MXPA02001754A - In vitro test system for gamma secretase from enriched membranes. - Google Patents

Abstract

The invention relates to a method for identifying specific ggr; secretase inhibitors which can be used for treating neuro degenerative diseases, in particular to a method which can be carried out in vitro. The invention also relates to a test kit which can be used for said method, in addition to the use of this test kit or method to identify substances which specifically inhibit ggr; secretase. A further embodiment relates to the use of these substances to produce a medicament for treating neuro degenerative diseases and to pharmaceutical formulations which contain said substances.

Description

IN VITRO ASSAY SYSTEM OF RANGE-SECRETASE Field of the invention The present invention pertains to the field of methods for the detection of specific inhibitors of β-secretase that can be used for the treatment of neurodegenerative diseases, especially procedures that can be carried out in vitro. Additionally, this invention relates to a test equipment with which the method can be carried out. according to the invention, as well as to the use of this test equipment or of the method for the detection of substances that specifically inhibit β-secretase. Another embodiment relates to the use of these substances for the preparation of a medicament for the treatment of neurodegenerative diseases and pharmaceutical formulations containing these substances. Background of the Invention t Protein aggregation and precipitation are involved in the establishment of different diseases neurodegenerative diseases such as Alzheimer's, Parkinson's and Veitstanz (in English, "Chorea Huntington"). In Alzheimer's disease, the amyloid-β peptide is added (Aß) and gives rise to insoluble senile plaques, which REF .: 136038 - "- • - - * - - represent a pathological marker of the disease (Selkoe et al., 1996) .Aß is formed by proteolytic cleavage of a precursor protein, the amyloid precursor protein (English:" amyloid precursor "). Protein ": APP: There are two metabolic pathways of APP, the non-amyloidogenic pathway and the amyloidogenic pathway (Selkoe, 1991, 1994). In the non-amyloidogenic metabolism of APP, a-secretase is cleaved within the Aβ region of the APP and thus leads to the secretion of the soluble N-terminal region of the protein (a-APP), as well as, after the cleavage of the? -secretase, to the release of p3. , the amyloidogenic pathway leads to the formation of Aβ, in which two proteases generate the N-terminal (β-secretase) or the C-terminus (? -secretase) of Aß (Haass, 1993; Selkoe, 1994). In vivo, Aß can be detected in human plasma and in the cephalo-rachidian fluid. Also in cell cultures, Aβ secreted in the cell culture supernatant can be detected in different cell types, which endogenously express or overexpress APP or fragments thereof. Both the production of Aß and the formation of amyloid plaque are affected by different genetic risk factors. They include mutations in the homologous proteins presenilin 1 and presenilin 2, as well as in the APP itself. The analysis of these mutations in fibroblasts of patients with Alzheimer's disease, with familially inherited Alzheimer's disease (English: "Family Alzheimer Disease" (FAD)), has shown the influence they have on the formation of Aß. This has been confirmed by investigations in transfected cells and transgenic animals. All the mutations increase the production of Aβ and give rise, in case of mutations of presenilin, to a selective increase of the longer variant of Aβ, the Aβ42 (Selkoe, nineteen ninety six; Price, 1998). This peptide is added to a greater extent than the shortest form, Aβ40, and is found in diffuse plaques and, together with Aβ40, in senile plaques.

(Lemere et al., 1996; Mann et al., 1996). In addition to this influence of the mutations, there are indications that also the wild type forms of the presenilins play a fundamental role in the physiological formation of Aß. In neurons of mouse embryos, in which the gene PS1 (PS: presenilin) is inactivated by means of a gene technique, a drastic reduction of Aβ40 and Aβ42 has been demonstrated. In addition, in these cells the C-terminal fragments of the APP accumulate, which gave rise to the opinion that presenilins activate the β-secretase or have, by themselves ? same, β-secretase activity (De Strooper et al., 1998; Sisodia et al., 1998). The first in vitro assay systems combined with studies of mutations in preserved aspartate of presenilin 1 allow us to suppose that the 5 presenilins could be special, autocatalytically active aspartate proteases, which are responsible for the cleavage of the? -secretase in the membrane (olfe). et al., 1999). The discussion on the identification of? -secretase as a decisive stage in the generation of Aß and, therefore, in the establishment of Alzheimer's has not yet ended. Independently of this, this protease represents an interesting objective to be able to intervene pharmacologically in the process of Aß formation, finding inhibitors that selectively reduce its activity. For this, it is important to develop, in addition to animal models and cell assays, in vitro test systems that, independently of the transport processes within the cell, make it possible to test specific active substances. 20 Wolfe et al. (1999) describe an in vitro system for the measurement of β-secretase activity. For the set-up of the system, membranes are processed from cells that express PS1 stably. These are mixed with the The HMMililb plasmid encodes the LC99 polypeptide and is subjected to an in vitro transcription / translation reaction in the presence of 35S-methionine, forming the substrate of the? -secretase C99. The mixture is then incubated under suitable conditions, where the C99 fragment of APP is proteolytically cleaved from the β-secretase and the cleavage products are detected by gel electrophoresis after immunoprecipitation. Within the framework of this invention, LC99 represents a fusion protein between the substrate of the β-secretase C99 and a signal sequence (L: "leader" (English); Shoji et al., 1992). Brief Description of the Invention The present invention pertains to the field of methods for the detection of specific inhibitors of? -secretase that can be used for the treatment of neurodegenerative diseases, especially of procedures that can be carried out in vitro. Aditionally, this invention relates to a test equipment with which the method according to the invention can be carried out, as well as to the use of this test equipment or the method for the detection of substances that specifically inhibit β-secretase. Another embodiment relates to the use of these substances for the preparation of a ,. medicine for the treatment of neurodegenerative diseases and pharmaceutical formulations containing these substances. Figures Fig. 1: Characterization of the SIII microsomal fraction. H4-ind / APP-LC99 cells were cultured in the absence of doxycycline for three days to induce the expression of LC99. PNS were prepared in the decipher form and continued to be concentrated with gradual gradients of sucrose (Taylor et al., 1997). A. Concentration of the substrate of? -secretase C99 in the misrosomic fraction SIII. Aliquots (in each case 30 μg) of the PNS and microscopic traction (SIII) were transferred to a 12% SDS-polyacrylamide gel. For this purpose, PNS was prepared from H4-ind / APP-LC99 cells, cultured in the presence and absence of doxycycline. The proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Poly Screen, New Lite Science) and C99 was detected with the polyclonal antibody 5818 directed against the last 20 amino acids of C99 (diluted to 1: 1000; equivalent action alternative: product number SAD 3138, Labgen).

B. Concentration of presenilins in the microsomal fraction SIII. Aliquots (in each case 30 μg) of the PNS and fractions of the gradients of sucrose were loaded onto a 12% SDS-paliacrylamide gel and the excised proteins were transferred to a PVDF membrane (Poly Screen, New Life Science ). For the detection of the two PS proteins, the monoclonal antibody BI.3D7 (specific for PSl CTF, 1: 3000, Steiner et al., 1999) or the monoclonal antibody BI-HFSC (specific for PS2 CTF; 1: 3000; Steiner et al., 1999). It could be demonstrated that the CTF of PS1 and PS2 had been concentrated in the SIII fraction. It could not be demonstrated whether the observed protein band, with a molecular weight of approximately 46-50 kDa, was PS1 or PS2 from full length. C. Characterization of the SIII fraction by detection of marker proteins, characteristics for certain compartments Aliquots (in each case 30 μg) were loaded.

