WO2003087842A1 - Method for the identification of modulators of a secretase activity - Google Patents

Method for the identification of modulators of a secretase activity Download PDF

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Publication number
WO2003087842A1
WO2003087842A1 PCT/IB2002/001342 IB0201342W WO03087842A1 WO 2003087842 A1 WO2003087842 A1 WO 2003087842A1 IB 0201342 W IB0201342 W IB 0201342W WO 03087842 A1 WO03087842 A1 WO 03087842A1
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Prior art keywords
protein
secretase
anyone
activity
invertase
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PCT/IB2002/001342
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French (fr)
Inventor
Alcide Barberis
Oliver Michael Middendorp
Urs LÜTHI
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Esbatech Ag
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Priority to CA002482824A priority Critical patent/CA2482824A1/en
Priority to AU2002253468A priority patent/AU2002253468B2/en
Priority to DE60210266T priority patent/DE60210266T2/en
Priority to EP02722595A priority patent/EP1495329B1/en
Priority to US10/512,001 priority patent/US20060068388A1/en
Priority to PCT/IB2002/001342 priority patent/WO2003087842A1/en
Publication of WO2003087842A1 publication Critical patent/WO2003087842A1/en

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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96472—Aspartic endopeptidases (3.4.23)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • 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
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to a in vivo method for the identification of modulators of secretase activities .
  • Protein secretion is central to the proper development and function of eukaryotic organisms. Moreover, several pathophysiological processes such as neuro- degeneration, oncogenesis, apoptosis and inflammation are associated with the malfunction or aberrant regulation of protein secretion. It has become clear that there is no single biosynthetic mechanism common to all secretory proteins. Secretion of proteins can occur through either the regulated or constitutive pathways and, in some cell types, this secretion can be polarized to distinct cellu- lar domains. An increasing number of proteins are now recognized as being derived from integral membrane proteins of type I and type II topology and, in this case, the secretory event involves their selective post- translational hydrolysis from the cell surface.
  • proteases known as secretases.
  • the cleavage of membrane proteins generally occurs near the extracellular face of the membrane, although in some cases it has been shown also to occur within the trans- membrane domain. Proteins secreted in this fashion in- elude membrane receptors and receptor ligands, ectoen- zymes, cell adhesion molecules and others. Examples of protein secretion through the action of secretases include the vasoregulatory enzyme ACE (angiotensin converting enzyme) , the tumor necrosis factor (TNF) ligand and receptor superfamilies, the transforming growth factor- ⁇ , certain cytokine receptors, the Alzheimer *s amyloid precursor protein (APP) and others (7) .
  • ACE angiotensin converting enzyme
  • TNF tumor necrosis factor
  • APP Alzheimer *s amyloid precursor protein
  • the involvement of secretases in the development of human diseases makes them potential drug target candidates in a variety of disease areas, including anti- cancer drugs, cardiovascular drugs, anti-neurode
  • US patent No. 5,942,400 and WO 96/40885 dis- close in vi tro methods for screening for candidate drugs for the ability to inhibit the activity of beta- ⁇ ecretase. Said methods are based on the detection of APP cleavage products using specific antibodies.
  • WO 98/13488 describes a method for determin- ing the activity of modulators of APP secretases that are active in cultured human cells. Said method involves transfection of tissue-culture cells with vectors that express cleavable reporter proteins. Upon cleavage by the endogenous secretases, a reporter domain is secreted and can be detected using standard biochemical and immuno- logical methods.
  • WO 01/49871 discloses an in vivo process for finding substances which specifically inhibit ⁇ -secre- tase.
  • Said process encompasses cells expressing a secre- tase activity and a fusion protein which comprises the substrate of said secretase with the specific cleavage site and a reporter.
  • Said cells are contacted with a test substance and the quantity of reporter cleaved is either measured directly or indirectly.
  • Suitable reporter pro- teins allowing direct detection are GFP, luciferase, ⁇ - galactosidase and secreted alkaline phosphatase.
