WO2001016355A2 - SYSTÈME DE TEST IN VITRO POUR LA η-SECRÉTASE - Google Patents

SYSTÈME DE TEST IN VITRO POUR LA η-SECRÉTASE Download PDF

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Publication number
WO2001016355A2
WO2001016355A2 PCT/EP2000/008340 EP0008340W WO0116355A2 WO 2001016355 A2 WO2001016355 A2 WO 2001016355A2 EP 0008340 W EP0008340 W EP 0008340W WO 0116355 A2 WO0116355 A2 WO 0116355A2
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Prior art keywords
secretase
membranes
test kit
cells
substrate
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PCT/EP2000/008340
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German (de)
English (en)
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WO2001016355A3 (fr
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Katja Fechteler
Klaus Fuchs
Marcus Kostka
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Boehringer Ingelheim Pharma Kg
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Priority to EP00962393A priority Critical patent/EP1212454A2/fr
Priority to MXPA02001754A priority patent/MXPA02001754A/es
Priority to CA002381952A priority patent/CA2381952A1/fr
Priority to JP2001520900A priority patent/JP2003508058A/ja
Publication of WO2001016355A2 publication Critical patent/WO2001016355A2/fr
Publication of WO2001016355A3 publication Critical patent/WO2001016355A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4709—Amyloid plaque core protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96472—Aspartic endopeptidases (3.4.23)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention is in the field of methods for finding specific ⁇ -secretase inhibitors which can be used for the treatment of neurodegenerative diseases, in particular methods which can be carried out in vitro. Furthermore, this invention relates to a test kit with which the method according to the invention can be carried out and the use of this test kit or the method for finding substances which specifically inhibit ⁇ -secretase. Another embodiment relates to the use of these substances for the manufacture of a medicament for the treatment of neurodegenerative diseases and pharmaceutical formulations which contain these substances.
  • Aß amyloid- ⁇ -peptide
  • APP amyloid precursor protein
  • the ⁇ -secretase In the non-amyloidogenic metabolism of the APP, the ⁇ -secretase cleaves within the Aß region of the APP and thus leads to the secretion of the soluble N-terminal region of the protein ( ⁇ -APPs) and, after a ⁇ -secretase cut, to the release of p3.
  • the amyloidogenic pathway leads to the formation of Aß in that two proteases generate the N-terminus (ß-secretase) and the C-terminus ( ⁇ -secretase) of Aß (Haass, 1993; Selkoe, 1994).
  • Aß can be detected in vivo in human plasma and cerebrospinal fluid.
  • Aß secreted Aß can be detected in the cell culture supernatant in various cell types that endogenously express or overexpress APP or fragments thereof. Both Aß production and thus amyloid plaque formation are influenced by various genetic risk factors. This includes mutations in the homologous proteins presenilin 1 and presenilin 2 as well as in the APP itself. The analysis of these mutations in fibroblasts from Alzheimer's patients with familial inherited Alzheimer's disease Disease "(FAD)) showed the influence that they have on the formation of Aß. This could be confirmed by studies on transfected cells and transgenic animals.
  • FAD familial inherited Alzheimer's disease Disease
  • PS Presenilin
  • the present invention is in the field of methods for finding specific ⁇ -secretase inhibitors which can be used for the treatment of neurodegenerative diseases, in particular methods which can be carried out in vitro. Furthermore, this invention relates to a test kit with which the method according to the invention can be carried out, and to the use of this test kit or the method for finding
  • Another embodiment relates to the use of these substances for the manufacture of a medicament for the treatment of neurodegenerative diseases and pharmaceutical formulations which contain these substances.
  • the H4-ind / APP-LC99 cells were grown in the absence of doxycycline for three days to induce the expression of LC99.
  • PNS was prepared as described and further enriched with sucrose step gradients (Taylor et al. 1997)
  • An anti-calreticulin antibody (Stressgen Biotechnologies; 1: 2000) was used to show the accumulation of membranes of the endoplasmic reticulum in the SBI fraction.
  • Antibodies Transduction Laboratories Inc. are shown.
  • Fig. 2 Cell-free generation of Aß 40 and Aß 42 from isolated microsomes
  • the specific antibodies BI.40 and BI.42 were used to precipitate the Aß40 and Aß42 peptides alone.
  • the precipitated proteins were separated with a Tris-Bicine gel, transferred to a nitrocellulose membrane and visualized with the antibodies 6E10 and 4G8, using a highly sensitive Western blot procedure (Ida et al., 1996).
  • immunoprecipitations were carried out directly with the microsomal fraction stored at - 80 ° C (lane c).
  • the Aß40 and Aß42 generated in vitro migrate with the synthetic Aß peptides. Lower part: Longer exposure
  • the SDI fraction was prepared as described and incubated at 37 ° C or 4 ° C under neutral conditions (pH 6.8) for the indicated time. Ate peptides were immunoprecipitated with the specific antibodies BI.40 and BI.42. The precipitated proteins were separated using Tris-Bicine gel electrophoresis (Klafki et al., 1996) and then using a highly sensitive Westemblot procedure with the antibodies 6E10 and 4G8 detected (Ida et al., 1996). The synthetic Aß40 and Aß42 peptides were used as standard. After 3-4 hours of incubation at 37 ° C, the de novo Aß generation reached a maximum.