PNS and fractions of the gradients of sucrose (9) on a 12% SDS-polyacrylamide gel and the excised proteins were transferred to a PVDF membrane (Poly.

Screen, New Life Science). To show the concentration of membranes An anti-calreticulin antibody (Stressgen Biotechnologies, 1: 2000) was used in the endoplasmic reticulum in the SIII fraction. The distribution of endosomal membranes in gradients was demonstrated with the help of anti-rab5 antibodies (Transduction Laboratories Inc.). Fig. 2: Acellular generation of Aß 40 and Aβ42 of isolated microsomes A. De novo generation of Aβ in an acellular? -secretase assay system with isolated microsomes of H4 cells, stably transfected with APP-LC99. Microsomes were prepared in the manner described (Taylor et al., 1997) (fraction SIII) and incubated at 37 ° C or 4 ° C for 4 hours under neutral conditions (pH 6.8). After incubation of the C99 substrate, the Aβ340 and Aβ42 products were immunoprecipitated with the 6E10 and 4G8 antibodies (Senetek, Great Britain, Galli et al., 1998). In this way, the substrate / product ratio could be calculated. For the precipitation of peptides-Aβ40 and Aβ42 alone7 specific antibodies BI.40 and BI.42 were used. The precipitated proteins were separated with a tris-bicine gel, transferred onto a nitrocellulose membrane and made visible with the 6E10 and 4G8 antibodies, using a highly sensitive Western blot procedure (Ida et al., 1996). To determine the baseline intracellular content of Aβ, immunoprecipitations were carried out directly with the microsomal fraction stored at -80 ° C (lane c). The Aß40 and Aβ42 generated in vi tro migrate with the synthetic Aβ peptides. Bottom: longer exposure. B. Dependence of the time of the generation ± n vitro of Aß The fraction SIII was prepared in the manner described and incubated at 37 ° C or 4 ° C under neutral conditions (pH 6.8) for the indicated time. The Aβ peptides were immunoprecipitated with the specific antibodies BI.40 and BI.42. Precipitated proteins were separated by tris-bicine gel electrophoresis (Klafki et al., 1996) and then detected with a highly sensitive Western blot procedure with antibodies 6E10 and 4G8 (Ida et al., 1996). As a standard synthetic peptides Aβ 40 and Aβ42 were used. After 3-4 hours of incubation at 37 ° C, the de novo generation of Aß reached a maximum. Fig. 3: The β-secretase Aβ dissociation product is degraded by a Ca 2+ -dependent protease The SIII fraction was created in the manner described and incubated under conventional conditions (37 ° C, pH 6.8; 4 hours) in the presence or absence of cationic chelators such as EDTA or BAPTA. Cone controls were incubated microsomes at 4 ° C in the presence of EDTA. The Aβ peptides were immunoprecipitated with the specific antibodies BI.40 and BI.42 and were detected by Western blotting with the 6E10 and 4G8 antibodies (Senetek, Great Britain, Galli et al., 1998) in the manner described. The synthetic Aβ 40 and Aβ42 peptides served as the standard. All reactions were performed in triplicate. In the absence of a chelator, the de novo production of Aß is drastically reduced. Both EDTA and BAPTA, which chelates only Ca2 + ions, determine a higher amount of Aβ de novo. This can be explained by the fact that Aβ is degraded again immediately after its creation by a Ca2 + -dependent protease. Fig. 4: The? -secretase is a transmembrane protease The SIII fraction was prepared in the manner described and incubated under conventional conditions (pH 6.8; EDTA mM; 4 hours) at 37 ° C or 4 ° C as control. The microsomal membranes were washed with saline (1M KCl) or extracted with Na 2 CO 3 to separate weakly bound proteins from the microsomal membranes. For this purpose, the granulated membranes were resuspended in a KCl buffer (1M KCl)., 250 mM sucrose, 20 mM Hepes, pH 6.8) and incubated for 30 min. at 4 ° C. For extraction by Na2CO3 the membranes were homogenized in 100 mM Na2CO3, pH 11.5 and incubated for 30 min. at 0 ° C. The membranes were incubated at 220,000 g for 1 hour at 4 ° C, carefully washed with cold water and with 1 ml of reaction buffer (9) during homogenization. The membranes were centrifuged as described above and resuspended in the reaction buffer. Aliquots were frozen in liquid nitrogen. The Aβ peptides were immunoprecipitated with the specific antibodies BI.40 and BI.42 and detected in the manner described by Western blotting with the 6E10 and 4G8 antibodies. As a standard synthetic peptides Aβ 40 and Aβ42 were used. All reactions were carried out in duplicate. Independently of the pretreatment of the membranes, the Aß content generated de novo was practically identical. These data allow us to presume that β-secretase is a protease that is firmly bound to the membrane or that it is an integral membrane protein. Fig. 5: The pH optimum for β-secretase activity is between pH 6.8 and 7.4 The SIII fraction was prepared in the manner described and incubated at the indicated pH values under standard conditions (pH 6.8).; 5 mM EDIA; 4 hours). The Aβ peptides were immunoprecipitated with the specific antibodies BI.40 and BI.42 and detected in the manner described by Western blotting with the 6E10 and 4G8 antibodies. All reactions were carried out in duplicate. As a control, the reaction was carried out in vi tro at 4 ° C and at pH 6.8. The pH optimum for the activity of the β-secretase is in the neutral range between pH 6.8 and pH 7.4. Under both slightly acidic and basic pH conditions, a strongly reduced β-secretase activity was found. Fig. 6: Effect of a possible β-secretase inhibitor in the α-secretase acellular assay system The Sil: fraction was prepared in the manner described and incubated at a pH value of 6.8 under conventional conditions in the presence or absence of different concentrations of compound A (Fig. A) (pH 6.8, 5 mM EDTA, 4 hours). The Aβ peptides were immunoprecipitated with the specific antibodies BI.40 and BI.42 and detected in the manner described by Western blotting with the 6E10 and 4G8 antibodies (Senetek, Great Britain, Galli et al., 1998). All reactions were performed in duplicate or triplicate. As a control, the reactions were carried out at 4 ° C.

A. Compound A showed a concentration-dependent inhibition of the ip-vitro generation of Aβ. The quantification of the Aß generated de novo was done with the Chemiluminescence Imaging System (Biorad) and it is represented in the lower part. B. Compound A showed a concentration-dependent reduction of extracellular Aβ 40 and Aβ42. Compound A was also active in a cellular assay system, in which the extracellular content of Aβ40 and Aβ42 secreted by the cell line U373 (U373: ATCC # HTB 14) is determined. This cell line U373 / APP751 is an astrocytoma cell line that overexpresses human APP751 and secretes large amounts of Aβ40 (~ 1000 pg / ml / 4 hours with 5 × 10 7 cells in 15 ml of medium) and Aβ42 (-100 pg / ml / 4 hours with 5xl07 cells in 15 ml of medium). The determination takes place on ELISA (ELISA: "Enzyme linked immunosorbent assay" (Steiner et al., 1998)). Compound A strongly reduced, in a concentration-dependent manner, the secretion of Aβ40, thereby slightly increasing the secretion of Aβ42 at sub-inhibitory doses, being similarly inhibited later at higher doses.