  • the cleaved reporter is a transcription factor or part of a transcription factor which migrates into the nucleus and induces expression of a reporter e.g. luciferase.
  • suitable eukaryotic host cells that are contacted with a test substance wherein said suitable host cells comprise: a) a fusion protein comprising a secretory protein, a membrane anchor domain and at least one secretase cleavage sequence, b) a protein comprising a secretase activity recognising said cleavage sequence of said fusion protein and c) at least one reporter gene under control of a transcriptional activation system wherein said transcriptional activation system is regulated by the release of said secretory protein from said fusion protein and its subsequent secretion then culturing said cells under suitable conditions such that said reporter gene allowing detection and/or survival of cells is only expressed or repressed in a manner that is dependent on an altered secretase activity due to said test substance.
  • the present invention relates to a method for the identification of a secretase inhibitor.
  • Said method is characterised in that a reduced or no release of said secretory protein from said fusion protein due to a reduced/inhibited secretase ac- tivity leads to a reduced or no secretion of said secretory protein and to the induction of expression of said reporter gene.
  • the induction of expression of said re- porter gene allows under suitable culturing conditions the detection and/or survival of cells which are in contact with a test compound having an inhibitory effect on the secretase activity.
  • Said reporter gene is preferably selected from genes conferring antibiotic resistance, genes complementing auxotrophies and genes encoding reporter molecules with an activity that can be detected by colorimet- ric or fluorescent methods such as genes selected from the group consisting of: lacZ, Luciferase gene, green fluorescence protein gene and chloramphenicol acetyl transferase gene.
  • the present invention relates to a method for the identification of stimulators of a secretase activity.
  • Said method is characterised in that the release of said secretory protein from said fusion protein due to an enhanced secretase activity leads to repression of said reporter gene expression thereby allowing detection and/or survival of cells which are in contact with a test substance having an stimulating effect on the secretase activity.
  • Appropriate culturing conditions of the cells must be used such that said fusion protein is not efficiently cleaved by the secretase in the absence of said test substance having a stimulating effect on the secretase activity.
  • Said reporter gene is preferably selected from genes such as CYH2 or CANl conferring sensitivity to a chemical .
  • said suitable host cells comprise a second reporter gene that is selected from the group consisting of: a) genes encoding reporter molecules with an activity that can be detected by colori etric or fluorescent methods such as genes selected from the group consisting of: lacZ, luciferase gene, green fluores- cence gene (GFP) and chloramphenicol acetyl trans- ferase gene (CAT) , b) genes conferring antibiotic resistance, c) genes conferring sensitivity to a chemical and d) genes complementing auxotrophies .
  • a) genes encoding reporter molecules with an activity that can be detected by colori etric or fluorescent methods such as genes selected from the group consisting of: lacZ, luciferase gene, green fluores- cence gene (GFP) and chloramphenicol acetyl trans- ferase gene (CAT)
  • GFP green fluores- cence gene
  • CAT chloramphenicol acetyl trans- ferase gene
  • any eukaryotic cell can be used, preferably a yeast cell.
  • membrane anchor domain refers to molecules and/or protein domains which are responsible for the membrane association of a protein and includes e.g. transmembrane domains and GPI anchors.
  • secretory protein as used herein en- compasses polypeptides or fragments thereof which are destined for export. Said secretory protein has preferably an enzymatic activity, more preferably it is a protein with invertase activity or functional fragments of a protein with invertase activity.
  • An especially preferred invertase is a yeast invertase or functional fragments thereof. Yet it is obvious for the man skilled in the art that other secretory proteins can be used in a method of the present invention.
  • Any recognition sequence of a known secretase can be used for the construction of said fusion protein of the present invention.
  • Preferred secretase recognition sites are selected from the ⁇ site and the ⁇ site of the human amyloid precursor protein (APP) and the S2 site of Notch 1 protein.
  • a much preferred recognition site is the ⁇ site of human APP in which the Lys595Asn and Met596Leu changes were introduced (Swedish APP mutant) .