  • the SHI fraction was generated as described and incubated under standard conditions (37 ° C; pH 6.8; 4 hours) in the presence or absence of cation chelators such as EDTA or BAPTA. As a control, the microsomes were incubated at 4 ° C in the presence of EDTA to.
  • a ⁇ peptides were immunoprecipitated with the specific antibodies BI.40 and BI.42 and detected by Westemblot with the antibodies 6E10 and 4G8 (Senetek, Great Britain; Galli et al., 1998) as described.
  • the synthetic peptides AJ340 and Aß42 served as standard. All reactions were carried out in triplicate. In the absence of a chelator, de novo Aß production is drastically reduced.
  • Fig. 4 The ⁇ -secretase is a transmembrane protease.
  • the SHI fraction was prepared as described and incubated under standard conditions (pH 0 6.8; 5 mM EDTA; 4 hours) at 37 ° C. or 4 ° C. as a control.
  • the microsomal membranes were either washed with high saline (IM KC1) or extracted with Na 2 CO 3 to remove weakly bound proteins from the microsomal membranes.
  • IM KC1 high saline
  • the pelleted membranes were resuspended in KCl-PufFer (1 M KC1, 250 mM sucrose, 20 mM
  • Fig. 5 The optimum pH for the ⁇ -secretase activity lies between pH 6.8 and 7.4
  • the SHI fraction was prepared as described and incubated at the specified pH values under standard conditions (pH 6.8; 5 mM EDTA; 4 hours).
  • Aß peptides were immunoprecipitated with the specific antibodies BI.40 and BI.42 and detected by Westemblot with the antibodies 6E10 and 4G8 as described. All reactions were carried out in duplicate.
  • the in vitro reaction was carried out at 4 ° C. and pH 6.8.
  • the pH optimum for the in vitro ⁇ -secretase activity lies in the neutral range between pH 6.8 and pH 7.4. Heavily reduced ⁇ -secretase activity was found under both slightly acidic and basic pH conditions.
  • the SIH fraction was prepared as described and incubated at pH 6.8 under standard conditions in the presence or absence of various concentrations of compound A (Fig .: A) (pH 6.8; 5 mM EDTA; 4 hours ).
  • Aß peptides were immunoprecipitated with the specific antibodies BI.40 and BI.42: and detected by means of Westemblot with the antibodies 6E10 and 4G8 (Senetek, Great Britain; Galli et al., 1998) as described. All reactions were carried out two or three times. As a control, the reactions were carried out at 4 ° C.
  • Compound A showed a concentration-dependent inhibition of the in vitro Aß generation.
  • the quantification of the de novo generated Aß was carried out with the Chemiluminescence Imaging System (Biorad) and is shown in the lower part.
  • Compound A was also active in a cellular test system in which the extracellular content of Aß40 and 42 is determined, which is secreted by the U373 cell line (U373: ATCC No. HTB 14).
  • This cell line U373 / APP751 is an astrocytoma cell line that overexpresses human APP 751 and large amounts of Aß40 ( ⁇ 1000pg / ml / 4 hours with 5x10 7 cells in 15 ml Medium) and Aß42 ( ⁇ 100pg / ml / 4 hours with 5x10 7 cells in 15 ml medium).
  • the determination is carried out by ELISA (ELISA: “Enzyme linked immunosorbent assay” (Steiner et al., 1998).
  • the Aß40 secretion was greatly reduced by the compound A in a concentration-dependent manner, the Aß42 secretion being slightly increased at subinhibitory doses and then also inhibited at higher doses.
  • the SÜI fraction was prepared as described and incubated at pH 6.8 under standard conditions in the presence or absence of various concentrations of the compound MG132 (Biomol order number: PI-102; De Strooper et al. 1999) (pH 6 , 8; 5 mM EDTA; 4 hours).
  • Aß peptides were immunoprecipitated with the specific antibodies BI.40 and BI.42 and detected by means of Western emblot with the antibodies 6E10 and 4G8 (Senetek, Great Britain; Galli et al., 1998) as described. All reactions were carried out in duplicate. As a control, the reactions were carried out at 4 ° C. or 37 ° C. without an inhibitor.
  • the compound MGI 32 showed a concentration-dependent inhibition of the in vitro Aß generation.
  • Fig. 8 Characterization of the microsomal fractionation of H4indLC99 cells
  • the H4-ind / APP-LC99 cells were grown in the absence of doxycycline for three days to induce the expression of LC99.
  • the fractions were prepared as described
  • An anti-PDI antibody (Stressgen Biotechnologies; 1: 2000) was used to show the accumulation of membranes of the endoplasmic reticulum in the Mi fraction.
  • the SHI fraction of the first microsomal fractionation was also applied.
  • PDI is enriched in the microsomal fraction.
  • microsomal fraction is free of lysosomal proteins.
  • PNS of the H4indLC99 cells was also plotted.
  • Microsomes (Mi fraction) were prepared as described and incubated at pH 6.8 under standard conditions (pH 6.8; 5 mM EDTA; 4 hours).