Fig. 7: Effect of a β-secretase inhibitor described in the α-secretase acellular assay system The SIII fraction was prepared in the manner described and incubated at a pH value of 6.8 under standard conditions in the presence or absence of various concentrations of compound MG132 (Biomol Order No.: PI-102; De Strooper et al., 1999) (pH 6.8; 5 mM EDTA; 4 hours). The Aβ peptides were immunoprecipitated with the specific antibodies BI.40 and BI.42 and detected in the manner described by Western blot with • antibodies 6E10 and 4G8 (Senetek, Great Britain, Galli et al., 1998). All reactions were carried out in duplicate. As controls, the reactions were carried out at 4 ° C or at 37 ° C without inhibitor. A) Compound MG132 showed a concentration-dependent inhibition of the in vitro generation of Aβ. B) The quantification of the Aß generated de novo was carried out with the Chemiluminescence Imaging System (Biorad Signature) and it is represented in the lower part. Fig. 8: Characterization of the microsomal fractionation of H4indLC99 cells. H4-ind / APP-LC99 cells were cultured in the absence of doxycycline for three days, to induce the expression of LC99. The fractions were prepared in the manner described (Schroter in al., 1999). A) Detection of marker proteins, characteristics for 5 certain compartments Aliquots (in each case 30 μg) of the fractions were loaded onto a 12% SDS-polyacrylamide gel and the separated proteins were transferred to a PVDF membrane (Poly Screen, New Life Science). To demonstrate the concentration of membranes of the endoplasmic reticulum in the Mi fraction, an anti-PDI antibody was used (Stressgen Biotechnologies; 1: 2000). For comparison, fraction SIII of the first microsomal fraction was co-transferred. PDI is concentrated in the first fraction microsomal. The distribution of lysosomal membranes in the fractions was demonstrated with the help of anticatepsin antibodies D (Transduction Laboratories) Inc .; 1: 1000). The microsomal fraction is free of lysosomal proteins. As a comparison, the PNS of the H4IndLC99 cells was co-transferred.

B) Formation of acellular Aβ in the microsomal fraction The microsomes (Mi fraction) were prepared in the manner described and incubated at a pH value of 6.8 under conventional conditions (pH 6.8, 5 mM EDTA, 4 hours). The Aβ peptides were immunoprecipitated with the c) MM ?iateMtf | ¡ß - specific antibodies BI.40 and BI.42 and were detected as described by Western blotting with antibodies 6E10 and 4G8 (Senetek, Great Britain, Galli et al., 1998). Incubations at 37 ° C were carried out by 5 duplicates. In the microsomal fraction, as well as in the endosomal fraction, a formation of Aβ takes place. Detailed Description of the Invention The mission of making an improved test system available has been solved by means of the present invention in the context of the description and claims. According to the invention, an in vitro test system is available for the discovery of substances capable of specifically inhibiting β-secretase. In a further embodiment of the invention, it is given to know a test kit for the discovery of substances capable of specifically inhibiting? -secretase. Another embodiment of the invention is the use of the method according to the invention or of the test equipment according to the invention for the discovery of substances capable of inhibiting specifically the? -secretase. In addition, substances that can be detected with the process according to the invention or with the test equipment according to the invention are made available. An additional form of embodiment is refers to the use of these substances for the preparation of a medicament for the treatment of neurodegenerative diseases and pharmaceutical formulations containing these substances. The method according to the invention uses purified membranes, isolated from cells, which detectably express a β-secretase substrate and show β-secretase activity. By? -secretase it is to be understood, within the framework of this invention, a protein having the property of proteolytically cleaving the APP or fragments thereof, in particular the C99 peptide (Shoji et al., 1992), in p3 or Aß. Therefore, for the method according to the invention all cells are suitable in which the specialist can detect, by Western blotting and the use of specific antibodies, a substrate of the β-secretase or its cleavage products. Suitable membranes are all membranes in which the specialist, by means of Western blotting and using specific antibodies, can detect a β-secretase substrate, preferably lysosomal and endosomal membranes and, most preferably, microsomal membranes. The technician knows procedures for the specific purification of membranes from "Methods in Enzymology", Vol. 219, title: "Reconstitution of intracellular Transport" and from the book "Biochemische Arbeits ethoden", T.G. Cooper, Editorial De Gruyter, 1981. For carrying out the method according to the invention, as described in a non-limiting embodiment in the Example, cells can be transfected with a DNA sequence (DNA: deoxyribonucleic acid) encoding a substrate of the? -secretase. In an exemplary embodiment, the expression of this substrate can be induced by the deprivation of doxycycline. The cells can be opened and a post-nuclear supernatant can be prepared (in English, "post nuclear supernatant", abbreviation PNS), which is further processed to isolate, for example, the microsomal fraction. This fraction can be incubated under suitable conditions and the formation of the product of the reaction of the β-secretase with a suitable substrate can be determined by immunoprecipitation and consequently detection by suitable antibodies in the Western blot procedure. The β-secretase substrate has been found in the PNS and, in a higher concentration, in the microsomal fraction (Fiq. LA). The C-terminal fragments (CTF) of PS1 and PS2 were also concentrated in the SIII fraction (Fig. IB). The microsomal fraction did not contain endosomal membranes, but the ER and Golgi compartments were concentrated (Fig. 1C). In the microsomal fractions, incubated at 37 ° C (Fig. 2A), Aβ generated de novo was detected. Incubation to 4 ° C prevented the de novo formation of Aß. Small amounts of Aβ were conditioned by the existence of intracellular Aβ in the freshly prepared microsomal fractions (Fig. 2A, lane c). The de novo formation of Aβ was time dependent and peaked after 3 to 4 hours of incubation (Fig. 2B). However, an evaluation of the substrate / product ratio showed that the generation of the Aβ in vitro did not constitute an effective proteolytic reaction (Fig. 2A). Incubation of the microsomal membranes in the absence of EDTA resulted in a drastic reduction in the generation of Aβ in vi (Fig.3). The BAPTA calcium chelator had the same effect. This makes it possible to assume that a Ca2 + -dependent protease, present in the same fraction, degrades Aß. The treatment of the microsomal membranes with 1M KCl in buffer or extraction of these membranes with 0.1M Na2C03 at pH 11.5 before carrying out the? -secretase assay system, demonstrates that? -secretase is stable against the membrane or , at least, it must be firmly attached to the membrane (Fig. 4). In contrast to previously published data (Wolfe et al., 1999), the pH optimum determined with this assay system for β-secretase activity is at a pH value between 6.8 and 7.4 (Fig. 5). The de novo generation of Aß did not occur at a more basic pH 5 (pH 8.0 to 8.5) or more acidic (pH 6.0 to 6.4). In an embodiment according to the invention, the purified membranes described, especially microsomal membranes, are mixed with a reaction buffer purified and a test substance. By substance . In the context of this invention, each assay that must be tested to determine whether it can act in an inhibitory manner on the activity of the? -secretase should be understood. Then the mixture is incubated under co-conditions in which the β-secretase substrate is cleaved by the absence of the test substance. Next, the amount of a formed product-cleavage is determined and the value obtained is compared with the value obtained in the absence of the substance of separation. trial. If the amount of cleavage product formed in the presence of the test substance is lower than in the absence of the test substance, this test substance inhibits the formation of the cleavage product and the Test substance is a? -secretase inhibitor. In the state of the art have been identified, among others, compounds that have been designated, as specific inhibitors of? -secretase and that exerted an inhibitory activity on the secretion of Aß40 and Aβ42, without influencing the formation of Aβ. With the? -secretase assay system according to the invention, it is now possible, advantageously, to identify and validate specific? -secretase inhibitors, differentiating them from the inhibitors that prevent the secretion of A? 40 and A? 42. As described in the Example, a compound was tested with the test system according to the invention. Compound A c [aS- (aR *, R *, dR *)] N-butyl-β-hydroxy-a- (1-methylethyl) -d [(4-methylpentyl) amino] cyclohexanehexanamide; see Example 20 of EP 778 266 A1) was prepared according to the data of Example 20 of European Patent Application EP 778 266 A1 and showed a concentration-dependent inhibition of Aβ generation, with an IC50 value of about of 6 μM (Fig. 6A). Similar results were calculated with a test system that measures secreted Aβ40 and Aβ42 in a quantitative ELISA, which is specific for both Aβ peptides (Steiner et al., 1998). In this case, an astrocytoma cell line (U373) is used that overexpresses the wild-type APP751 and secretes detectable amounts of both Aβ species. A concentration-dependent reduction in the amount of extracellular Aβ40 and Aβ42 was observed after overnight treatment with compound A (Fig. 6B). In one embodiment of the invention, cells expressing an endogenous polypeptide which is a substrate for β-secretase are cultured. By the term endogenous it should be understood, within the framework of this invention, that this cell or cell line expresses the indicated polypeptide in sufficient quantity, without further manipulations being necessary, for example by gene technology methods. In the context of the invention, a sufficient amount of the β-secretase substrate should be understood as an amount which, in an established biochemical detection method (eg ELISA, Western blot), using specific antibodies, of a detectable signal that is above the bottom. In a particularly preferred embodiment of the invention, cells expressing an exogenous polypeptide which is a substrate of the β-secretase are cultured. By the term "exogenous" in the context of this invention it should be understood that this cell or cell line is manipulated by means of gene technology methods in such a way that it expresses the β-secretase substrate. If the cell or cell line contains the β-secretase substrate also without the aforementioned manipulations, it should be expressed by that term that the amount of the α-secretase substrate is measurably high with respect to the value without manipulation. For the preparation of a cell that exogenously expresses the β-secretase substrate, a nucleotide sequence encoding the amino acid sequence of the β-secretase substrate can be incorporated into a suitable expression cassette of a eukaryotic expression vector. Suitable expression cassettes have a functional promoter, in eukaryotic hosts such as, for example, the cytomegalovirus promoter (CMV promoter) and a functional polyadenylation signal, for example of the SV40 virus (in English: "Simian Virus"; abbreviation: SV). Suitable expression vectors are vectors that can replicate in eukaryotic hosts, ie possess a functional origin of replication (in English: "Origin of replication"). These expression vectors can be located, after transfection, episomally or integrated into the genome when they carry the appropriate sequences that allow integration.

