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WO1998015828A1 - METHODE D'IDENTIFICATION DES INHIBITEURS DIRECTS DE L'ENZYME η-SECRETASE PRODUISANT LE β-AMYLOIDE - Google Patents

METHODE D'IDENTIFICATION DES INHIBITEURS DIRECTS DE L'ENZYME η-SECRETASE PRODUISANT LE β-AMYLOIDE Download PDF

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WO1998015828A1
WO1998015828A1 PCT/US1997/016988 US9716988W WO9815828A1 WO 1998015828 A1 WO1998015828 A1 WO 1998015828A1 US 9716988 W US9716988 W US 9716988W WO 9815828 A1 WO9815828 A1 WO 9815828A1
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app
cells
amyloid
secretase
terminal fragments
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PCT/US1997/016988
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English (en)
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Barbara Cordell
Jeffrey N. Higaki
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Scios Inc.
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Priority to AU45892/97A priority Critical patent/AU4589297A/en
Publication of WO1998015828A1 publication Critical patent/WO1998015828A1/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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • 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
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present invention relates to assays useful for identifying agents for the 10 treatment of Alzheimer's disease.
  • Alzheimer's disease is a common age-related brain degenerative disease.
  • the 15 disease is characterized by progressive dementia together with the presence of characteristic neuropathological features.
  • the formation of ⁇ -amyloid deposits or plaques is a hallmark and diagnostic feature of Alzheimer's disease (Khachaturian (1985) Arch. Neurol. 42:1097-1 105).
  • a significant body of evidence suggests that the process of ⁇ -amyloid formation and deposition is directly linked to the development of this disease.
  • individuals with mutations in the gene encoding the ⁇ -amyloid precursor protein ( ⁇ -APP) invariably develop Alzheimer's disease (Goate et al. (1991) Nature 353:844-846; Mullan et al. (1992) Nature Genet. 1 :345-347; Murrell et al. (1991) Science 254:97-99; Van Broeck oven (1995) Eur. Neurol. 35:8-19).
  • the ⁇ -amyloid peptide is a 39-43 amino acid protein (Glenner et al. (1984)
  • ⁇ -amyloid peptide is derived from a larger Type I membrane spanning protein, ⁇ -APP, which has several alternatively spliced transcripts (Kang et al. (1987) Nature 325:530-532; Ponte et al.
  • a secreted form of ⁇ -APP is normally generated by proteolytic cleavage (Weidernann et al. (1989) Cell 57:1 15-126). This proteolytic cleavage occurs within the ⁇ -amyloid domain precluding ⁇ -amyloid formation (Esch et al. (1990) Science 248: 1122- 1 124; Sisodia et al. (1990) Science 248:492-495). As a result of the cleavage, the bulk of ⁇ -APP is released from the cell and a carboxyl-terminal fragment of ⁇ 8 kDa remains bound to the cell membrane. The enzyme(s) responsible for this non-amyloidogenic processing of ⁇ -APP is termed -secretase.
  • ⁇ -amyloid peptide The formation of ⁇ -amyloid peptide is a normal physiological process.
  • the peptide has been found to be naturally produced by cultured cells in vitro (Haass et al. (1992) Nature 359:322-325; Seubert et al. (1992) Nature 359:325-327; Shoji et al. (1992) Science 258:126-129; and in vivo (Seubert et al. (1992) Nature 359:325-327; Vigo-Pelfrey et al. (1993) J. Neurochem. 61 :1965-1968; Tabaton et al. (1994) Biochem. Biophys. Res. Commun. 200:1598-1603; Teller et al.
  • the ⁇ -amyloid peptide appears to be a degradation by-product of intracellular catabolism of the non-secreted form of ⁇ -APP (Higaki et al. (1995) Neuron 14:651-659) and inhibiting its formation has no apparent deleterious consequences in vitro.
  • ⁇ -secretase processing of ⁇ -APP occurs first as evidenced by the intracellular production of ⁇ -APP carboxyl-terminal fragments harboring the intact ⁇ -amyloid domain which have been demonstrated to be the direct precursors to the ⁇ -amyloid peptide (Higaki et al. (1995) Neuron 14:651-659; Golde et al. (1992) Science 255:726-730). These -10-12 kDa ⁇ -APP carboxyl-terminal fragments are subsequently processed by ⁇ -secretase to yield ⁇ -amyloid.
  • the -secretase, non-amyloidogenic ⁇ -APP carboxyl-terminal fragments are processed together with the amyloidogenic fragments by ⁇ -secretase resulting in a -3 kDa partial ⁇ -amyloid peptide (roughly spanning residues 17 to 39-43 of the ⁇ -amyloid domain).
  • active agents are likely to affect ⁇ -amyloid formation by a number of different mechanisms such as inhibition of ⁇ -secretase, inhibition of ⁇ -secretase, alteration of intracellular compartments in which ⁇ -amyloid processing occurs (Busciglio et al. (1993) Proc. Natl. Acad. Sci. USA 90:2092-2096; Higaki et al. (1995) Neuron 14:651-659), inhibition of ⁇ -APP transcription or translation, inhibition in ⁇ -APP intracellular transport; as well as may other undefined processes.