  • Aß peptides were immunoprecipitated with the specific antibodies BI.40 and BI.42 and by means of Western emblot with the antibodies 6E10 and 4G8 (Senetek, Great Britain; Galli et al.,
  • an in vitro test system for finding substances which can specifically inhibit ⁇ -secretase is provided.
  • a test kit for finding substances which can specifically inhibit ⁇ -secretase is disclosed.
  • a further embodiment of the invention is the use of the method according to the invention or the test kit according to the invention for finding substances which can specifically inhibit ⁇ -secretase.
  • substances are provided that can be found using the method according to the invention or the test kit according to the invention. Another embodiment relates to the use of these substances for the manufacture of a medicament for the treatment of neurodegenerative diseases and pharmaceutical formulations which contain these substances.
  • the method according to the invention uses purified membranes which are isolated from cells which detectably express a substrate of ⁇ -secretase and which have ⁇ -secretase activity.
  • ⁇ -secretase is to be understood as a protein which has the property of proteolytically cleaving APP or fragments thereof, in particular the C99 peptide (Shoji et al., 1992), in p3 or Aß.
  • all cells are suitable for the method according to the invention in which the person skilled in the art can detect a substrate of ⁇ -secretase or its cleavage products by means of Western blot and the use of specific antibodies.
  • Suitable ⁇ Membranes are all membranes in which the person skilled in the art can detect a substrate of ⁇ -secretase using Western blot and the use of specific antibodies, preferably lysosomal and endosomal membranes, particularly preferably microsomal membranes.
  • Methods for the specific purification of membranes are known to the person skilled in the art from Methods in Enzymology, Vol. 219, title: Reconstitution of intracellular Transport, and the book “Biochemical Working Methods”, TG Cooper, De Gruyter Verlag, 1981.
  • cells are transfected with a DNA sequence (DNA: deoxyribonucleic acid) which codes for a substrate of the ⁇ -secretase
  • DNA deoxyribonucleic acid
  • the expression of this substrate can be induced by the removal of doxycycline
  • the cells can be opened and a post-nuclear supernatant (abbr .: PNS) can be prepared, which is further processed, for example to isolate the microsomal fraction.
  • This fraction can be incubated under suitable conditions and the formation of the product of the reaction of the ⁇ -secretase with a suitable substrate can be determined by immunoprecipitation and subsequent detection by means of suitable antibodies in the Western blot method.
  • the ⁇ -secretase substrate could be found in the PNS and in a higher concentration in the microsomal fraction (FIG. 1A).
  • the C-terminal fragments (CTF) of PSl and PS2 were also enriched in the SDI fraction (Fig. 1B).
  • the microsomal fraction contained no endosomal membranes, but ER and Golgi compartments were enriched (Fig. IC).
  • De novo generated Aß could be found in the microsomal fractions which had been incubated at 37 ° C (Fig. 2A). Incubation at 4 ° C prevented the formation of de novo Aß. Small amounts of Aß were due to the existence of intracellular Aß in the freshly prepared microsomal fractions (Fig. 2A, lane c).
  • the cleaned membranes described, in particular microsomal membranes are mixed with a suitable reaction buffer and a test substance.
  • a test substance is to be understood as any substance which is to be tested for whether it could have an inhibitory effect on the ⁇ -secretase activity.
  • the mixture is then incubated under conditions under which the substrate of the ⁇ -secretase is cleaved in the absence of the test substance.
  • the amount of a fission product formed is then determined and the value obtained is compared with the value obtained in the absence of the test substance.
  • the test substance inhibits the formation of the cleavage product and the test substance is an inhibitor of ⁇ -secretase.
  • compounds were identified which were designated as specific ⁇ -secretase inhibitors and had an inhibitory activity on the secretion of Aß40 and Aß42 without influencing the formation of Aß.
  • specific ⁇ -secretase inhibitors can now advantageously be identified and validated and differentiated from inhibitors which prevent the secretion of Aß40 and Aß42. As described in the example, a compound was tested with the test system according to the invention.
  • astrocytoma cell line (U373) is used which overexpresses wild type APP751 and secretes detectable amounts of both Aß species.
  • a concentration-dependent reduction in the amount of extracellular Aß40 and Aß42 was observed after overnight treatment with compound A (FIG. 6B).
  • cells are cultivated which express an endogenous polypeptide which is a substrate of the ⁇ -secretase.
  • endogenous is understood to mean that this cell or cell line expresses the designated polypeptide in a sufficient amount without further manipulations, for example by means of genetic engineering methods, being necessary.
  • a sufficient amount of substrate of the ⁇ -secretase is to be understood as an amount which, in an established biochemical detection method (for example ELISA, Western Blot) using specific antibodies, results in a detectable signal lying above the background.
  • cells are cultivated which express an exogenous polypeptide which is a substrate of the ⁇ -secretase.
  • exogenous means that this cell or cell line is manipulated by means of genetic engineering methods in such a way that it expresses the ⁇ -secretase substrate.
  • the cell or cell line contains the ⁇ -secretase substrate even without said manipulations, this term is intended to express that the amount of the ⁇ -secretase substrate is measurably increased compared to the value without manipulation.
  • a nucleotide sequence which codes for the amino acid sequence of the ⁇ -secretase substrate can be introduced into a suitable expression cassette of a eukaryotic expression vector.