In a preferred embodiment, an expression system is used that allows the expression of the exogenous polypeptide to be induced, and in this case different systems can be used, such as, for example, the "Tet-on" or "Tet-off" system (US Pat. U.S. Patent 5,464,758, Gossen and Bujard, 1992, 1995, distributed by Clontech, Heidelberg), or the LacSwitch system (see U.S. Patent 5,589,392, distributed by Stratagene). In a particularly preferred embodiment of the invention, the expression of the β-secretase substrate is induced by the withdrawal of tetracycline or doxycycline ("Tet-off" system), for which the nucleotide sequence encoding the amino acid sequence is cloned. of the β-secretase substrate, behind at least one position of binding of the tetracycline repressor Additionally, it is in the same plasmid, in an additional plasmid or integrated in the chromosome another nucleotide sequence that encodes a protein Fusion factor between the Tet repressor and an activating domain, acid, which can be expressed constitutively. Acid activating domain is understood to mean a protein domain that possesses a high fraction of acidic amino acids and that possesses the property of inducing the transcription of a gene when the domain is incorporated in a suitable position in the transcription complex located in front of the gene. This property can be determined through the so-called "Test of a hybrid". With this method, a domain that binds to the DNA is fused with a domain to be investigated, where the domain that binds to the DNA is fixed to a sequence that is in front of a reporter protein, measuring the activity of the aforementioned reporter protein (Clontech, Heidelberg). In the case described above, the expression of the β-secretase substrate will not occur when the concentration of tetracycline or a tetracycline derivative such as, for example, doxycycline in the cell exceeds a certain value, since the repressor of Tetracycline fixes the tetracycline or its derivative, as a result of which it does not fixate on its binding position in the DNA and does not, therefore, induce the transcription of the gene located behind this binding position, which encodes the substrate of the? -secretase. In the absence of tetracycline or a derivative thereof, the fusion protein binds between the Tet repressor and the acid activation domain at its DNA binding position and induces transcription of the gene located behind, which codes for the substrate of the? -secretase. In this way, it is ensured that a directed and controlled expression of the β-secretase substrate takes place.