  • said fusion protein comprises amino acid residues 1- 532 of yeast invertase, amino acid residues 590-695 of human APP and optionally an ER retention signal.
  • said fusion protein comprises amino acid residues 1-532 of yeast invertase, amino acid residues 1714-1876 of human Notch 1 and optionally an ER retention signal.
  • Said fusion protein can be expressed from an extrachromosomal gene construct e.g. from an episomal vector enabling expression of the fusion protein in a host cell.
  • the nucleic acid construct encoding the fusion protein is integrated into the genome of the host cell.
  • the nucleic acid can be introduced into the cell by any transfection method leading to uptake of the nucleic acid sequence into the cell. Such methods are know to the man skilled in the art and are e.g described in Sambrook et al . , Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory, 2001) .
  • secretase activity encompasses proteolytic enzymes or functional fragments thereof which cleave membrane associated proteins, integral membrane proteins of type I and type II topology.
  • said protein comprising a secretase activity further comprises an ER sig- nal sequence.
  • Preferred secretase activities for the present invention are selected from ⁇ -secretase and ⁇ - secretase of human APP.
  • said protein comprising a ⁇ -secretase activity further comprises an ER signal sequence and amino acid residues 616-695 of human APP.
  • the protein comprising said secretase activity can be expressed endogenously by the host cell or it can be encoded by a nucleic acid construct e.g. an episomal expression vector or by a nucleic acid construct that is stably integrated into the genome of the host cell.
  • a nucleic acid construct e.g. an episomal expression vector or by a nucleic acid construct that is stably integrated into the genome of the host cell.
  • Another object of the present invention are compounds identified by a method of the present inven- tion.
  • Fig. 1 shows the primary structure and cellular processing of APP.
  • APP is processed by a sequential cleavage reaction during trafficking through the secretory pathway.
  • the ⁇ -secretase pathway is the preferred, constitutive pathway releasing, in combination with the ⁇ -secretase, a non-toxic p3 fragment.
  • the ⁇ -secretase pathway is competed by the amy- loidogenic ⁇ -secretase pathway, where, upon ⁇ -secretase cleavage, secretion of the A ⁇ -peptide leads to formation of extracellular plaques;
  • Fig. 2A shows the invertase selection system.
  • a portion of APP harboring the ⁇ - and the ⁇ -site is fused to the enzyme invertase.
  • the ⁇ -secretase (BACE) is co- expressed with the invertase-APP fusion protein.
  • Cleavage of the invertase-APP reporter at the ⁇ -site liberates the invertase.
  • Secretion of the invertase leads to cleavage of sucrose into glucose and fructose, thus enabling growth on sucrose plates;
  • Fig. 2B shows a schematic drawing of the in- vertase-APP reporter.
  • the invertase-APP reporter was constructed by fusing the full-length invertase (aa 1-532) to a portion of APP (aa 590-695) harboring the secretase sites, the transmembrane domain and the cytoplasmic tail. Fusion of the ER retention signal DEKKMP (Seq. Id. No. 1) to the C-term further retards growth of clones that do not express an active secretase;
  • Fig. 2C shows a schematic drawing of the engineered BACE.
  • the signal sequence of BACE was replaced by the SUC2 signal sequence (aa 1-19) to ensure efficient translocation into the yeast ER.
  • the C-term of BACE was substituted by a portion of APP har- boring the transmembrane domain and the cytosolic tail (aa 616-695) ;
  • Fig. 2D shows that BACE enhances growth on sucrose plates.
  • the invertase-APP fusion protein harbor- ing the Swedish mutation was co-expressed with the indicated constructs [BACE, empty vector or Yap3p (a yeast secretase that specifically cleaves APP at the ⁇ -site) ] in a yeast strain deficient for the suc2 gene and the endogenous ⁇ -secretases mkc7 and yap3. Growth on sucrose plates was monitored after 2.5 days and 3 days, respectively;
  • Fig. 3 shows principles of a cellular selection system to identify modulators of secretases.