  • Suitable expression cassettes have a promoter which is functional in eukaryotic hosts, such as, for example, the cytomegalovirus promoter (CMV promoter) and a functional polyadenylation signal, for example from the SV40 virus (English: "Simian virus”; abbr .: SV).
  • Suitable expression vectors are vectors which can replicate in eukaryotic hosts, ie have a functional origin of replication. These expression vectors can be episomal after transfection or can be integrated into the genome if they carry suitable sequences which enable integration.
  • an expression system which allows the expression of the exogenous polypeptide to be induced, various systems being able to be used here, e.g. the "Tet-on” or “Tet-ofP” system (US Patent 5,464,758; Gossen and Bujard, 1992, 1995, sold by Clontech, Heidelberg) or the LacSwitch system (see US Patent. 5,589,392; sold by Stratagene
  • the expression of the ⁇ -secretase substrate is induced by withdrawal of tetracycline or doxycycline (“tet-off” system)
  • a further nucleotide sequence can also be found on the same plasmid, a further plasmid or integrated chromosomally, which codes for a fusion protein between the tet repressor and an acidic, activating domain which is constitutively expressible.
  • An acidic, activating domain is to be understood as a protein domain which has a high proportion of acidic amino acids and which has the property of mediating the transcription of a gene if the domain is introduced in a suitable position in the transcription complex located in front of the gene.
  • This property can be determined by the known "one-hybrid assay".
  • a DNA-binding domain is fused to a domain to be examined, the DNA-binding domain binding to a sequence which is located in front of a reporter protein, and the activity of said reporter protein is measured (Clontech, Heidelberg).
  • expression of the ⁇ -secretase substrate will not take place if the concentration of tetracycline or a derivative of tetracycline such as doxycycline in the cell exceeds a certain value because the tetracycline repressor binds tetracycline or the derivative thereof, consequently, does not bind to its binding site in the DNA and therefore does not induce the transcription of the gene behind this binding site which codes for the ⁇ -secretase substrate.
  • the fusion protein between the Tet repressor and the acidic activation domain binds to its DNA binding site and induces the transcription of the gene behind it, which codes for the ⁇ -secretase substrate. This ensures that a controlled and targeted expression of the ⁇ -secretase substrate takes place.
  • the ⁇ -secretase substrate is the amyloid precursor protein (APP) or a fragment thereof, provided that it contains the ⁇ -secretase cleavage site.
  • the ⁇ -secretase substrate the C99 fragment of the amyloid precursor protein (Shoji et al., 1992).
  • the ⁇ -secretase substrate can be membrane-associated in general, but preferably membrane-attached.
  • membrane-bound should be understood to mean that the substrate is an integral part of the membrane.
  • membrane-associated should be understood to mean that the substrate is bound to the surface of the membrane or to integral membrane proteins.
  • This definition for membrane-associated substrates is also intended to include substrates which interact via chemical groups with the hydrophobic part of the membrane, caused by post-translational Modifications have been added.
  • membrane-associated substrates should also include substrates that interact with the hydrophobic part of the membrane via amino acid side chains, albeit to a lesser extent than the integral membrane proteins.
  • prostaglandin synthetase should be mentioned here.
  • the ⁇ -secretase substrate is a fusion protein of a reporter protein with the amyloid precursor protein or a fragment thereof, provided that it contains the ⁇ -secretase cleavage site.
  • the ⁇ -secretase substrate is a fusion protein between a reporter phrotein and the C99 fragment.
  • a reporter protein is to be understood as a protein which has the property of generating an easily detectable signal and whose amount correlates with the amount of the cleavage product of interest. The signals are generated either by determining the enzymatic activity of the reporter protein with easily detectable substrates or by measuring the intensity of the fluorescence of the reporter protein.
  • report proteins are the green fluorescent protein (GFP; green fluorescent protein; see e.g. WO95 / 07463) or derivatives thereof fluorescent in other wavelength ranges or enzymes such as luciferase, the secretory alkaline phosphatase or the ⁇ -galactosidase.
  • GFP green fluorescent protein
  • the fusion protein of the cleavage product with the reporter phrotein is separated from the fusion protein of the uncleaved ⁇ -secretase substrate with the reporter phrotein e.g. separated by immunoprecipitation. This can be done with selectively recognizing the cleavage product.
  • the amount of the report protein is then determined using the methods mentioned, which are dependent on the properties thereof.
  • the fusion proteins mentioned can be produced by conventional genetic engineering methods (Sambrook et al., 1989) if the DNA coding for the reporter phrotein and the ⁇ -secretase substrate is available.
  • the DNA encoding the report proteins can e.g. obtained from commercial providers, such as Clontech, Heidelberg, and can be used in the desired vectors by standard methods (Sambrook et al., 1989).
  • the DNA coding for the ⁇ -secretase substrate or for the C99 fragment can be obtained using standard methods from suitable gene banks (Sambrook et al., 1989).
  • Cells or cell lines known to the person skilled in the art are suitable for carrying out the invention, in particular eukaryotic cells or cell lines.
  • Cells or cell lines which are used in neurological or neurobiological research are preferred, for example Mammalian cells or cell lines such as H4, U373, NT2, HEK 293, PC12, COS, CHO, fibroblasts, myeloma cells, neuroblastoma cells, hybridoma cells, oocytes, embryonic stem cells.