^ ".. In one embodiment of the invention, the? -secretase substrate is the amyloid precursor protein (APP) or a fragment thereof, provided that it contains the cut-off position of the? secretase In a preferred embodiment of the invention, the β-secretase substrate is the C99 fragment of the amyloid precursor protein (Shoji et al., 1992). The β-secretase substrate may be associated, in general, with the membrane, but preferably forms part thereof. Because it is part of the membrane, It should be understood, in the context of this invention, that the substrate is an integral component of the membrane. By membrane associated it is to be understood, within the scope of this invention, that the substrate is attached to the surface of the membrane or is bound to integral protein of the membrane. This definition of substrates associated with the membrane should also include substrates that "interact through chemical clusters with the hydrophobic part of the membrane, contributed by post-translational modifications." Additionally, within the expression substrates associated with the membrane should also be included. substrates that interact through amino acid side chains with the hydrophobic part of the membrane, albeit to a lesser extent than the integral proteins of the membrane, by way of example, the prostaglandin synthetase should be mentioned here. the invention, the? -secretase substrate is a fusion protein of a reporter protein with an amyloid precursor protein or fragment thereof, provided that it contains the cleavage position of? -secretase. In a preferred embodiment, the β-secretase substrate is a fusion protein between a reporter protein and the C99 fragment. In the context of this invention, a reporter protein should be understood as a protein that has the property of generating an easily detectable signal, the amount of which correlates with the amount of the excision product of interest. The generation of the signal takes place through the determination of the enzymatic activity of the reporter protein with easily detectable substrates, or by the measurement of the fluorescence intensity of the reporter protein. Examples of reporter proteins are green fluorescent protein (GFP; English: "green fluorescent protein"; see, for example, WO95 / 07463), or fluorescent derivatives thereof at other wavelengths, or enzymes such as luciferase, secretory alkaline phosphatase or β-galactosidase. In this embodiment of the invention, the fusion protein of the cleavage product with the reporter protein of the fusion protein of the? -secretase substrate not cleaved with the reporter protein, is separated, for example, by immunoprecipitation. This can be carried out with antibodies that recognize the cleavage product selectively. Next, the amount of reporter protein is determined with the mentioned procedure, which depends on the properties thereof. The cited fusion proteins can be prepared by customary gene technology methods (Sambrook et al., 1989) when the DNA encoding the reporter protein and the β-secretase substrate is available. The DNA, which encodes the reporter protein, can be obtained, for example, from commercial suppliers such as, for example, Clontech, Heidelberg, by introducing it by conventional methods (Sambrook et al., 1989) into the desired vectors. The DNA encoding the β-secretase substrate or the C99 fragment can be obtained with conventional methods from suitable gene libraries (Sambrook et al., 1989). All cells or cell lines known to a person skilled in the art, especially eukaryotic cells or cell lines, are suitable for carrying out the invention. Cells or cell lines that are used in neurological or neurobiological research are preferred, for example cells or mammalian cell lines such as H4, U373, NT2, HEK 293, PC12, COS, CHO, fibroblasts, myeloma cells, cells of neuroblastoma, hybridoma cells, oocytes or embryonic stem cells. Additionally, the insect cell lines (e.g., using baculovirus vectors such as pPbac or pMbac (Stratagene, La Jolla, CA)), yeasts (for example, using yeast expression vectors such as pYESHIS (Invitrogen, CA)) and fungi. Cells or cell lines of neuronal or glial origin are especially preferred. In a very especially preferred embodiment of the invention, in the case of the cells used, it is H4 cells, human brain neuroglioma cells, deposited under the number of ATCC HTB-148 in the "American Type Culture Collection (ATCC)", in Manassas, Virginia, USA. In a preferred embodiment of the invention, purified cell membranes are used, preferably intracellular membranes and, in particular, -a > ^ * - * -a preferred, lysosomal or purified endosomal membranes. Especially preferably, purified microsomal membranes are used from the cells used for lysis of the cells, separation of the cell nuclei, purification on gradients of saccharose density, renewed sedimentation by ultracentrifugation, homogenization, renewed sedimentation by ultracentrifugation and renewed homogenization. . Conventional methods for membrane purification are described in Methods in Enzymology, Vol. 219, and in the book "Biochemische Arbeitsmethoden", T.G. Cooper, Editorial De Gruyter, 1981. Fundamentally, ultracentrifugation with gradients of sucrose, metrizamide, ficoll and yodoxanol is suitable for the purification of membranes. In a preferred embodiment of the present invention, the reaction buffer has a pH value in the range of 5-10, preferably in the range of 6.0 to 8.0, and especially preferably 6.8 to 7.4 and also contains , at least, a membrane stabilizer. By membrane stabilizer, it is to be understood within the scope of this invention that a substance prevents the aggregation of the membranes. Membrane aggregation should be understood, within the framework of this fiBni- '- caking or the aggregation and eventually subsequent fusion of vesicles or liposomes, or also the multilamellar formations. This can be determined by an experimentally detectable increase in the turbidity or by the dispersion of the light of a solution or suspension to be investigated, it being possible to establish with this, also, the property of a substance in relation to its membrane stabilizing activity. The membrane stabilizer within the framework of this invention is preferably sucrose or sorbitol, although the specialist can replace them with substances with the same action. Preferably, the concentration of the membrane stabilizer in the reaction buffer is between 200 and 1000 mM, preferably between 200 and 500 mM and, especially preferably, between 200 and 300 mM. In a particularly preferred embodiment of the process according to the invention, the reaction buffer additionally contains a complexing agent, preferably for bivalent ions. This can be, for For example, ethylene-diamino-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 complex trainer is The complexing agents are designated compounds capable of acting as ligands, preferably in a concentration of 0.1 to 20 mM, preferably 5 to 10 mM. for the formation of complexes, that is to say especially for the complexation and the masking of metals, especially bivalent metal ions.This designation is often used as a synonym for chelate formers.In the method according to the invention, other Complex Formers 10 In an embodiment of the invention, an ATP regenerative system is additionally added to the reaction batch.This system contains adenosine triphosphate (ATP), guanosine triphosphate (GTP), phosphocreatine, creatine phosphokinase. , preferably in a concentration of ATP 1 mM, 0.1 mM GTP, 8 mN phosphocreatine, 31 mM creatine phosphokinase at a neutral range pH value, preferably between 6.8 and 7.2, especially preferably at a pH value of 7.0. In a preferred embodiment of the invention, The determination of the amount of the cleavage product is carried out by immunoprecipitation or Western blotting, preferably by a combination of immunoprecipitation and Western blotting. The realization of immunoprecipitation and Western transfer has ** - * »* ** - * < ** place as described by Ida et al. (nineteen ninety six). By Western blotting is meant a method in which the proteins are separated according to their charge in the native state or, most of the time, denatured by means of gel electrophoresis, most of the time, polyacrylamide gel electrophoresis, they are transferred to carriers such as, for example, nitrocellulose or poly (vinylidene difluoride) and then detected by means of antibodies. In another embodiment of the invention, the determination of the amount of the cleavage product is carried out by enzyme-immunoassay (English: Enzyme-linked immunosorbent assay, abbreviation: ELISA) (Steiner et al., 1999), or by spectrometry. masses. In another embodiment of the invention, the determination of the amount of the cleavage product is carried out by determining the amount of the reporter protein fused to the cleavage product, after it has been purified from the fusion protein between the β-secretase substrate. excised and the reporter protein. The amount of luciferase, secretory alkaline phosphatase or β-galactosidase fused to the cleavage product. it is determined by calculating the enzymatic activity of the reporter protein after the addition of the substrate. In another embodiment of the invention, the determination of the amount of the green fluorescent protein or a derivative thereof takes place by measuring the intensity of the fluorescence light. A preferred embodiment of the invention is a test kit according to the invention for the detection of substances capable of specifically inhibiting β-secretase. A test set is a composition of all the components for the process according to the invention. Non-limiting examples of other elements for carrying out the process according to the invention are containers such as, for example 96-well plates or microtiter plates, reaction flasks, other suitable containers, surfaces and substrates, membranes such as nitrocellulose filters, washing reagents and buffers or the like. Additionally, a test kit may contain reagents, which can detect fixed antibodies, __ such as, for example, labeled secondary antibodies, chromophores and enzymes (eg, conjugated to the antibody), and their substrates or other substances capable of binding antibodies. The assay kit according to the invention contains at least cell membranes purified from cells showing β-secretase activity and containing a β-secretase substrate, and a reaction buffer.