  • the present invention provides a method for identifying direct acting ⁇ -secretase inhibitors of ⁇ -amyloid formation using intact or semi-intact cultured cells.
  • the method can be used to identify preferred therapeutic compounds or compositions for the treatment of Alzheimer's disease.
  • the method of the invention comprises the steps of:
  • the method of the invention can be carried out using a culture of intact cells or semi-intact cells prepared by standard methods such as hypotonic buffer treatment and mechanical shearing or with cells whose cell membranes have been rendered permeable using known agents such as digitonin or no-ionic detergents.
  • ⁇ -APP carboxyl-terminal fragments and/or ⁇ -amyloid peptide may include immunoprecipitation or Western blot coupled with polyacrylamide gel electrophoretic separation of protein, radioimmunoassay, or immunodisplacement assay.
  • immunoprecipitation followed by gel electrophoresis is used to quantify ⁇ -APP accumulated fragments and ⁇ -amyloid peptide, both of which are metabolically radiolabeled during the assay procedure.
  • the preferred embodiment is to use 35S-methionine radiolabeling but alternative embodiments may use J H- or l ⁇ C-amino acid radiolabeling.
  • Figure 1 is a schematic illustration of the assay method of the invention.
  • Figure 2 is an autoradiograph of polyacrylamide gels showing the ability of substrate accumulating compound to cause the accumulation of ⁇ -APP carboxyl-terminal fragments and inhibit the formation of ⁇ -amyloid peptide.
  • Figure 3 is a graphical representation of the analysis of polyacrylamide gels for a test substance which is a direct ⁇ -secretase ⁇ -amyloid inhibitor.
  • Figure 4 is a graphical representation of the analysis of polyacrylamide gels for a test substance which is inactive as a direct ⁇ -secretase ⁇ -amyloid inhibitor.
  • the invention involves a method of identifying agents capable of inhibiting the formation of the carboxyl-terminus of the ⁇ -amyloid peptide by directly inhibiting the activity of ⁇ -secretase.
  • agents capable of inhibiting the formation of the carboxyl-terminus of the ⁇ -amyloid peptide by directly inhibiting the activity of ⁇ -secretase.
  • Such agents can be used to intervene in the process of Alzheimer's disease amyloidosis.
  • ⁇ -amyloid peptide or protein refers to any of the ⁇ -amyloid species of -4 kDa known to have several different amino-termini and heterogenous carboxyl-termini based on characterization of the peptide from Alzheimer's disease tissue and from cultured cells (Glenner and Wong (1984) Biochem. Biophys. Res. Commun. 120:885-890; Joachim et al. (1988) Brain Res. 474: 100-1 1 1; Prelli et al. (1988) J. Neurochem. 51 :648-651 ; Mori et al. (1992) J. Biol. Chem. 267: 17082- 17806; Seubert et al.
  • the ⁇ -amyloid peptide has been shown to end at position 39, 40, 41, 42, 43, or 44 where position 1 is the aspartate of the ⁇ -amyloid sequence as defined by Glenner and Wong (1984) Biochem. Biophys. Res. Commun. 120:885-890.
  • This assay method applies to all ⁇ -amyloid species with various carboxyl-termini.
  • ⁇ -amyloid precursor protein or " ⁇ -APP” refers to any of the differentially spliced isoforms of this protein including the 695, 714, 751, and 770 amino acid isoforms (Kang et al. (1987) Nature 325:530-532; Ponte et al. (1988) Nature 331 :525-527;Tanzi et al.(1988) Nature 331 :528-530; Kitaguchi et al. (1988) Nature 331 : 530-532; de Savage et al. (1989) Science 245:651-653). This term also applies to naturally occurring human mutants of ⁇ -APP (Goate et al.
  • ⁇ -APP carboxyl-terminal fragments refers to carboxyl-terminal derivatives of ⁇ -APP which result from ⁇ -secretase and/or ⁇ -secretase processing and which generate fragments bearing the ⁇ -amyloid domain at or near the amino-terminus of the fragment or partial ⁇ -amyloid domain sequences.
  • substrate accumulating compound refers to an agent which has been shown, when contacted with a cell line expressing ⁇ -APP, to cause the intracellular accumulation of ⁇ -APP carboxyl-terminal fragments, which simultaneously blocks ⁇ -amyloid formation, and the activity of which can be reversed upon removal of the compound.
  • agents ammonium chloride, monensin and the compound MDL 28,170 (Higaki et al. (1995) Neuron 14:651-659), which is described below.
  • the present invention provides a method for identifying a ⁇ -secretase inhibitory substance wherein a compound which is known to act as a ⁇ -secretase inhibitor (the substrate accumulating compound) is applied to a cell line that is expressing ⁇ -APP, thus allowing ⁇ - and ⁇ -secretase processing of ⁇ -APP to occur in the absence of ⁇ -secretase processing. This results in the accumulation of C-terminal fragments of ⁇ -APP which serve as a substrate for ⁇ -secretase processing.
  • the known ⁇ -secretase inhibitor is then removed and replaced by a test substance.
