibody to SEAP. In addition to the identification of the secreted reporter, Fig. 2 also exhibits proof for the sequence-specific processing of the
SEAPsecαTM constructs. The antibody R1736 (Dr. Selkoe/Dr. Haass, Brigham and Woman's Hospital, 221 Long Wood Ave, Boston, MA 02119) recognises the C- terminal amino acid of APP, which is released after α-secretase activity. Immuno- reactivity with R1736 thus indicates sequence-specific α-secretase activity. As can be seen in Fig. 2, track α, only the SEAPsecαTM 1 and SEAPsecαTM2 cells secrete significant amounts of SEAP with the R1736 epitope. The small secretion of SEAP with the R1736 epitope in the case of the SEAPsecβTM2 cells results in a residual activity of α-secretase compared with the mutagenised recognition sequence (for details see above under expression vectors). The reporter fusion construct without secretase recognition site (SEAPTM= negative control) shows no reporter activity in the medium. Example 4
Verification of the regulation of the secretase activities with the fusion constructs as substrate
It is known that β-secretase activity can be inhibited by compounds that render alkaline endosomal/lysosomal compartments (NH4CI, chloroquine, methylamine etc.), whilst the α-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 SEAPsecβTM2 but not of SEAPsecαTMl 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. In contrast to NH4CI, phorbol ester (PMA) stimulates α-secretase activity without having an effect on β-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 SEAPαTM cells can be stimulated by PMA by approximately 400%, which agrees very well with the hitherto published data. Under those conditions no, or only a slight stimulation, is observed in the case of the secretion control (SEAPsec cells) and the SEAPsecβTM cells. Also, the specific inhibition of β-secretase activity in the presence of NH4CI is reproduced as shown by the SEAP assay in Fig. 4.
Example 5
Overview of the substances tested in the SEAP assay
Using the cellular test system, various compounds were examined for their activity during the processing of the reporter fusion constructs. The substances tested in the SEAP assay were divided into different classes as shown in the following Table. Substance Incubation Effect on the processing of sec-fusion proteins
Acetylcholinesterase 2 h, 5 h, 16 h inhibitors
Tacrin 10-30 μM at 30 μM 20-30 % inhibition of the SEAP secretion in all constructs
Galanthamin 10-30 μM no effect
Physostigmin 10-30 μM no effect
Growth factors 1-22 h
PDGF 50 ng/ml no effect, no action on starved cells
NGF 50 ng/ml no effect bFGF 5-50 ng/ml, 4 h no effect
IGF-1 100 ng/ml, 2 h no effect
EGF 100 ng/ml, 2 h no effect
Protease inhibitors 1-2 h
Pefabloc 200-400 μM no effect
Aprotinin 0.02-2 μg/ml no effect
Pepstatin 15 μM, 70* μM no effect
Leupeptin 10-100 μM no effect
Phenanthrolin 5-500* μM, 3 h 50 μM inhibits SEAP secretion of all constructs by -30 %
CGA 0.1-10μM, 2 h, 5 h no effect
Phosphoramidon 2, 20 μM no effect
NH4CI 10 mM, 2.5 h inhibits SEAP secretion of SEAPβsec and SEAPβsecsw by -40 %
BafilomycinAI 0.2 μM inhibits SEAP secretion of SEAPβsec and SEAPβsecsw by -35 %
Methylamine 10-30 mM inhibits SEAP secretion of SEAPβsec and SEAPβsecsw by -40 %
Cytokine 2 h
IL-1β 10 ng/ml no effect on SEAPased
IL-4 10 ng/ml -20 % inhibition of SEAP secretion in all constructs
TNF-α 200 ng/ml -30 % inhibition of SEAP secretion in all constructs
Con. medium Mikroglia -/+ LPS stim. no effect arachidonic acid 0.3-3 μM no effect
Figure imgf000024_0001
For each substance group, the range for the incubation time for all substances of the group is quoted, with incubation times that differ therefrom being noted after the amount Concentrations marked with * resulted in a visually detectable change in the morphology of the Sy5y cells
Key
SEAPαsec corresponds to SEAPαsecTM 1 and SEAPsecαTM2
SEAPased . corresponds to SEAPsecaTMI
SEAPasec2 corresponds to SEAPsecaTM2
SEAPβsec. corresponds to SEAPβsecTMl and SEAPβsecTM2
SEAPβsed : corresponds to SEAPsecβTMl
SEAPβsec2. corresponds to SEAPsecβTM2
SEAPβsecsw corresponds to SEAPβsecswTMl and SEAPβsecswTM2
SEAPβsecswl : corresponds to SEAPsecβswTMl
SEAPβsecsw2. corresponds to SEAPsecβswTM2 Example 6
Detection of pharmacological modulators using the process according to the invention and the plasmids and cells according to the invention
The process according to the invention is used as described above for screening for pharmacological modulators of amyloid βA4 release.