  • Fig. 3A-C show schematic drawings of yeast cells.
  • the re- porter gene is either the LacZ or different selectable marker genes, that can act either positively or negatively on growth. These genes are under the control of the Gall/10 promoter (denoted by the GAL4 transcription factor) ;
  • Fig. 3A shows default readout (without BACE) :
  • the reporter gene is induced.
  • Biscrel cells harboring the invertase-APP fusion protein in the absence of any secretase activity express the reporter gene when grown on 5% sucrose/2% galactose.
  • Galactose activates transcription of the reporter gene, whereas sucrose is inert to the system in the absence of secreted invertase;
  • Fig. 3B shows that in the presence of an active BACE the reporter gene is repressed. Cleavage of the invertase-APP (Sw) by the ⁇ -secretase leads to liberation and subsequent secretion of invertase.
  • invertase hydrolyses sucrose into glucose and fructose. The yielded glucose is taken up by the cell where it dominantly represses transcription of the reporter system;
  • Fig.3C shows that in the presence of an inhibited BACE the reporter gene is induced. If the action of BACE is blocked by a potent inhibitor the readout of the system is the same as in the absence of BACE (described in 3A) ;
  • Fig. 4A shows a schematic representation of the reporter gene.
  • the HIS3 open reading frame (ORF) was fused to the 47 N-terminal amino acids of GAL10.
  • the expression of the GAL10-HIS3 fusion protein is under the control of the GAL10 promoter, the expression of the LacZ ORF is controlled by the GAL1 promoter.
  • the arrows indicate the transcription start points.
  • the boxes labeled I, II, III and IV represent GAL4 binding sites;
  • Fig. 4B shows LacZ assays.
  • Biscrel cells harboring the indicated constructs were cultivated in 5% sucrose 2% galactose -ura -trp drop out medium. Expression of the LacZ reporter gene was quantified using the ⁇ - galactosidase substrate ONPG. The columns represent the average ⁇ -galactosidase units of three samples each. Error bars are indicated.
  • Invertase soluble and secreted invertase
  • Invertase-APP displayed in figure 2B
  • Invertase-APP (Sw) the same as invertase-APP, but harboring the Swedish mutation at the ⁇ -site
  • BACE displayed in figure 2C
  • BACEi the same as BACE, but harboring a point mutation in the active center, which renders the enzyme inactive.
  • the constructs named "empty" only carry the yeast marker genes necessary to grow in the drop out me- dium,
  • Fig. 4C shows liquid growth assays.
  • Biscrel cells harboring the indicated constructs were cultivated in non-selective -ura -trp 2% glucose medium as well as in selective -his -ura -trp 5% sucrose 2% galactose me- dium. After 24 h, the cell density of the cultures was determined by measuring the optical density (OD600) .
  • the cell density in the selective medium is an indicator for the expression of the HIS3 gene.
  • the selective cultures the average OD600 of three cultures is displayed. The standard deviation is indicated by an error bar .
  • Fig. 6 shows a schematic drawing of the in- vertase-Notch 1 reporter.
  • the invertase-Notch 1 reporter was constructed by fusing the full-length invertase (aa 1-532) to a portion of Notch 1 (aa 1714-1876) harboring the S2 site of Notch 1 protein, the transmembrane domain and the cytoplasmic tail. Fusion of the ER retention signal DEKKMP (Seq. Id. No. 1) to the C-term further retards growth of clones that do not express an active secretase and Fig.
  • a reporter gene for the use in the present invention is under control of a transcriptional activation system.
  • a preferred transcriptional activation system is a GAL gene regulatory system, more preferably a yeast GAL gene regulatory system.
  • said reporter gene is under control of the yeast GALl-10 gene regulatory region.
  • the use of said system in a preferred embodiment of the present invention has the following theoretical background:
  • the preferred carbohydrate of yeast cells is glucose since it can be metabolized directly.