  • the ability of the test substance to inhibit ⁇ -secretase activity can then be determined by quantifying the conversion of the C-terminal fragments of ⁇ -APP to ⁇ -amyloid peptide.
  • ⁇ - and ⁇ -secretase cleavage of ⁇ -APP in the presence of a substrate accumulating compound gives rise to the accumulation of four C-terminal fragments due to the existence of two ⁇ -processing sites and two ⁇ -processing sites.
  • the shaded segments in the two C-terminal fragments produced by ⁇ -secretase cleavage represent the ⁇ -amyloid peptide segment.
  • the shaded segments in two C-terminal fragments produced by ⁇ -secretase cleavage represent a non-amyloidogenic fragment of the ⁇ -amyloid peptide.
  • the substrate accumulating compound is the compound MDL 28,170, which has the following formula (Medhi S. (1991) Trends Biochem. Sci. 16: 150-153; Higaki et al. (1995) Neuron 14:651-659).
  • any compound which is known to accumulate substrate whether by direct or indirect inhibition of ⁇ -secretase activity, including ⁇ -secretase inhibitor compounds identified using the method of this invention, can be employed as the substrate accumulating compound provided it has reversible activity.
  • the substrate accumulating compound is removed and replaced by a test substance.
  • the processing (or absence of processing) of C-terminal fragments by ⁇ -secretase cleavage is then monitored by quantitating ⁇ -APP carboxyl-terminal fragments in the cells, ⁇ -amyloid peptide in the medium or both.
  • Graph (a) of Fig. 1 illustrates a typical result when the test compound exhibits no ⁇ -secretase inhibitory activity. The presence of C-terminal fragments diminishes and the presence of ⁇ -amyloid peptide increases as ⁇ -secretase processing occurs.
  • Graph (b) illustrates a typical result when the test compound exhibits strong ⁇ -secretase inhibitory activity. Little or none of the C-terminal fragments are converted to ⁇ -amyloid due to the inhibition of ⁇ -secretase activity.
  • the assay method may employ primary or cultured cell lines of any type from any mammalian species. Since ⁇ -amyloid peptide is normally produced only at low levels in cultured mammalian cells, a preferred embodiment is to use a cell line which has been transfected with a ⁇ -APP encoding cDNA or gene operably linked to a promoter which is capable of effecting its expression in the host cell line, resulting in increased levels of the ⁇ -amyloid peptide.
  • Standard vectors for recombinant mammalian cell expression (available from commercial sources such as InVitrogen or Stratagene) of the exogenous ⁇ -APP DNA are suitable such as those, for example, using the ⁇ -actin or cytomegalovirus promoters combined with a selectable drug marker such as neomycin or zeomycin.
  • Standard methods can be used for introducing exogenous DNA into the cell such as lipofectamine, calcium phosphate or electroporation. Isolation of single cell clones carrying the recombinant DNA is performed by standard methods usually making use of a drug-resistance gene harbored on the expression plasmid. These methods are described in detail by the commercial manufacturer of the expression plasmid selected.
  • a Chinese hamster ovary (CHO) cell line is used in conjunction with an expression plasmid in which a ⁇ -APP695 encoding cDNA is driven by a cytomegalovirus promoter and which possesses the neomycin-resistance gene.
  • the expression plasmid is purchased from a commercial source, such as InVitrogen.
  • Lipofectamine obtained from Gibco BRL, is used to introduce the DNA plasmid into the CHO cells and the drug G418 to select the CHO cells harboring the plasmid. Manufacturers directions are applied for the use of lipofectamine and for drug selection of cells with expression plasmid.
  • Antibodies to the ⁇ -amyloid peptide and to the carboxyl-terminus of ⁇ -APP are useful for analysis, isolation and quantitation of ⁇ -APP carboxyl-terminal fragments and ⁇ -amyloid peptide in the assay of the invention.
  • synthetic or natural peptides can be used for the generation of the specific antibodies. Synthetic peptides are preferred since they can be used to raise antibodies to select regions of ⁇ -amyloid or ⁇ -APP and because of their general ease of preparation. Synthetic peptides are prepared by either the phosphotriester method as described by Edge et al. (1981) Nuc. Acids Res. 292:756 and Duckworth et al. (1981) Nuc. Acids Res. 9: 1691 or the phosphoramidite method as described by Beaucage and Caruthers (1981) Tet. Lett.
  • the synthetic or natural peptides may be conjugated to a carrier protein for animal immunization or they may be used as prepared.
  • a synthetic peptide is coupled to a carrier such as keyhole limpet hemocyanin and to immunize rabbits according to standard methods.
  • the peptide conjugate can be used to immunize mice to prepare monoclonal antibodies.
  • synthetic peptides conjugated to a carrier Preferred peptides correspond to residues 1-13, 1-28, or 1-40 of ⁇ -amyloid.
  • synthetic peptides To prepare antibodies specific to the carboxyl-terminal cytoplasmic domain of ⁇ -APP, synthetic peptides have been demonstrated to yield reagents with the desired features. Peptides of 20 to 30 residues in length corresponding to sequences spanning residues 713 to 770 of the 770 amino acid ⁇ -APP isoform are preferred.