In a preferred embodiment, the membranes are purified intracellular membranes, preferably lysosomal or endosomal, and, most preferably, microsomal membranes. Especially preferably, the membranes have been purified from cells that exogenously express a β-secretase substrate. In a preferred embodiment of the invention, the reaction buffer has a pH value in the range of 5-10, preferably in the range of 6.0- 8.0 and, especially preferably, in the range of 6.8 to 7.4, and contains as additional components at least one membrane stabilizer according to the invention, as described above. Preferably, the concentration of the membrane stabilizer, which is preferably sucrose or sorbitol, in the reaction buffer is between 200 and 1000 mM, preferably between 200 and 500 mM, and particularly preferably between 200 and 300 mM. In a particularly preferred manner, the reaction buffer additionally contains a complexing agent according to the invention, preferably for bivalent ions. As described above, this may be, for example, EDTA, BAPTA or EGTA, or a salt thereof. The complex former is in a concentration of 0.1 to 20 mM, preferably 5 to 10 mM. In an embodiment of the invention, the test kit additionally contains an ATP regenerative system according to the invention as described above, which can be added to the reaction batch. In yet another embodiment of the invention, the 4 'assay kit contains antibodies that allow determination of the caritity of the cleavage product by immunoprecipitation or Western blotting, preferably by a combination of immunoprecipitation and Western blotting. In a preferred embodiment of the invention, the method according to the invention or the test equipment according to the invention is used to detect substances capable of specifically inhibiting β-secretase. In another embodiment, a substance which can be detected with the method according to the invention or with the test equipment according to the invention and which specifically inhibits the proteolytic cleavage of a β-secretase substrate is made available. The substance according to the invention can be used for the preparation of a medicament for the treatment of neurodegenerative diseases, in particular of Alzheimer's disease. Additionally, pharmaceutical formulations containing the **** substance according to the invention and a pharmaceutically acceptable vehicle. A pharmaceutically acceptable carrier can contain physiologically acceptable compounds which, for example, increase the stability or absorption of the substance according to the invention. Such physiologically acceptable compounds include, for example, carbohydrates such as glucose, sucrose or dextrans, antioxidants such as ascorbate or glutathione, chelating agents, low molecular weight proteins or other stabilizers (see, for example, Remington's Pharmaceutical Sciences (1990). )). The technician knows that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration.

EXAMPLE 1 - Carrying out the test system 1.1 Preparing a suitable cell line Neurogymal H4 cells were transfected (deposit number HTB 148 in the "American Type Culture Collection" Manassas, Virginia, USA) under conventional conditions with the regulatory plasmid pUHD15-lneo (pUHD15-l-with neomycin resistance gene), which carries the gene for a repressible tetracycline transactivator (Gossen and Bujard, 1992, 1995). Transient transfection experiments were selected for the second stable transfection with the APP-LC99 construct an individual clone that showed a strictly regulated and strongly inducible transient expression of a reporter gene (pUHD10-3 / SEAP; SEAP: secretory alkaline phosphatase). For the preparation of the APP-LC99 construct, a sequence containing the N-terminal signal sequence and the last 99 amino acids of the APP (Sho i et al., 1992) was cloned on the BamHl and BamHl restriction cleavage positions. Sacll in the vector, controlled expression of tetracycline pUHD10-3. This construction was designated pUHD10-3 / APP-LC99. The cell clone obtained in the manner described above was co-transfected with 10 μg of pUHD10-3 / APP-LC99 and 1 μg of pTK-Hyg (Clontech, Heidelberg, Accession number of Genbank U40398) and selection was carried out. with the Fugene transfection system from Boehringer Mannheim according to the manufacturer's instructions. Individual, hygromycin-resistant cell clones were investigated by removal of doxycycline and subsequent immunofluorescence and / or Western blotting with an antibody specific for APP-CTF on the inducible expression of LC99. The selected clone was designated H4-ind / APP-LC99. 1. 2 Treatment of a microsome fraction from cells H4 LC99 The H4-ind / APP-LC99 cells were grown to confluence at 37 ° C, 5% C02 with DMEM medium (DMEM: "Dulbecco's Modified Eagle Medium" (English) supplied by the BioWittacker) Signature and fetal calf serum (English: "fetal calf serum" FCS) 10%, 1% glutamine, 1% penicillin and streptomycin, in the absence of doxycycline on Petri dishes of 15 cm. The expression of the fusion protein was induced by the withdrawal of doxycycline. All the steps of the preparation of the post-nuclear supernatant were carried out on ice or at 4 ° C. The cells were separated with a scraper of cells from the Petri dishes after the addition of 2 ml of PBS per Petri dish. 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, subsequently, they were resuspended in 300 μl of HIS + buffer (HIS buffer with 5 mM EDTA) per Petri dish. The cell homogenate was compressed with a 22 gauge needle using a 1 ml syringe and cell lysis was monitored by phase contrast microscopy. The used cell was centrifuged at 2500 g for 10 min, "to separate the supernatant from the intact cells and from the cell fragments." The PNS was further processed with a gradient gradient of sucrose (Taylor et al., 1997), adjusting it to first place to 100 mM KH2P04 / K2HPO4, pH 6.8. All sucrose solutions contained 100 mM KH2P04 / K2HP04, pH 6.8, 5 mM MgCl2 and the protease inhibitors leupeptin (10 μg / ml) and aprotinin (10 μg / ml). The gradient contained 1.3 M, 0.86 M, and 0.5 M sucrose stages, which were coated with the PNS adjusted and then centrifuged at 100,000 g for 1.5 hours at 2 ° C. The intermediate layer between sucrose 1.3 M and 0.86 M is the microsomic fraction SIII. To granulate the membranes, the SIII fraction was adjusted to 250 mM sucrose with 100 mM KH2HP04 / K2HP04. pH 6.8, and centrifuged at 220,000 g for 20 min. at 4 ° C. Membranes were washed with reaction buffer (250 mM sucrose, 50 mM KCl, magnesium acetate 2.5 mM, 20 mM HEPES, pH 6.8, 5 mM EDTA), centrifuged as described above forma_ and resuspended to homogeneity in 1 ml reaction buffer. HE small aliquots of the microsomal fraction were frozen in liquid nitrogen and stored at -80 ° C.

M il l 'i i liiii-litlr rtiilÉi i ii- 1.3 Embodiment system inhibitor assay -? secretase The microsomal fraction of cells frozen H4 ind / APP-induced LC99 was thawed on ice and 5 used respectively 10 μl for each cell-free reaction. The samples were diluted with reaction buffer to 30 μl and incubated at temperatures, pH values and determined times. After incubation, the samples were adjusted to 2% SDS and heated at 95 ° C for 5 hours. minutes. To the samples were added 1 ml of IP buffer (NaCl 150 mM, 10 mM Tris, pH 7.4, 1 mM EDTA, 0.2% NP40 and aprotinin protease inhibitors (10 μg / ml), leupeptin (10 μg / ml), 5 μg / ml of pepstatin, 1 mM pefabloc) and, respectively, 6 μg / ml of specific antibodies BI.40 and BI.42 (equivalent alternative antibodies can be obtained from QCB, Quality Control Biochemicals, Inc., Hopkinton, USA, catalog numbers 44-438 and 44-344). After one hour at 4 ° C, 20 μl of pre-washed spheres of Gammabind-Sepharose G (Pharmacia) were added.