  • Insect cell lines for example using baculovirus vectors such as pPbac or pMbac (Stratagene, La Jolla, CA)
  • yeast for example using yeast expression vectors such as pYESHIS (Invitrogen, CA)
  • fungi are also suitable.
  • Cells or cell lines of neuronal or glial origin are particularly preferred.
  • the cells used are H4 cells, human neuroglioma cells from the brain, which are listed under the ATCC number HTB-148 at the "American Type Culture Collection (ATCC)" in Manassas, Virginia, USA.
  • ATCC American Type Culture Collection
  • purified cellular membranes are used, preferably intracellular membranes, particularly preferably purified lysosomal or endosomal membranes.
  • Microsomal membranes are particularly preferably used, which are purified from the cells used by lysing the cells, removing the cell nuclei, cleaning via sucrose density gradients, renewed sedimentation by ultracentrifuge, homogenization, renewed sedimentation by ultracentrifuge and renewed homogenization. Standard procedures for the purification of membranes are described in Methods in Enzymology, Vol. 219, and in the book “Biochemical Working Methods", TG Cooper, De Gruyter Verlag, 1981.
  • the reaction buffer has a pH in the range from 5-10, preferably in the range from 6.0 to 8.0, particularly preferably from 6.8 to 7.4 and also contains at least one membrane stabilizer
  • membrane stabilizer is to be understood as meaning a substance which prevents the aggregation of the membranes.
  • membrane aggregation is to be understood to mean clumping or aggregation and possibly subsequent fusion of vesicles or liposomes or else multilamellar structures the experimentally determinable increase in turbidity or light Scattering of a solution or suspension to be examined can be determined, whereby the property of a substance can also be determined with regard to its membrane-stabilizing effect.
  • the membrane stabilizer in the context of this invention is preferably sucrose or sorbitol, although those skilled in the art can replace them with substances having the same effect.
  • the concentration of the Membrane stabilizer in the reaction buffer between 200 and 1000 mM, preferably between 200 and 500 mM, particularly preferably between 200 and 300 mM.
  • the reaction buffer additionally contains a complexing agent, preferably for divalent ions.
  • a complexing agent preferably for divalent ions.
  • This can e.g. Ethylene diamine tetraacetic acid (EDTA) or a salt thereof, 1,2-bis (o-aminophenoxy) ethane-N, N, N ', N'-tetraacetic acid (BAPTA) or a salt thereof or ethylene glycol bis (b- aminoethyl) -N, N, N ', N'-tetraacetic acid (EGTA) or a salt thereof.
  • the complexing agent is preferably present in a concentration of 0.1 to 20 mM, preferably 5 to 10 mM.
  • Complexing agents are compounds that are capable of forming complexes as ligands, i.e. especially for complexing and masking metals, especially divalent metal ions.
  • the term is often used synonymously for chelating agents.
  • Further complexing agents can also be used in the process according to the invention.
  • an ATP-regenerating system is additionally added to the reaction mixture.
  • This system contains adenosine triphosphate (ATP), guanosine triphosphate (GTP), phosphocreatine, creatine phosphokinase, preferably in a concentration of 1 mM ATP, 0.1 M GTP, 8 mM phosphocreatine, 31 mM creatine phosphokinase at a pH in the neutral range, preferably between 6.8 and 7.2, particularly preferably at a pH of 7.0.
  • the amount of the cleavage product is determined by immunoprecipitation or western blot, preferably by a combination of immunoprecipitation and western blot. The immunoprecipitation and the Western blot are carried out as described by Ida et al. (1996).
  • Western blot is a method in which proteins are separated according to their charge in the native or mostly in the denatured state by means of gel electrophoresis, mostly polyacrylamide gel electrophoresis, are transferred to carriers, e.g. Nitrocellulose or polyvinylidene difluoride and then detected using antibodies.
  • the amount of the cleavage product is determined by enzyme-linked immunosorbent assay (abbr .: ELISA) (Steiner et al., 1999) or by mass spectrometry.
  • the amount of the cleavage product is determined by determining the amount of the report protein fused to the cleavage product after it has been separated from the fusion protein between uncleaved ⁇ -secretase ⁇ r Substrate and Reporter ⁇ rotein was cleaned.
  • the amount of luciferase, secretory alkaline phosphatase or ⁇ -galactosidase fused to the cleavage product is determined by determining the enzymatic activity of the report protein after addition of the substrate.
  • the amount of the green fluorescent protein or a derivative thereof is determined by measuring the intensity of the fluorescent light.
  • a preferred embodiment of the invention is a test kit according to the invention for finding substances which can specifically inhibit ⁇ -secretase.
  • a test kit is a compilation of all components for the method according to the invention.
  • Non-exhaustive examples of further elements for carrying out the method according to the invention are vessels such as e.g. B. 96-hole plates or microtitre plates, reaction vessels, other suitable vessels, surfaces and substrates, membranes such as nitrocellulose filters, washing reagents and buffers or the like.
  • a test kit can contain reagents that can detect bound antibodies, such as. B. labeled secondary antibodies, chromophores, enzymes (z. B. conjugated to Antikö ⁇ er) and their substrates or other substances that can bind Antikö ⁇ er.