  • One specific peptide consisting of residues 723-749 of ⁇ -APP 770 (or 704-730 of ⁇ -APP 751) has been effective (Higaki et al.
  • ⁇ -APP carboxyl-terminal and ⁇ -amyloid antibodies may be characterized for reactivity against their respective cognate immunogen in an enzyme- linked immunoadsorption sandwich assay. Further characterization of the polyclonal or monoclonal antibodies for the ability to react with native ⁇ -amyloid can be conducted using a variety of techniques known to the art, including, for example, immunoprecipitation of radiolabeled products, Western blot analysis, radioimmunoassay, or immunodisplacement assay. Identification of ⁇ - Amyloid Inhibitors
  • the method of the invention can be employed to determine the ⁇ -secretase inhibitory activity of any test substance.
  • Preferred candidates for testing are substances which have previously been identified as inhibitors of ⁇ -amyloid formation.
  • Inhibitors of ⁇ -amyloid production may be identified by several methods such as by application of compounds to cultured cells, semi-intact or cells whose membranes have been rendered permeable that produce ⁇ -amyloid, and measuring the reduction of ⁇ -amyloid over a period of hours, preferably 1-8 hours. Measurement of ⁇ -amyloid may be made by immunoaffinity reagents using a variety of techniques, for example, immunoprecipitation, Western blot, enzyme-linked immunoadsorption or immunodisplacement assays. Active inhibitors are compounds which partially or fully reduce levels of ⁇ -amyloid peptide. Active compounds may be of a wide array of chemical and/or protein structures.
  • Cultured mammalian cells which produce and release ⁇ -amyloid peptide into the culture medium preferably at levels of 1-50 ng per ml may be used for the assay.
  • cells in serum- free medium are treated with a pre-determined non-toxic but effective dose of substrate accumulating compound and cellular protein is simultaneously metabolically radiolabeled for example by adding - ⁇ S-methionine (100-200 ⁇ Ci per ml) to the serum-free medium.
  • - ⁇ S-methionine 100-200 ⁇ Ci per ml
  • protein labeling is allowed to proceed for about 1 hour.
  • the medium containing the substrate accumulating compound and ⁇ S-methionine is removed, the cells are washed with an appropriate solution such as saline, and serum- free medium containing the agent to be tested is applied.
  • the test substance has been previously identified as an active inhibitor of ⁇ -amyloid formation.
  • the test substance is allowed to remain in contact with the cells for about 1 to 6 hours, most preferably about 4 hours.
  • Cells and medium are harvested and each is analyzed. All of the cells and medium may be harvested at once at the end of the treatment period or aliquots may be harvested at various times during the treatment in order to provide a time course of conversion of ⁇ -APP carboxyl terminal fragments to ⁇ -amyloid peptide.
  • the harvested cells may be lysed to enable protein extraction by any of a number of standard methods but treatment with mild detergent is preferred.
  • Protein can be extracted from the lysed cells by the method of Gabuzda et al. (1991) J. AIDS 4:34-40 or by other conventional methods. An aliquot of the protein extracted from the cells is reacted with a ⁇ -APP cytoplasmic domain-specific antibody to measure the levels of carboxyl-terminal fragments present after treatment with the test substance.
  • Immunoprecipitation is a preferred method of analysis.
  • the immunoaffinity purified ⁇ -APP fragments are analyzed by polyacrylamide gel electrophoresis. Presence or absence of ⁇ -APP carboxyl-terminal fragments in the sample treated with the test substance is assessed by comparing the levels to that in cells having no substrate accumulating compound or test substance added, as well as to cells continuously treated with only the substrate accumulating compound. Quantitation may be made by direct measurement of radioactivity present in the gel region of ⁇ -APP fragments or indirectly by computer integration of the exposed regions from the radiolabeled fragments.
  • Quantitation of the ⁇ -APP carboxyl-terminal fragments in the cells treated with the test substance, relative to the amounts present in the controls indicates the ability of the test substance to inhibit cleavage of the ⁇ -APP carboxyl-terminal fragments by ⁇ -secretase.
  • the collected medium may be assessed for levels of ⁇ -amyloid protein, which is another indicator of the conversion of accumulated ⁇ -APP carboxyl-terminal fragments by ⁇ -secretase cleavage. It is known that, after ⁇ -amyloid protein is formed in the cell, it is immediately released into the extracellular environment (Haass et al. (1992) Nature 359:322-325; Seubert et al. (1992) Nature 359:325-327; Shoji et al. (1992) Science 258: 126-129; Busciglio et al. (1993) Proc. Natl. Acad. Sci. USA 90:2092-2096; Vigo-Pelfrey et al.
  • the cell culture medium in this case, the cell culture medium.
  • An aliquot of medium is reacted with ⁇ -amyloid- specific antibodies, immunoprecipitated and analyzed by polyacrylamide gel analysis.
  • an aliquot of the medium is electrophoresed on polyacrylamide gel and then Western blotted.
  • the medium can also be analyzed by an enzyme-linked immunoadsorption or immunodisplacement assay. The preferred mode of analysis is to use immunoprecipitation.