  • Other carbohydrates are converted to the glycolytic substrate glu- cose-1-phosphate in several steps.
  • Addition of galactose to yeast cultures grown on glycerol induces expression of the GAL genes at least 1000-fold. If glucose is added to the galactose-containing media, the GAL genes are induced to only 1% of the levels obtained with galactose alone. This phenomenon is known as glucose repression.
  • GA 2 , GAL1-GAL7-GAL10, and MEL1 are the GAL structural genes. Their products transport galactose into the cell and convert it to glucose-1-phosphate.
  • Gal4p the product of the GAL4 gene
  • UAS G specific regulatory DNA elements
  • Gal ⁇ Op is a direct repressor of Gal4p
  • Gal3p mediates the galactose-dependent release of Gal ⁇ Op inhibition of Gal4p (for review see [5] .
  • the glucose repression is exerted through several mechanisms [5] .
  • the GALl-10 regulatory region contains four UAS G elements located between the divergent GALl and GAL10 promoters. These UAS G elements are bound by Gal4p in a cooperative manner. Due to this cooperativity, the system is rather sensitive towards changes in the concentration of Gal4p, which is reduced 3- to 5-fold in the presence of glucose. Gal ⁇ Op significantly contributes to glucose repression of the GALl-10 genes by binding to Gal4p to mask its activation domain, thereby preventing expression of these GAL genes [6].
  • the GALl promoter contains an additional regulatory element, which is the binding site for the re- pressor Miglp.
  • the activity of this repressor is also regulated by glucose.
  • a Miglp site is also present in the GAL4 promoter .
  • the construction of suitable host cells and the other molecular biological reagents for the use in the present invention e.g. fusion protein constructs can be done using standard molecular biology techniques as described e.g. in Sambrook et al . , Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory, 2001) .
  • the man skilled in the art is as well able to determine suitable culturing conditions allowing the de- tection and/or survival of the used cells. Said conditions are dependent on the used genetic constructs and the host cells.
  • Chemical libraries consist of structural analogues of known compounds.
  • Natural product libraries are collections of microorganism, animals, plants or marine organisms which are used to create mixtures for screening by for example fermentation and extraction of broths from soil, plant or marine microorganisms or extraction of plants or marine organisms.
  • Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic com- pounds as a mixture. They are relatively easy to prepare e.g. by traditional synthesis methods, PCR or cloning.
  • test compound in a screening test of the present invention can e.g. be added to the culture medium of the host cells or it can be expressed by the host cells e.g. from an expression construct.
  • the expression of test substances within the host cells is e.g. suitable for the screening of peptide libraries.
  • invertase secretion of invertase enables yeast to use sucrose as a carbon source.
  • the invertase hydrolyses su- crose to yield fructose and glucose.
  • the endogenous gene encoding the invertase (the SUC2 gene) was knocked out.
  • a recombinant invertase was fused to the N-terminus of a portion of APP harboring the transmembrane domain as well as the ⁇ - and ⁇ -sites (residues 590-695) .
  • an ER retention signal was added to the C-terminus of this fusion construct (Fig. 2A, B) .
  • yeast endogenous secretases described in the literature which can cleave APP at the ⁇ - site: Yap3p and Mkc7 [1 2] . Since these proteins have ⁇ - secretase activity that constitutively cleaves APP, their respective genes had to be knocked out in order to investigate ⁇ -secretase activity in yeast using the invertase reporter system described in WO 01/75068.
  • the protein had to be modified. Since trans- membrane and cytosolic sequences contribute to the sub- cellular localization of transmembrane proteins, the transmembrane and cytosolic portion of BACE was substi- tuted by the transmembrane and cytosolic portion of APP. In this way, co-localization of the enzyme with its substrate was facilitated. To ensure efficient translocation into the yeast ER, the signal sequence of BACE was sub- stituted with the SUC2-signal peptide at the N-terminus of the protein (Fig. 2C) .
  • FIG. 2D shows the growth effect on sucrose plates due to cleavage of the invertase-APP (Sw) reporter protein by BACE or Yap3p.