  • the test kit according to the invention contains at least purified cellular membranes from cells which have ⁇ -secretase activity and contain a substrate of ⁇ -secretase, and reaction buffer.
  • the membranes are purified intracellular membranes, preferably lysosomal or endosomal, particularly preferably microsomal membranes.
  • the membranes were particularly preferably purified from cells which exogenously express a substrate of the ⁇ -secretase.
  • the reaction buffer has a pH in the range from 5-10, preferably in the range from 6.0-8.0, particularly preferably in the range from 6.8 to 7.4, and contains as further components at least one inventive membrane stabilizer as described above.
  • the concentration of the membrane stabilizer, which is preferably sucrose or sorbitol, in the reaction buffer is preferably between 200 and 1000 mM, preferably between 200 and 500 mM, particularly preferably between 200 and 300 mM.
  • the reaction buffer particularly preferably additionally contains a complexing agent according to the invention, preferably for divalent ions. As described above, this can be, for example, EDTA, BAPTA or EGTA or a salt thereof.
  • the complexing agent is present in a concentration of 0.1 to 20 mM, preferably 5 to 10 mM.
  • the test kit additionally contains an ATP-regenerating system according to the invention as described above, which can be added to the reaction mixture.
  • the test kit contains antibodies which make it possible to determine the amount of the cleavage product by immunoprecipitation or western blot, preferably by a combination of immunoprecipitation and western blot.
  • the method according to the invention or the test kit according to the invention is used to find substances which can specifically inhibit ⁇ -secretase.
  • a substance is provided which can be found using the method according to the invention or the test kit according to the invention and which specifically inhibits the proteolytic cleavage of a ⁇ -secretase substrate.
  • the substance according to the invention can be used to produce a medicament for the treatment of neurodegenerative diseases, in particular Alzheimer's disease.
  • pharmaceutical formulations are provided which contain a substance according to the invention and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier may contain physiologically acceptable compounds, e.g. increase the stability or absorption of the substance according to the invention.
  • physiologically acceptable compounds include e.g. Carbohydrates such as glucose, sucrose or dextrans, antioxidants such as ascorbate or glutathione, chelating agents, low molecular weight proteins or other stabilizers (see e.g. Remington's Pharmaceutical Sciences (1990)).
  • physiologically acceptable compounds include e.g. Carbohydrates such as glucose, sucrose or dextrans, antioxidants such as ascorbate or glutathione, chelating agents, low molecular weight proteins or other stabilizers (see e.g. Remington's Pharmaceutical Sciences (1990)).
  • a pharmaceutically acceptable carrier including a physiologically acceptable compound e.g. depends on the administration route.
  • H4 neuroglioma cells (accession number HTB 148 with the "American Type Culture Collection", Manassas, Virginia, USA) were transfected under standard conditions with the regulato ⁇ lasmid pUHD15-lneo (pUHD15-l with neomycm resistance gene), which carries the gene for a tetracycline-repressible transactivator (Gossen and Bujard, 1992, 1995.)
  • pUHD15-l with neomycm resistance gene carries the gene for a tetracycline-repressible transactivator
  • an individual clone was selected for the second stable transfection with the APP-LC99 construct, which had a strictly regulated and a strongly inducible transient expression of a reporter gene (pUHD10-3 / SEAP; SEAP : Secretory alkaline phosphatase)
  • a sequence containing the N-terminal signal sequence and the last 99 amino acids of the APP was controlled via B
  • the cell clone obtained as described above was treated with 10 ⁇ g pUHD10-3 / APP-LC99 and 1 ⁇ g pTK-Hyg (Clontech, Heidelberg; Genbank accession number U40398) and the selection was carried out using the Boehringer Mannheim Fugene transfection system according to the manufacturer's instructions. Individual hygromycin-resistant cell clones were examined for the inducible expression of LC99 by withdrawal of doxycycline and subsequent immunofluorescence and / or Western blot with an APP-CTF-specific antibody. The selected clone was named H4-ind / APP-LC99.
  • the H4-ind / APP-LC99 cells were left at 37 ° C., 5% CO 2 with DMEM medium (DMEM: "Dulbecco's Modified Eagle Medium” (Engl.), Sold by Bio Wittacker) and 10% fetal calf serum ( Engl .: “Fetal Calf Serum", FCS), 1% glutamine, 1% penicillin and streptomycin in the absence of doxycycline on 15 cm Petri dishes to confluence. Expression of the fusion protein was induced by withdrawal of doxycycline. All steps of the preparation of the post-nuclear supernatant were carried out on ice or at 4 ° C.
  • the cells were removed from the Petri dishes with a cell scraper. After centrifugation at 500 g for 10 min, the cells were carefully resuspended in HIS buffer (250 mM sucrose, 5 mM imidazole, 10 mM HEPES pH 6.8), centrifuged again at 1400 g for 10 min and then in 300 ⁇ l HIS + Buffer ⁇ S (HIS buffer with 5 mM EDTA) resuspended per petri dish. The cell homogenate was pressed through a 22 gauge needle using a 1 ml syringe and the lysis of the cells was checked by phase contrast microscopy.