  • Quantitation of ⁇ -amyloid can be made by comparing levels to cells in which the no substrate accumulating compound and agent were added and to cells persistently treated with only the substrate accumulating compound. Quantitation may be made by direct measurement of radioactivity present in the gel region with ⁇ -APP fragments or indirectly by computer integration of the exposed regions from the radiolabeled fragments.
  • Analysis of ⁇ -amyloid peptide and ⁇ -APP carboxyl-terminal fragments will indicate whether an agent is a direct ⁇ -secretase inhibitor or not.
  • An agent which is not a direct ⁇ -secretase inhibitor displays a conversion of radiolabeled accumulated substrate to ⁇ -amyloid peptide.
  • An agent which is a direct ⁇ -secretase inhibitor will display continued high levels of accumulated substrate and an absence of ⁇ -amyloid formation.
  • a Chinese hamster ovary (CHO) cell line expressing recombinant human ⁇ -APP was used in the assay method.
  • This cell line was prepared with the 695 amino acid ⁇ -APP isoform cDNA and a commercially available expression plasmid from InVitrogen.
  • the expression plasmid, pcDNA3 has an immediate early gene cytomegalovirus promoter and a neomycin-resistance gene.
  • the expression plasmid has the necessary sequences for bacterial replication plus an ampicillin resistance gene to allow for selection of bacteria containing plasmids.
  • a -2.3 kilobase pair Smal-Xmnl fragment of the ⁇ -APP cDNA which contains the entire coding region was cloned into the polylinker region of the plasmid.
  • Bacterial clones were selected and evaluated for those possessing the ⁇ -APP cDNA in a sense orientation. Standard methods, including DNA sequence analysis, were used to isolate and characterize the appropriate expression construct.
  • CsCl banded plasmid DNA was made and used to transfect wild-type CHO cells.
  • ⁇ -APP and ⁇ -amyloid peptide were assessed for levels of ⁇ -APP and ⁇ -amyloid peptide. This was achieved by metabolically radiolabeling a collection of random clones with 150 ⁇ Ci/ml - ⁇ S-methionine in serum-free medium for 6 hours. The medium from each clone was harvested and the clone monolayers were washed with saline then prepared according to the method of Gabuzda et al. (1991) J. AIDS 4:34-40. To measure ⁇ -APP and ⁇ -amyloid levels, immunoprecipitation was used.
  • each cell lysate was diluted to 5 ml with RIPA buffer (7 ml: 50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% deoxycholate, 0.1% SDS, 1% Triton X-100), was precleared with 4 ⁇ l normal rabbit serum and 100 ⁇ l protein A-Sepharose by rocking at 23°C for 2.5 hours or 4°C overnight followed by centrifugation at low speed.
  • RIPA buffer 7 ml: 50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% deoxycholate, 0.1% SDS, 1% Triton X-100
  • the mixture was centrifuged at low speed and the pellet was washed twice with 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, twice with 50 mM Tris-HCl (pH 7.5) 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, and twice with 10 mM Tris-HCl (pH 7.5).
  • the pellet was resuspended in 100 ⁇ l SDS Laemmli sample reducing buffer and 20 ⁇ l were applied to a 8% SDS polyacrylamide gel for electrophoresis.
  • the final pellet was resuspended in 50 ⁇ l Laemmli SDS reducing buffer and applied to a 16.5% Tris-Tricine-SDS polyacrylamide gel.
  • Gel autoradiograms were developed and quantitative densitometry of the ⁇ 4 kDa ⁇ -amyloid protein band was made using a Phosphorimager from Molecular Dynamics.
  • a comparative analysis of the clones identified a single clone with the highest levels of both ⁇ -APP and ⁇ -amyloid. This clone was selected as the assay cell line.
  • FIG. 2 is an autoradiograph showing the effects of the substrate accumulating compound, MDL 28,170, in which ⁇ -amyloid formation is blocked and a concomitant increase in C-terminal ⁇ -APP fragments is observed (upper panel).
  • the gel pattern on the left, designated by (+) indicates application of the substrate accumulating compound and the gel pattern on the right, designated (-) is the untreated control.
  • the upper panel shows that the cell lysate from the cells cultured in the presence (+) of MDL 28,170 for 4 hours contains substantial amounts of accumulated C-terminal fragments, whereas the lysate from the untreated control cells exhibit only normal levels of C-terminal fragments.
  • the lower panel shows that the medium from the cells cultured in the presence (+) of MDL 28,170 for 4 hours does not contain detectable ⁇ -amyloid, whereas the medium from the untreated control cells contains ⁇ -amyloid peptide.
  • Agents which block ⁇ -amyloid formation were identified by applying random chemical agents to the CHO assay cell line and measuring ⁇ -amyloid production.
  • Agents X and Y were identified as active ⁇ -amyloid inhibitors in the following manner.