Biotech) and incubated overnight at 4 ° C. The sepharose immunocomplex was washed with IP buffer and the precipitated proteins were eluted with 20 ml of Tris-bicma sample buffer (Klafki et al., 1996). The samples - «- '* - * ****. **** -. they were separated by means of electrophoresis in tris-bicine polyacrylamide gel in the manner described (Klafki et al., 1996). The already described highly sensitive Western blot method with antibodies 6E10 and 4G8 (mAb product numbers 200-10 and mAb 300-10, Senetek, Great Britain, Galli et al., 1998) was used to detect immunoprecipitated Aβ species (Ida et al., 1996). The chemiluminescence was detected with the Western Star substrate (Tropix) and quantified with a chemiluminescence detection system from BioRad. 1. 4 Obtaining the hepatocyte cytosol ~ from guinea pig A post-nuclear supernatant (SNP) was obtained from the liver of a guinea pig by homogenization and centrifugation (Taylor et al., 1997) in the manner already described. This supernatant is applied on a gradual sucrose 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 lxKPi buffer over 250 mM sucrose and centrifuged at 200,000 g and 4 ° C for 20 minutes. The supernatant is the cytosol (Jones et al., 1998). 1. 5 Alternative embodiment of the β-secretase inhibitor assay system with an ATP regenerator system A purified microsomal fraction of these recombinant H4 cells is incubated at 37 ° C with a suitable buffer system (50-150 mM KCl, magnesium acetate 1.5-5 mM, 250 mM sucrose, 20 mM Hepes, pH 6.8), an ATP regenerative system (1 mM ATP, 0.1 mM GTP, pH 7.0, 8 mM phosphocreatine, 31 mM creatinine-phosphokinase) and cytosol, treated with described in 1.2, and then the activity of β-secretase is measured through the detection of the product Aβ by means of Western blotting, as described above. Example - realization of the test system 1. 6 Alternative treatment of a microsomal fraction from H4 LC99 cells The same cell line (neuroglysis cell clone 114 with construction APP-LC99) was used as described in 1.1. H4-ind / APP-LC99 cells were grown to confluence at 37 ° C, 5% C02 with DMEM medium (DNEM: "Dulbecco's Modified Eagle Mediun" (English), supplied by the BioWittaker Firm) and fetal calf serum (English: "Fetal Calf serum", FCS) at 10%, 1% glutamine, 1% penicillin and streptomycin, in the absence of doxycycline, on Petri dishes 15 cm in size. After the withdrawal of doxycycline, expression of the fusion protein was induced for three days. All stages of preparation of the post-nuclear supernatant were carried out on ice or at 4 ° C. For a preparatory round, 15 Petri dishes were treated every 10 times. The cells were removed from the Petri dish after the addition of 2 ml of ice-cold PBS per Petri dish, by means of a cell scraper. All other work was carried out in the manner described in Schroter et al. After centrifugation at 500 g for 10 min. the cells were carefully resuspended in ST buffer (250 mM sucrose, 10 mM Tris, pH 7.4), were centrifuged again at 1400xg for 10 min. and, then, all the cells were resuspended in 5 ml of ST buffer. Cells were homogenized with a 5 ml crucible (Firma Braun, Melsungen) at 500 rpm and cell lysis was monitored by phase contrast microscopy. The cell lysate was first centrifuged at 2000xg for 2 min., To pellet intact cells and large cell fragments. Next, the supernatant was centrifuged at 4000xg for 2 min., To separate cell membranes and cell nuclei (PN fraction). The pellet composed of cell nuclei - and plasma membranes was washed twice with ST buffer and centrifuged in the manner previously described. The supernatants were pooled and centrifuged in a new vessel at 10000xg for 2 min to separate mitochondria, lysosones and endosomes (EL fraction). To pellet, then, the collected microsomes, the supernatant was centrifuged at 400,000 xg. For the separation of lysosomes and endosomes, the EL fraction continued to be treated. The lysosomes were burst by a 10-minute hypotonic lysis on ice (Bohley et al., 1969) and the intact endosomes sedimented at lOOOOOxg for 2 min. (lysosomes = fraction L, endosomes = fraction E). For the characterization of the separation 30 μg of the total protein of each fraction was deposited on a polyacrylamide gel and the distribution of the different marker proteins of the different compartments in the fractions was detected in the Western blot procedure. The endosomal and microsomal membranes were harvested with reaction buffer (250 mM sucrose), 50 mM KCl, 2.5 mM magnesium acetate, 20 mM HEPES, pH 6.8, 5 mM EDTA) and resuspended to homogeneity in 1 ml of reaction buffer. Small aliquots of the various fractions were frozen in liquid nitrogen and stored at -80 ° C. The β-secretase inhibitor assay system is then carried out unchanged in the manner described in 1.3.

Bibliography: Bohley et al. (1969), FEBS Lett. 5, 233-236 De Strooper et al. (1998). Nature 391, 387-390 De Strooper et al. (1999). Nature 398, 518-522 Galli et al. (1998). Proc. Nati Acad. Sol. USA, 95, 1247-1252 Gossen, M. and Bujard, H. (1992). Proc. Nati Acad. Sci. 89, 5547-51 Gossen, M. and Bujard, H. (1995). Biotechniques 19, 213-216 Haass, C. and Selkoe, D.J. (1993). Cell 75, 1039-1042 Ida et al. (nineteen ninety six). J. Biol. Chem. 271, 22908-22914 Jones, S.M. et al. (1998). Science 279, 573-577 Klafki et al. (1996): Anal. Biochem. 237, 24-29 Lemere et al. (1996): Nat. Med. 2, 1146-1150 Mann et al. (nineteen ninety six). Ann. Neurol. 40, 149-156 Price, D. and Sisodia, S. (1998). Ann. Rev. Neuroscience 21, 479-505 Remington's Pharmaceutical Sciences (1990). 18th edition.