The following compounds could be found as modulators
1 , 1 ,2,2-cyclopropane tetracarbonitrile (1) and 9,10-dihydro-12-methylene-ethane anthraceπ-11 -one (2)
Compound (1) and its preparation are described by Wideqvist (Ark. Kemi, 20 B No. 4, p 6, 1945) and Hart et al. (J. Org. Chem. 31 , 2784-2789, 1966).
Compound (2) and its preparation is described by E. Snyder (J. Org. Chem. 25, 1328-
1330, 1960), Hart et al. (J. Amer. Chem. Soc, 95, 6294, 1973) and Varecht et al. (J. Chem. Res. Miniprint, 1979).
SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT.
(A) NAME: Scheπng Aktiengesellschaft
(B) STREET Mϋllerstraβe 172-178
(C) CITY: Berlin (E) COUNTRY. Germany
(F) POSTAL CODE (ZIP). D-13342
(G) TELEPHONE. (030)-4681 2085 (H) TELEFAX (030)-4681 2058
(ii) TITLE OF INVENTION" Process for the determination of APP secretase modulators and the use thereof as agents in the treatment of Alzheimer's disease
(in) NUMBER OF SEQUENCES 16
(iv) COMPUTER READABLE FORM
(A) MEDIUM TYPE. Floppy disk
(B) COMPUTER IBM PC compatible (C) OPERATING SYSTEM PC-DOS/MS-DOS
(D) SOFTWARE Patentin Release #1 0, Version #1.25 (EPO)
(vi) PRIOR APPLICATION DATA
(A) APPLICATION NUMBER DE 19641 180 7 (B) FILING DATE 24-SEP-1996
(2) INFORMATION FOR SEQ ID NO 1
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH. 20 base pairs (B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(xi) SEQUENCE DESCRIPTION SEQ ID NO. 1
CGACTCACTA TAGGGAGACC
(2) INFORMATION FOR SEQ ID NO: 2-
(i) SEQUENCE CHARACTERISTICS. (A) LENGTH. 28 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
CCATCGATCC CCGGGTGCGC GGCGTCGG
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
TTCCGACATA GTAGCAGTGC TCTCATTGGC
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
ACTGCTACTA TGTCGGAATT CTGCATCCAT
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
AAATTGGTGC CACTGCGGGA GGACTTCAGT (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
CCGCAGTGGC ACCAA I I I I I GATGATGAAC
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
CTCCTTATCG ATAATGGAGA GTTC
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
CTGAGTATCG ATGCTCTCAT TGGCCTG
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
CCGCTCGAGC TAAATCTGCA TCTGCTCCAG (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
CGAATCGATT CAGGATATGA AGTTCAT
(2) INFORMATION FOR SEQ ID NO: 1 1 :
(i) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS. single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 1
GTGGGTTCAA GCAGTGCTCT CATTGGCCTG
(2) INFORMATION FOR SEQ ID NO: 12
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH. 30 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS. single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
AGCACTGCTT GAACCCACAT CTTCTGAAAA
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS (A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: CTCCCGCAGG AACACCAATT TTTGATGATG
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS. single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION- SEQ ID NO: 14:
TTGGTGTTCC TGCGGGAGGA CTTCAGTCTG
(2) INFORMATION FOR SEQ ID NO 15.
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH. 29 base pairs
(B) TYPE' nucleic acid (C) STRANDEDNESS single
(D) TOPOLOGY linear
(xi) SEQUENCE DESCRIPTION. SEQ ID NO: 15.
GGACTAGTGG CCCACTGCGG GAGGACTTC
(2) INFORMATION FOR SEQ ID NO 16
(i) SEQUENCE CHARACTERISTICS (A) LENGTH 32 base pairs
(B) TYPE- nucleic acid
(C) STRANDEDNESS. single
(D) TOPOLOGY linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16.
CCGCTCGAGC CTAAATCTGC ATCTGCTCCA GG

Claims

Patent Claims
1. Process for the determination of the activity of APP secretase in intact cells and for the identification of modulators of APP secretase activity, 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.
2. Process for the quantitative and differential determination of the APP secretases involved in amyloid βA4 release and for the direct isolation of corresponding modulators, 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 α- or β-secretase, with ii) a suitable transmembrane anchor sequence and iii) expression under the control of a suitable promoter, b) eukaryotic ceils 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 α- or β-secretase activity are determined.
3. Process according to claim 2, characterised in that, in accordance with step b) human neuroblastoma cells are stably transfected with the expression vector so produced.