  • Monolayers of CHO cells (-10 ⁇ cells per well) plated in a 6 multi-well plate in a 1 : 1 mixture of Dulbecco's minimum essential medium and Coon's F12 medium with 10% fetal calf serum were grown for about 24 hours after which the cell monolayers were washed twice with saline then placed in 2 mis of Delbecco's minimal essential medium 21 containing 150 ⁇ Ci/ml -"S-methionine. Individual agents were applied to the cells at 25 ⁇ M for 4 hours. After the treatment period, the medium was harvested and assayed for ⁇ -amyloid peptide using immunoprecipitation with a ⁇ -amyloid-specific antiserum as detailed in the previous section.
  • Both agents X and Y displayed complete inhibitory activity for ⁇ -amyloid production at 25 ⁇ M as well at concentrations of 50 ⁇ M and 100 ⁇ M. No gross toxicity was detected based on morphology of the cells, determination of general protein radiolabeling by examining ⁇ -APP in the cell lysates as described above, and by using a MTT (3-[4,5-dimethylthiazol-2-yl]-2,5,-diphenyltetrazolium bromide) reduction assay (Hansen et al. (1989) J. Immunol. Methods 1 19:203-210).
  • MTT 3-[4,5-dimethylthiazol-2-yl]-2,5,-diphenyltetrazolium bromide
  • the CHO cell line was plated into a 6 multi-well plate with -10 ⁇ cells per well.
  • the cell monolayers were washed twice with saline and 2 ml of serum-free medium (Dulbecco's minimum essential medium 21) containing 100 ⁇ Ci/ml - ⁇ S-methionine and 200 ⁇ M of the substrate accumulating compound, MDL 28,170 (Higaki et al. (1995) Neuron 14: 651-659) were added. Radiolabeling and compound treatment was for 1 hour. After this period, the cell monolayers were washed free of label and substrate accumulating compound by rinsing the cells three times with saline. Two ml of fresh serum-free medium containing 25 ⁇ M agent X was added to the monolayers and then incubated for 4 hours.
  • serum-free medium Dulbecco's minimum essential medium 21
  • MDL 28,170 Haigaki et al. (1995) Neuron 14: 651-659
  • Fig. 3 results of the densitometry scans are presented graphically in Fig. 3, in which the results of the scans are reported in arbitrary pixel units (A.U.) generated by the computerized scanning.
  • the upper panel demonstrates the lack of ⁇ -amyloid production during the treatment period with agent X indicating inhibition of ⁇ -amyloid formation.
  • the lower panel of Fig. 3 reveals the lack of utilization of radiolabeled ⁇ -APP carboxyl- terminal substrate, indirectly confirming inhibition of ⁇ -amyloid production by direct inhibition of ⁇ -secretase.
  • Agent Y was tested in the assay method in an identical manner as agent X.
  • the upper panel reveals that agent Y, while it is an active inhibitor of ⁇ -amyloid formation (Example 1 ; Section C), is not a direct ⁇ -secretase inhibitor since the agent did not inhibit ⁇ -amyloid formation: radiolabeled ⁇ -amyloid was found present in the culture medium.
  • the lower panel of Fig. 4 shows that the radiolabeled ⁇ -APP carboxyl-terminal substrates are depleted indirectly confirming the lack of ⁇ -secretase inhibition by agent Y.
  • Agent Y was next reconfirmed as an active general ⁇ -amyloid inhibitor according to the procedure described in Example 1 Section C.

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Abstract

L'invention porte sur une méthode d'identification des inhibiteurs directs de l'enzyme η-secrétase. Une lignée de cellules exprimant β-APP est mise en culture en contact avec un composé connu pour inhiber l'activité de la η-secrétase, ce qui entraîne l'accumulation dans la cellule de fragments carboxyle terminaux de β-APP. Le composé inhibiteur connu de la η-secrétase est extrait et remplacé par une substance d'essai. L'activité inhibitrice directe de η-secrétase de la substance d'essai dépend de la quantité de fragments carboxyle terminaux de β-APP dans les cellules et/ou de la quantité de peptide β-amyloïde présente dans le milieu de culture pendant un temps donné.