  • a reporter construct was cloned which expresses the LacZ gene under the control of the GALl promoter and the divergently oriented HIS3 gene under the control of the GAL10 promoter. As in the case of the endogenous GALl and GAL10 genes, four UAS G elements are located between these two promoters.
  • This reporter construct was integrated in yeast cells deficient for suc2 , mkc7 and yap3 to create the strain Biscrel. If this strain is grown in the presence of galactose, expression of the LacZ gene and the HIS3 gene is induced by the UAS G -binding Gal4p activator.
  • the newly generated glucose dominantly represses the HIS3 gene expressed from the GAL10 promoter and growth on -his medium is inhibited.
  • Biscrel cultured on sucrose and galactose and expressing the invertase-APP (Sw) fusion protein provides a tool to select for inhibited BACE. Indeed, if BACE expressed in Biscrel is active, the membrane-anchored invertase-APP (Sw) fusion protein is cleaved at the ⁇ site and the invertase moiety is secreted. The secreted invertase hydrolyses sucrose into fructose and glucose, the latter of which represses the HIS3 and the LacZ reporter genes (Fig 3B) .
  • BACE is inhibited, either by mutations or by an inhibiting compound, the invertase moiety re- mains anchored to the ER-membrane via the APP domain, the sucrose in the medium is not hydrolyzed, and expression of the HIS3 and LacZ genes is induced by galactose (Fig 3C) .
  • Expression of the product of the LacZ gene, a ⁇ -galactosidase can be measured by using the substrate analogue o-nitro-phenyl- ⁇ -galactopyranosid (ONPG) .
  • ONPG substrate analogue o-nitro-phenyl- ⁇ -galactopyranosid
  • a cleavage product of ONPG has a yellow colour.
  • OD measure- ment at 420 nm allows quantification of the reaction catalyzed by ⁇ -galactosidase, and indirectly of the LacZ- expression. This assay allowed us to investigate the effect of invertase-APP cleavage by BACE in the context of Biscrel .
  • Biscrel was transformed with plasmids expressing different ⁇ -secretase activities together with the invertase-APP (Sw) construct, or its wildtype variant (invertase-APP) .
  • Empty plasmids and an invertase- expressing plasmid were used as negative and positive controls, respectively.
  • Liquid cultures were grown in 5 % sucrose, 2 % galactose, 0.1 % glucose drop-out medium (The medium contained histidine for this assay as the cultures should grow equally) .
  • the maximal level of LacZ expression in this assay was obtained with transformants harboring two empty plasmids.
  • Free invertase was generated either by direct expression of the natural SUC2 gene or by libera- tion from a membrane-bound fusion protein. The liberation and subsequent secretion of the invertase moiety only took place efficiently when the active BACE secretase was co-expressed with the invertase fusion protein harboring the Swedish mutation, thus underlying the specificity of the system.
  • the expression of the HIS3 gene was quantified by a growth assay in liquid medium.
  • Biscrel cells were cultivated in liquid -his medium containing 5% sucrose and 2% galactose (selective condi- tions) .
  • Two-ml cultures were inoculated with equal amounts of cells transformed with the constructs of interest.
  • Cell density was measured after 24 h.
  • the same amounts of cells were used to inoculate cultures in non- selective medium.
  • This control experiment showed that none of the constructs per se had an effect on cell growth under non-selective conditions (Fig. 4C, columns 1-4) .
  • the cell densities measured after growth in selective medium are displayed in figure 4C, columns 5-8.
  • the screening system is as well suitable to screen for modulators of other secretases .
  • Figure 5 shows the results of such an experiment.
  • Yap3p is a yeast secretase which cleaves APP at the ⁇ -site.
  • the experimental procedures were the same as described for fig. 4B.
  • the mutated Yap3p as well as the wildtype version were provided on a yeast expression vector.
  • Yap3p cleaves the in- vertase-APP fusion protein at the ⁇ -site thereby liberating the invertase.