  • HIS buffer 250 mM sucrose, 5 mM imidazole, 10 mM HEPES pH 6.8
  • the cell lysate was centrifuged at 2500 g for 10 min to separate the supernatant from the intact cells and the cell debris.
  • the PNS was worked up further with a sucrose step gradient (Taylor et al., 1997), whereby this was first adjusted to 100 mM KHJO K 2 HPO, pH 6.8. All sucrose solutions contained 100 mM KHJO K 2 HPO 4 , pH 6.8, 5 mM MgCl 2 and the protease inhibitors leupeptin (10 ⁇ g / ml) and aprotinin (10 ⁇ g / ml).
  • the gradient contained levels of 1.3 M, 0.86 M and 0.5 M sucrose, which were overlaid with the set PNS and then centrifuged at 100,000 g for 1.5 hours at 2 ° C.
  • the intermediate layer between 1.3 M and 0.86 M sucrose is the microsomal fraction SIH.
  • SIÜ fraction was adjusted to 250 mM sucrose with 100 mM KH ⁇ O ⁇ K 2 HPO, pH 6.8, and centrifuged at 220,000 g for 20 min at 4 ° C.
  • reaction buffer 250 mM sucrose, 50 mM KCl, 2.5 mM magnesium acetate, 20 mM HEPES, pH 6.8, 5 mM EDTA
  • reaction buffer 250 mM sucrose, 50 mM KCl, 2.5 mM magnesium acetate, 20 mM HEPES, pH 6.8, 5 mM EDTA
  • the frozen microsomal fraction from the induced H4-ind / APP-LC99 cells was thawed on ice and 10 ⁇ l was used for each cell-free reaction.
  • the samples were diluted to 30 ⁇ l with reaction buffer and incubated at certain temperatures, pH values and times. After incubation, the samples were adjusted to 2% SDS and heated at 95 ° C for 5 minutes.
  • IP buffer 150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 0.2% NP40 and the protease inhibitors aprotinin (10 ⁇ g / ml), leupeptin (10 ⁇ g / ml), 5 ⁇ g / ml pepstatin, 1 mM Pefabloc) and 6 ⁇ g ml each of the specific antibodies BI.40 and BI.42 (alternative antibodies having the same effect are from QCB, Quality Control Biochemicals, Inc., Hopkinton, USA; catalog numbers 44-348 and 44 -344 available) added.
  • a post-nuclear supernatant (PNS) from the liver of a guinea pig is obtained by homogenization and centrifugation as described (Taylor et al., 1997).
  • This supernatant o is applied to a sucrose step gradient (Taylor et al., 1997) and centrifuged at 100,000 g and 4 ° C. for 1.5 h.
  • the fraction with 500 mM sucrose is diluted with 1 x KPi buffer to 250 mM sucrose and centrifuged at 200,000 g and 4 ° C for 20 minutes.
  • the supernatant is the cytosol (Jones et al., 1998).
  • a purified microsome fraction of these recombinant H4 cells is mixed with a suitable buffer system (50-150 mM KCl, 1.5-5 mM gastroesium acetate, 250 mM sucrose, 20 mM Hepes pH 6.8), an ATP regenerating system (1 mM ATP, 0 , 1 mM GTP pH 7.0; 8 0 mM phosphocreatine, 31 mM creatine phosphokinase) and cytosol, which was worked up as described under 1.2, incubated at 37 ° C. and then the ⁇ -secretase activity by detecting the product Aß by means of Western blot measured as described above.
  • a suitable buffer system 50-150 mM KCl, 1.5-5 mM gastroesium acetate, 250 mM sucrose, 20 mM Hepes pH 6.8
  • an ATP regenerating system (1 mM ATP, 0 , 1 mM GTP pH 7.0; 8 0
  • the same cell line (H4 neuroglioma cell clone with APP-LC99 construct) was used as described under 1.1.
  • the H4-in ⁇ V APP-LC99 cells were left at 37 ° C., 5% CO 2 with DMEM medium (DMEM: "Dulbecco's Modified Eagle Medium” (Engl.), Sold by BioWittacker) and 10% 0 fetal calf serum (Engl .: “Fetal Calf Serum” (FCS), 1% glutamine, 1% penicillin and streptomycin in the absence of doxycycline on 15 cm petri dishes until they reach confluence. By withdrawing doxycycline, the expression of the fusion protein for
  • the cells were homogenized with a 5 ml Potter (Braun, Melsungen) at 500 rpm and the lysis of the cells was checked using phase contrast microscopy.
  • the cell lysate was first centrifuged at 2000xg for 2 min to sediment intact cells and large cell debris. The supernatant was then centrifuged at 4000 ⁇ g for 2 min in order to separate cell membranes and cell nuclei (fraction PN).
  • the sediment consisting of cell nuclei and plasma membranes was washed twice with ST buffer and centrifuged as described above. The supernatants were pooled and centrifuged in a new tube at 10000xg for 2 min to separate mitochondria, lysosomes and endosomes (EL fraction).
  • the supernatant would be centrifuged at 400000xg.
  • the EL fraction was further processed to separate the lysosomes and the endosomes.
  • the lysosomes were burst on ice by a 10-minute hypotonic lysis (Bohley et al. 1969) and the intact endosomes were then sedimented at 10000 g for 2 min.