PCT/US1997/016988 1996-10-07 1997-09-19 METHODE D'IDENTIFICATION DES INHIBITEURS DIRECTS DE L'ENZYME η-SECRETASE PRODUISANT LE β-AMYLOIDE WO1998015828A1 (fr)

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WO2000028331A1 (fr) * 1998-11-12 2000-05-18 Du Pont Pharmaceuticals Company Utilisation de radioligands pour detecter des inhibiteurs de la production de peptides beta-amyloides
WO2000034511A3 (fr) * 1998-12-07 2000-11-16 Aventis Pharma Gmbh ANALYSE SELECTIVE DE Aβ-PEPTIDE
DE19941039A1 (de) * 1999-08-28 2001-03-01 Boehringer Ingelheim Pharma gamma-Sekretase in vitro Testsystem
WO2001029563A1 (fr) * 1999-10-21 2001-04-26 Smithkline Beecham Plc Procede de depistage des inhibiteurs de asp2
WO2001031054A1 (fr) * 1999-10-22 2001-05-03 Smithkline Beecham P.L.C. Procede de depistage des inhibiteurs de asp1
US6245884B1 (en) 1998-10-16 2001-06-12 Vivian Y. H. Hook Secretases related to alzheimer's dementia
WO2001049871A2 (fr) * 2000-01-06 2001-07-12 Boehringer Ingelheim Pharma Kg Methode de recherche d'un inhibiteur de protease
WO2001053255A1 (fr) * 2000-01-24 2001-07-26 Merck Sharp & Dohme Limited Inhibiteurs de la gamma-secretase
US6313268B1 (en) 1998-10-16 2001-11-06 Vivian Y. H. Hook Secretases related to Alzheimer's dementia
WO2002004644A2 (fr) * 2000-07-07 2002-01-17 Boehringer Ingelheim Pharma Kg Aspartyl-protease
US6392787B1 (en) * 2000-09-01 2002-05-21 Agere Systems Guardian Corp. Process for fabricating article comprising photonic band gap material
WO2001075435A3 (fr) * 2000-04-03 2002-08-08 Bristol Myers Squibb Co Isolement d'un complexe proteinique gamma-secretase fonctionnelement actif ; methodes de detection d'activite et inhibiteurs de cette activite
WO2003008635A2 (fr) * 2001-07-04 2003-01-30 Boehringer Ingelheim Pharma Gmbh & Co. Kg Essai de criblage in vitro de $g(g)-secretase
WO2001085924A3 (fr) * 2000-05-11 2003-02-27 Scios Inc Modulation de l'activité des $g(g)-sécrétases
US6545127B1 (en) 1999-06-28 2003-04-08 Oklahoma Medical Research Foundation Catalytically active recombinant memapsin and methods of use thereof
WO2003064681A3 (fr) * 2002-01-31 2003-12-24 Eisai London Res Lab Ltd Essais
US6753410B2 (en) 2000-03-02 2004-06-22 Merck & Co., Inc. Investigational compounds
US6870030B2 (en) 1997-01-28 2005-03-22 Smithkline Beecham Corporation Asp2
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
US6962913B2 (en) 1998-08-07 2005-11-08 Bristol-Myers Squibb Company Benzo-1,4-diazepin-2-ones as inhibitors of Aβ protein production
WO2005075632A3 (fr) * 2004-01-29 2005-11-10 Cellzome Ag Traitement de maladies neurodegeneratives au moyen de atp7a
EP1392340A4 (fr) * 2001-03-30 2006-04-26 Buck Inst Peptides cytotoxiques, peptidomimetiques bases sur ces peptides, et methodes d'utilisation
US7153491B2 (en) 1998-11-12 2006-12-26 Bristol-Myers Squibb Pharma Company Use of small molecule radioligands to discover inhibitors of amyloid-beta peptide production and for diagnostic imaging
US7304056B2 (en) 1998-08-07 2007-12-04 Bristol-Myers Squibb Pharma Company Succinoylamino lactams as inhibitors of Aβ protein production
US7335632B2 (en) 2001-10-23 2008-02-26 Comentis, Inc. Beta-secretase inhibitors and methods of use thereof
US7354914B2 (en) 2000-06-01 2008-04-08 Bristol-Myers Squibb Pharma Company Lactams substituted by cyclic succinates as inhibitors of Aβ protein production
US7544855B2 (en) 2004-04-23 2009-06-09 Buck Institute Transgenic mouse whose genome comprises an APP having a mutation at amino acid 664
CN1795203B (zh) * 2003-05-26 2010-06-16 塞诺菲-安万特德国有限公司 筛选γ-分泌酶抑制剂的方法

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US6870030B2 (en) 1997-01-28 2005-03-22 Smithkline Beecham Corporation Asp2
US7507815B2 (en) 1998-08-07 2009-03-24 Bristol-Myers Squibb Pharma Company Succinoylamino lactams as inhibitors of a-β protein production
US7304055B2 (en) 1998-08-07 2007-12-04 Bristol-Myers Squibb Pharma Company Succinoylamino lactams as inhibitors of Aβ protein production
US7304056B2 (en) 1998-08-07 2007-12-04 Bristol-Myers Squibb Pharma Company Succinoylamino lactams as inhibitors of Aβ protein production
US7101870B2 (en) 1998-08-07 2006-09-05 Bristol-Myers Squibb Pharma Company Succinoylamino lactams as inhibitors of A-β protein production
US6962913B2 (en) 1998-08-07 2005-11-08 Bristol-Myers Squibb Company Benzo-1,4-diazepin-2-ones as inhibitors of Aβ protein production
US6245884B1 (en) 1998-10-16 2001-06-12 Vivian Y. H. Hook Secretases related to alzheimer's dementia