  • the observed effect on LacZ expression is comparable to the effect observed when soluble and secreted invertase was expressed (last column) .
  • invertase a different target, namely the human Notch 1 protein, was fused to invertase (Fig. 6) .
  • the features of this new construct are identical to those of the invertase-APP fusion protein, except that the APP portion is replaced by the amino acids 1714-1876 of the Notch 1 protein (NCBI Protein Databank accession number AAG33848) , which include the transmembrane domain.
  • the invertase-Notch construct was co-expressed with Yap3p ( ⁇ - secretase) or with an empty vector in the Biscrel strain and a ⁇ -gal assay was performed. The results of this ex- periment are displayed in figure 7.
  • the first column indicates the value obtained with the empty vector (0.4532 ⁇ -gal units) ; the second column indicates the value obtained for Yap3p (0.0724 ⁇ -gal units).
  • the cleavage of the invertase-Notch fusion protein was confirmed by de- tecting the cleavage products in a Western blot (data not shown) .

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Abstract

The present invention relates to a cellular screening method for the identification of modulators of secretases. Said method comprises contacting suitable host cells with a test substance wherein said suitable host cells comprise: a) a fusion protein comprising a secretory protein, a membrane anchor domain and a secretase cleavage sequence, b) a protein comprising a secretase activity recognising said cleavage sequence of said fusion protein anda) at least one reporter gene under control of a transcriptional activation system wherein said transcriptional activation system is regulated by the release of said secretory protein from said fusion protein by said secretase activity and its subsequent secretion then culturing said cells under suitable conditions such that said reporter gene allowing detection and/or survival of cells is only expressed or repressed in a manner that is dependent on an altered secretase activity due to said test substance.

Description

Method for the identification of modulators of a secretase activity
Technical Field The present invention relates to a in vivo method for the identification of modulators of secretase activities .
Background Art Protein secretion is central to the proper development and function of eukaryotic organisms. Moreover, several pathophysiological processes such as neuro- degeneration, oncogenesis, apoptosis and inflammation are associated with the malfunction or aberrant regulation of protein secretion. It has become clear that there is no single biosynthetic mechanism common to all secretory proteins. Secretion of proteins can occur through either the regulated or constitutive pathways and, in some cell types, this secretion can be polarized to distinct cellu- lar domains. An increasing number of proteins are now recognized as being derived from integral membrane proteins of type I and type II topology and, in this case, the secretory event involves their selective post- translational hydrolysis from the cell surface. This se- cretion is catalyzed by proteases known as secretases. The cleavage of membrane proteins generally occurs near the extracellular face of the membrane, although in some cases it has been shown also to occur within the trans- membrane domain. Proteins secreted in this fashion in- elude membrane receptors and receptor ligands, ectoen- zymes, cell adhesion molecules and others. Examples of protein secretion through the action of secretases include the vasoregulatory enzyme ACE (angiotensin converting enzyme) , the tumor necrosis factor (TNF) ligand and receptor superfamilies, the transforming growth factor-α, certain cytokine receptors, the Alzheimer *s amyloid precursor protein (APP) and others (7) . The involvement of secretases in the development of human diseases makes them potential drug target candidates in a variety of disease areas, including anti- cancer drugs, cardiovascular drugs, anti-neurodegenera- tive drugs and anti-inflammatory drugs.
In the past, different in vi tro/ in vivo and biochemical methods have been used to identify modulators of secretease enzymes .
US patent No. 5,942,400 and WO 96/40885 dis- close in vi tro methods for screening for candidate drugs for the ability to inhibit the activity of beta- εecretase. Said methods are based on the detection of APP cleavage products using specific antibodies.
WO 98/13488 describes a method for determin- ing the activity of modulators of APP secretases that are active in cultured human cells. Said method involves transfection of tissue-culture cells with vectors that express cleavable reporter proteins. Upon cleavage by the endogenous secretases, a reporter domain is secreted and can be detected using standard biochemical and immuno- logical methods.