Mack Publ., Easton. Schróter et al. J, of Im unological Methods 227 (1999) 161-168 Selkoe, D.J. (1991). Neuron 6, 487-498 Selkoe, D.J. (1994) Annu. Rev. Cell Biol. 10, 373-403 Selkoe, D.J. (nineteen ninety six). J. Biol. Chem. 271, 18295-18298 Shoji et al. (1992). Science 158, 126-129 Sisodia et al. (1998). Neuron 21, 1213-1221 Steiner et al. (1999). J. Biol. Chem. 273, 32322-32331 Steiner et al. (1999) J. Biol. Chem. 274, 7615-7618 Taylor et al, (1997). Mol. Biol. Cell 8, 1911-1931 Wolfe et al. (1999). Nature 398, 513-517 It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Claims (43)

1. RE IVINDI CAC I ON ES Having described the invention as above, the content of the following claims is claimed as property: 1. Procedure for the detection of substances capable of specifically inhibiting β-secretase, characterized in that: a) purified membranes are prepared from cells showing β-secretase activity and containing a β-secretase substrate, b) these membranes are mixed with reaction buffer and a test substance, c) this mixture is incubated under conditions under which the substrate the? -secretase is cleaved by absence of the test substance, d) the amount of a cleavage product formed is determined, and e) - the value obtained is compared to the value that is obtained in the absence of the test substance.
2. 2. Method according to claim 1, characterized in that cells expressing an endogenous polypeptide which is a substrate of the β-secretase are cultured.
3. 3. Method according to claim 1, characterized in that cells expressing an exogenous polypeptide which is a substrate of the β-secretase are cultured.
4. 4. Method according to claim 3, characterized in that the expression of the exogenous polypeptide is inducible.
5. The method according to claim 4, characterized in that the expression of the β-secretase substrate can be induced by the withdrawal of tetracycline or a derivative of tetracycline.
6. 6. Method according to one of claims 1 to 5, characterized in that the β-secretase substrate is the amyloid precursor protein or a fragment thereof.
7. 7. The method according to claim 6, characterized in that the fragment of the amyloid precursor protein is the C99 fragment.
8. 8. Method according to one of claims 1 to 5, characterized in that the β-secretase substrate is a fusion protein of the amyloid precursor protein or a fragment thereof, in particular of the C99 fragment, with a reporter protein.
9. The method according to claim 8, characterized in that the reporter protein is the green fluorescent protein or a derivative thereof, luciferase, secretory alkaline phosphatase or β-galactosidase.
10. 10. Method according to one of claims 1 to 9, characterized in that the cells are of neuronal or glial origin.
11. 11. The method according to claim 10, characterized in that the cells are H4 cells.
12. Method according to one of claims 1 to 11, characterized in that the membranes are cell membranes, preferably intracellular membranes.
13. 13. Process according to claim 12, characterized in that the membranes are lysosomal or endosomal membranes.
14. 14. Process according to claim 12, characterized in that the membranes are microsomal membranes.
15. The method according to claim 14, characterized in that the microsomal membranes are purified by the following steps: a) lysis of the cells, b) separation of the cell nuclei, c) purification on sucrose density gradients, d) sedimentation renewed by ultracentrifugation, e) homogenization, f) renewed sedimentation by ultracentrifugation, and g) renewed homogenization.
16. Method according to one of claims 1 to 15, (characterized in that the pH value of the reaction buffer is in the range of 5-10, preferably in the range of 6.0-8.0, particularly preferably 6.8 to 7.4 and contains as additional components, at least one membrane stabilizer
17. 17. Method according to claim 16, characterized in that the membrane stabilizer is sucrose or sorbitol
18. 18. Method according to claim 16 or 17, characterized in that the concentration of the stabilizer of the membrane is between 200 and 1000 mM, preferably between 200 and 500 mM, and particularly preferably between 200 and 300 mM
19. 19. Method according to one of claims 1 to 18, characterized in that the reaction buffer contains, additionally, , a complex former
20. 20. Method according to claim 19, characterized in that the complex former is -EDTA, EGTA or BAPTA, or a salt thereof.
21. 21. Process according to claim 20, characterized in that EDTA, EGTA or BAPTA, or the salt thereof, is present in a concentration of 0.1 to 5 20 mM, preferably 5 to 10 mM.
22. 22. Method according to one of claims 1 to 21, characterized in that, additionally, an ATP regenerative system is added to the reaction batch.
23. Method according to one of claims 1 to 10, characterized in that the determination of the amount of the cleavage product takes place by immunoprecipitation or Western blotting, preferably by a combination of immunoprecipitation and Western blotting.
24. 24. Method according to one of claims 1 to 15, characterized in that the determination of the amount of the cleavage product takes place by ELISA.
25. 25. Method according to one of claims 1 to 22, characterized in that the determination of the amount of the cleavage product takes place by mass spectrometry.
26. 26. Method according to one of claims 8 to 22, characterized in that the determination of the amount of the cleavage product takes place by determining the amount of reporter protein fused to the cleavage product. * ---•*** to .
27. 27. Process according to claim 26, characterized in that the amount of luciferase, secretory alkaline phosphatase or β-galactosidase fused to the cleavage product takes place by determining the enzymatic activity after the addition of the substrate.
28. The method according to claim 26, characterized in that the determination of the quantity of the green fluorescent protein or a derivative thereof takes place by measuring the intensity of the fluorescence light.
29. 29. Test equipment for the detection of substances capable of specifically inhibiting β-secretase, characterized in that the test kit contains, at least, membranes purified from cells that have β-secretase activity and contain a substrate of the α-secretase. secretase, and reaction buffer.
30. 30. Test kit according to claim 29, characterized in that the membranes are lysosomal or endosomal membranes, preferably microsomal.
31. 31. Test kit according to claims 29 to 30, characterized in that the cells exogenously express a β-secretase substrate.
32. 32. Test kit according to one of claims 29 to 31, characterized in that the reaction buffer has a pH value in the range of 5-10, preferably in the range of 6.0-8.0, particularly preferably in the range of 6.8 to 7.4 and, as additional components, it contains, at least, a membrane stabilizer.
33. 33. Test kit according to claim 32, characterized in that the membrane stabilizer is sucrose or sorbitol.
34. 34. Test kit according to claim 32 or 33, characterized in that the concentration of the membrane stabilizer in the reaction buffer is between 200 and 1000 mM, preferably between 200 and 500 mM, and especially preferably between 200 and 300. mM.
35. 35. Test kit according to one of claims 29 to 34, characterized in that the reaction buffer contains, in addition, a complexing agent.
36. 36. Test kit according to claim 35, characterized in that the complexing agent is EDTA, EGTA or BAPTA, or a salt thereof.
37. 37. Test kit according to claim 36, characterized in that EDTA, EGTA or BAPTA, or a salt thereof is in a concentration of 0.1 to 20 mM, preferably 5 to 10 mM.
38. 38. Test equipment according to one of claims 29 to 37, characterized in that the test equipment additionally contains an ATP regeneration system.
39. 39. Test kit according to one of claims 29 to 38, characterized in that the determination of the amount of the cleavage product takes place by immunoprecipitation or Western blotting, preferably by a combination of immunoprecipitation and Western blotting.
40. 40. Use of a method according to one of claims 1 to 28, or of an assay kit according to one of claims 29 to 39 for the detection of substances capable of specifically inhibiting? -secretase.
41. 41. Detectable substance with a method according to one of claims 1 to 28, or a test kit according to one of claims 29 to 39, characterized in that it specifically inhibits the proteolytic cleavage of a β-secretase substrate.
42. 42. Use of a substance according to claim 41 for the preparation of a medicament for the treatment of neurodegenerative diseases, especially Alzheimer's disease.
43. 43. Pharmaceutical formulation containing a substance according to claim 41.