US6313268B1 (en) 1998-10-16 2001-11-06 Vivian Y. H. Hook Secretases related to Alzheimer's dementia
US6331408B1 (en) * 1998-11-12 2001-12-18 Robert Zaczek Use of small molecule radioligands to discover inhibitors of amyloid-beta peptide production
US7153491B2 (en) 1998-11-12 2006-12-26 Bristol-Myers Squibb Pharma Company Use of small molecule radioligands to discover inhibitors of amyloid-beta peptide production and for diagnostic imaging
WO2000028331A1 (fr) * 1998-11-12 2000-05-18 Du Pont Pharmaceuticals Company Utilisation de radioligands pour detecter des inhibiteurs de la production de peptides beta-amyloides
US7109027B2 (en) 1998-12-07 2006-09-19 Sanofi-Aventis Deutschland Gmbh Aβ-Peptide screening assay
WO2000034511A3 (fr) * 1998-12-07 2000-11-16 Aventis Pharma Gmbh ANALYSE SELECTIVE DE Aβ-PEPTIDE
US7244708B2 (en) 1999-06-28 2007-07-17 Oklahoma Medical Research Foundation Inhibitors of memapsin 2 and use thereof
US6545127B1 (en) 1999-06-28 2003-04-08 Oklahoma Medical Research Foundation Catalytically active recombinant memapsin and methods of use thereof
DE19941039A1 (de) * 1999-08-28 2001-03-01 Boehringer Ingelheim Pharma gamma-Sekretase in vitro Testsystem
WO2001016355A2 (fr) * 1999-08-28 2001-03-08 Boehringer Ingelheim Pharma Kg SYSTÈME DE TEST IN VITRO POUR LA η-SECRÉTASE
WO2001016355A3 (fr) * 1999-08-28 2001-08-30 Boehringer Ingelheim Pharma SYSTÈME DE TEST IN VITRO POUR LA η-SECRÉTASE
WO2001029563A1 (fr) * 1999-10-21 2001-04-26 Smithkline Beecham Plc Procede de depistage des inhibiteurs de asp2
WO2001031054A1 (fr) * 1999-10-22 2001-05-03 Smithkline Beecham P.L.C. Procede de depistage des inhibiteurs de asp1
WO2001049871A3 (fr) * 2000-01-06 2002-03-28 Boehringer Ingelheim Pharma Methode de recherche d'un inhibiteur de protease
WO2001049871A2 (fr) * 2000-01-06 2001-07-12 Boehringer Ingelheim Pharma Kg Methode de recherche d'un inhibiteur de protease
WO2001053255A1 (fr) * 2000-01-24 2001-07-26 Merck Sharp & Dohme Limited Inhibiteurs de la gamma-secretase
US6756511B2 (en) 2000-01-24 2004-06-29 Merck Sharp & Dohme Limited Gamma-secretase inhibitors
US6753410B2 (en) 2000-03-02 2004-06-22 Merck & Co., Inc. Investigational compounds
WO2001075435A3 (fr) * 2000-04-03 2002-08-08 Bristol Myers Squibb Co Isolement d'un complexe proteinique gamma-secretase fonctionnelement actif ; methodes de detection d'activite et inhibiteurs de cette activite
US6713248B2 (en) 2000-04-03 2004-03-30 Bristol-Myers Squibb Company Methods for detection of gamma-secretase activity and identification of inhibitors thereof
US6579689B2 (en) 2000-05-11 2003-06-17 Scios Inc. Modulation of γ-secretase activity
WO2001085924A3 (fr) * 2000-05-11 2003-02-27 Scios Inc Modulation de l'activité des $g(g)-sécrétases
US7456278B2 (en) 2000-06-01 2008-11-25 Bristol-Myers Squibb Pharma Corporation Lactams substituted by cyclic succinates as inhibitors of Aβ protein production
US7354914B2 (en) 2000-06-01 2008-04-08 Bristol-Myers Squibb Pharma Company Lactams substituted by cyclic succinates as inhibitors of Aβ protein production
WO2002004644A3 (fr) * 2000-07-07 2002-05-10 Boehringer Ingelheim Pharma Aspartyl-protease
WO2002004644A2 (fr) * 2000-07-07 2002-01-17 Boehringer Ingelheim Pharma Kg Aspartyl-protease
US6392787B1 (en) * 2000-09-01 2002-05-21 Agere Systems Guardian Corp. Process for fabricating article comprising photonic band gap material
EP1392340A4 (fr) * 2001-03-30 2006-04-26 Buck Inst Peptides cytotoxiques, peptidomimetiques bases sur ces peptides, et methodes d'utilisation
WO2003008635A3 (fr) * 2001-07-04 2004-01-29 Boehringer Ingelheim Pharma Essai de criblage in vitro de $g(g)-secretase
WO2003008635A2 (fr) * 2001-07-04 2003-01-30 Boehringer Ingelheim Pharma Gmbh & Co. Kg Essai de criblage in vitro de $g(g)-secretase
US7335632B2 (en) 2001-10-23 2008-02-26 Comentis, Inc. Beta-secretase inhibitors and methods of use thereof
WO2003064681A3 (fr) * 2002-01-31 2003-12-24 Eisai London Res Lab Ltd Essais
CN1795203B (zh) * 2003-05-26 2010-06-16 塞诺菲-安万特德国有限公司 筛选γ-分泌酶抑制剂的方法
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
WO2005075632A3 (fr) * 2004-01-29 2005-11-10 Cellzome Ag Traitement de maladies neurodegeneratives au moyen de atp7a
US7544855B2 (en) 2004-04-23 2009-06-09 Buck Institute Transgenic mouse whose genome comprises an APP having a mutation at amino acid 664

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