4. Process according to claim 1 or 2, characterised in that the expression vectors that may be used in process step a) are the vectors pCEP/SEAPαsecTM1 , pCEP/SEAPαsecTM2, pCEP/SEAPβsecTM1 , pCEP/SEAPβsecTM2, pCEP/SEAPβsecswTM1 and pCEP/SEAPβsecswTM2.
5. Process according to any one of claims 2 to 4, characterised in that the secretory protein that may be used in process step a) i) is the secretory form of alkaline phosphatase or urokinase.
6. Process according to any one of claims 2 to 5 characterised in that the transmembrane anchor sequences that may be used in process step a) ii) are the sequences of human APLP2.
7. Process according to any one of claims 2 to 6, characterised in that the promoter that may be used in process step a) iii) renders possible expression in eukaryotic systems.
8. Process according to any one of claims 2 to 7, characterised in that the promoter that may be used in process step a) iii) is a eukaryotic or viral promoter.
9. Process according to any one of claims 2 to 8, characterised in that the promoter that may be used in process step a) iii) is the Cytomegalovirus promoter.
10. Process according to any one of claims 1 to 9, characterised in that the cells used in process step b) are Syδy cells.
11. Process according to any one of claims 2 to 10, characterised in that the selection marker used in process step c) is hygromycin.
12. Process according to any one of claims 2 to 11 , characterised in that the vectors suitable for use in process step e) are, for the negative control, the vector pCEP/SEAPTM and, for the positive control, the vector pCEP/SEAPsec.
13. Modulator, obtainable in accordance with the process according to any one of claims 1 to 12.
14. Modulator, obtained in accordance with the process according to any one of claims 1 to 12.
15. Modulator according to claim 13 or 14, characterised in that it is the compound 1 ,1,2,2-cyclopropane tetracarbonitrile or 9,10-dihydro-12-methylene-ethane anthracen-11-one.
16. Pharmaceutical agent, comprising at least one modulator according to any one of claims 13 to 15 with customary formulation substances and additives.
17. Pharmaceutical agent according to claim 16, for the treatment of Alzheimer's disease.
18. Use of a modulator obtainable by the process according to any one of claims 1 to 15 in the manufacture of an agent for the treatment of Alzheimer's disease.
19. Use of a modulator obtained by the process according to any one of claims 1 to 15 in the manufacture of an agent for the treatment of Alzheimer's disease.
20. Expression vector prepared by i) recombinant fusion of the secretory form of a protein (reporter), which secretory form is fused to the recognition sites for APP α- or β- secretase, with ii) a suitable transmembrane anchor sequence and iii) expression of the protein under the control of a suitable promoter.
21 . Expression vector according to claim 20, characterised in that the vector is pCEP/SEAPαsecTM1 , pCEP/SEAPαsecTM2, pCEP/SEAPβsecTM1 , pCEP/SEAPβsecTM2, pCEP/SEAPβsecswTM1 or pCEP/SEAPβsecswTM2.
22. Cell, transfected with an expression vector according to claim 20 or 21.
23. Human neuroblastoma cell, transfected with an expression vector according to claim 20 or 21.
24. Sy5y cell, transfected with an expression vector according to claim 20 or 21.
25. Use of the transfected cells according to any one of claims 22 to 24 in the identification of APP secretase modulators.
26. Kit for the determation of the activity of APP secretase in intact cells and for the identification of modulators of APP secretase activity, 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.
27. Kit, according to claim 26, for the quantitative and differential determination of the APP secretases involved in amyloid βA4 release and for the direct isolation of corresponding modulators, 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 α- or β-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 ceils 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 α- or β-secretase activity are determined.
28. Kit, according to claim 27, characterised in that in b) human neuroblastoma cells are stably transfected with the expression vector so produced.
29. Kit, according to claim 27, characterised in that in b) Sy5y cells are stably transfected.
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DE19920514A1 (en) * 1999-05-05 2000-11-16 Boehringer Ingelheim Pharma Methods for finding proteases that specifically cleave membrane-bound substrates
US6245884B1 (en) 1998-10-16 2001-06-12 Vivian Y. H. Hook Secretases related to alzheimer's dementia
WO2001075088A1 (en) * 2000-04-05 2001-10-11 Esbatech Ag Method for identify polypeptides with protease activity
US6313268B1 (en) 1998-10-16 2001-11-06 Vivian Y. H. Hook Secretases related to Alzheimer's dementia
US6420534B1 (en) 1998-09-24 2002-07-16 Pharmacia & Upjohn Company Alzheimer's disease secretase, APP substrates therefor, and uses thereof
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US6627739B1 (en) 1999-02-10 2003-09-30 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
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