  • 30 ⁇ g total protein of each fraction was applied to a polyacrylamide gel and the distribution of different brand proteins of individual compartments in the fractions was verified using the Western emblot method.
  • reaction buffer 250 mM sucrose, 50 mM KCl, 2.5 mM magnesium acetate, 20 mM HEPES, pH 6.8, 5 mM EDTA
  • reaction buffer 250 mM sucrose, 50 mM KCl, 2.5 mM magnesium acetate, 20 mM HEPES, pH 6.8, 5 mM EDTA
  • Small aliquots of the different fractions were frozen in liquid nitrogen and stored at -80 ° C. Then, as described in 1.3, the ⁇ -secretase inhibitor test system is carried out unchanged.
  • Literature 250 mM sucrose, 50 mM KCl, 2.5 mM magnesium acetate, 20 mM HEPES, pH 6.8, 5 mM EDTA

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Abstract

L'invention concerne des procédés pour l'identification d'inhibiteurs de η-secrétase spécifiques servant au traitement d'affections neurodégénératives, en particulier des procédés pouvant être exécutés in vitro. L'invention concerne également un nécessaire pour tests permettant d'exécuter le procédé selon l'invention, ainsi que l'utilisation de ce nécessaire pour tests ou du procédé pour détecter des substances inhibant la η-secrétase de manière spécifique. L'invention concerne en outre l'utilisation de ces substances pour la production d'un médicament servant au traitement d'affections neurodégénératives, ainsi que des formulations pharmaceutiques contenant ces substances.
PCT/EP2000/008340 1999-08-28 2000-08-25 SYSTÈME DE TEST IN VITRO POUR LA η-SECRÉTASE WO2001016355A2 (fr)

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EP00962393A EP1212454A2 (fr) 1999-08-28 2000-08-25 Systeme de test in vitro pour la gamma-secretase a partir de membranes enrichis
MXPA02001754A MXPA02001754A (es) 1999-08-28 2000-08-25 Sistema de ensayo in vitro de gama-secretasa.
CA002381952A CA2381952A1 (fr) 1999-08-28 2000-08-25 Systeme de test in vitro pour la .gamma.-secretase
JP2001520900A JP2003508058A (ja) 1999-08-28 2000-08-25 γ−セクレターゼインビトロテストシステム

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DE1999141039 DE19941039A1 (de) 1999-08-28 1999-08-28 gamma-Sekretase in vitro Testsystem
DE19941039.9 1999-08-28

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WO2001049871A2 (fr) * 2000-01-06 2001-07-12 Boehringer Ingelheim Pharma Kg Methode de recherche d'un inhibiteur de protease
WO2001085924A2 (fr) * 2000-05-11 2001-11-15 Scios Inc. Modulation de l'activité des $g(g)-sécrétases
US6713248B2 (en) 2000-04-03 2004-03-30 Bristol-Myers Squibb Company Methods for detection of gamma-secretase activity and identification of inhibitors thereof
WO2005031363A1 (fr) * 2003-09-24 2005-04-07 University Of Chicago Preparation membranaire tiree de pichia pastoris pour tester l'activite gamma-secretase
WO2005074971A1 (fr) * 2004-01-29 2005-08-18 Cellzome Ag Traitement de maladies neurodegeneratives a l'aide de molecules interagissant avec le cgi-13
WO2005075632A2 (fr) * 2004-01-29 2005-08-18 Cellzome Ag Traitement de maladies neurodegeneratives au moyen de atp7a

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DE10131899A1 (de) * 2001-07-04 2003-02-27 Boehringer Ingelheim Pharma In vitro-Screening-Assay für gamma-Secretase

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001049871A2 (fr) * 2000-01-06 2001-07-12 Boehringer Ingelheim Pharma Kg Methode de recherche d'un inhibiteur de protease
WO2001049871A3 (fr) * 2000-01-06 2002-03-28 Boehringer Ingelheim Pharma Methode de recherche d'un inhibiteur de protease
US6713248B2 (en) 2000-04-03 2004-03-30 Bristol-Myers Squibb Company Methods for detection of gamma-secretase activity and identification of inhibitors thereof
WO2001085924A2 (fr) * 2000-05-11 2001-11-15 Scios Inc. Modulation de l'activité des $g(g)-sécrétases
WO2001085924A3 (fr) * 2000-05-11 2003-02-27 Scios Inc Modulation de l'activité des $g(g)-sécrétases
US6579689B2 (en) 2000-05-11 2003-06-17 Scios Inc. Modulation of γ-secretase activity
WO2005031363A1 (fr) * 2003-09-24 2005-04-07 University Of Chicago Preparation membranaire tiree de pichia pastoris pour tester l'activite gamma-secretase
WO2005074971A1 (fr) * 2004-01-29 2005-08-18 Cellzome Ag Traitement de maladies neurodegeneratives a l'aide de molecules interagissant avec le cgi-13
WO2005075632A2 (fr) * 2004-01-29 2005-08-18 Cellzome Ag Traitement de maladies neurodegeneratives au moyen de atp7a
WO2005075632A3 (fr) * 2004-01-29 2005-11-10 Cellzome Ag Traitement de maladies neurodegeneratives au moyen de atp7a

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