WO 01/49871 discloses an in vivo process for finding substances which specifically inhibit γ-secre- tase. Said process encompasses cells expressing a secre- tase activity and a fusion protein which comprises the substrate of said secretase with the specific cleavage site and a reporter. Said cells are contacted with a test substance and the quantity of reporter cleaved is either measured directly or indirectly. Suitable reporter pro- teins allowing direct detection are GFP, luciferase, β- galactosidase and secreted alkaline phosphatase. In case of an indirect detection of the reporter, the cleaved reporter is a transcription factor or part of a transcription factor which migrates into the nucleus and induces expression of a reporter e.g. luciferase.
Although the prior art already discloses screening methods for secretase modulators, there is a need for improved reliable in vivo screening systems for the identification of modulators of a secretase activity.
Disclosure of the Invention
Hence, it is a general object of the present invention to provide a method for the identification/ isolation of modulators of a secretase activity. Said method is defined by the following steps: suitable eukaryotic host cells that are contacted with a test substance wherein said suitable host cells comprise: a) a fusion protein comprising a secretory protein, a membrane anchor domain and at least one secretase cleavage sequence, b) a protein comprising a secretase activity recognising said cleavage sequence of said fusion protein and c) at least one reporter gene under control of a transcriptional activation system wherein said transcriptional activation system is regulated by the release of said secretory protein from said fusion protein and its subsequent secretion then culturing said cells under suitable conditions such that said reporter gene allowing detection and/or survival of cells is only expressed or repressed in a manner that is dependent on an altered secretase activity due to said test substance. In a preferred embodiment, the present invention relates to a method for the identification of a secretase inhibitor. Said method is characterised in that a reduced or no release of said secretory protein from said fusion protein due to a reduced/inhibited secretase ac- tivity leads to a reduced or no secretion of said secretory protein and to the induction of expression of said reporter gene. The induction of expression of said re- porter gene allows under suitable culturing conditions the detection and/or survival of cells which are in contact with a test compound having an inhibitory effect on the secretase activity. Said reporter gene is preferably selected from genes conferring antibiotic resistance, genes complementing auxotrophies and genes encoding reporter molecules with an activity that can be detected by colorimet- ric or fluorescent methods such as genes selected from the group consisting of: lacZ, Luciferase gene, green fluorescence protein gene and chloramphenicol acetyl transferase gene.
In another preferred embodiment, the present invention relates to a method for the identification of stimulators of a secretase activity. Said method is characterised in that the release of said secretory protein from said fusion protein due to an enhanced secretase activity leads to repression of said reporter gene expression thereby allowing detection and/or survival of cells which are in contact with a test substance having an stimulating effect on the secretase activity. Appropriate culturing conditions of the cells must be used such that said fusion protein is not efficiently cleaved by the secretase in the absence of said test substance having a stimulating effect on the secretase activity.
Said reporter gene is preferably selected from genes such as CYH2 or CANl conferring sensitivity to a chemical .
In a further preferred embodiment of the in- vention said suitable host cells comprise a second reporter gene that is selected from the group consisting of: a) genes encoding reporter molecules with an activity that can be detected by colori etric or fluorescent methods such as genes selected from the group consisting of: lacZ, luciferase gene, green fluores- cence gene (GFP) and chloramphenicol acetyl trans- ferase gene (CAT) , b) genes conferring antibiotic resistance, c) genes conferring sensitivity to a chemical and d) genes complementing auxotrophies .
Illustrative examples of suitable reporter genes that can be used in the present invention are mentioned above .
In a method of the present invention any eukaryotic cell can be used, preferably a yeast cell. The term membrane anchor domain as used herein refers to molecules and/or protein domains which are responsible for the membrane association of a protein and includes e.g. transmembrane domains and GPI anchors. The term secretory protein as used herein en- compasses polypeptides or fragments thereof which are destined for export. Said secretory protein has preferably an enzymatic activity, more preferably it is a protein with invertase activity or functional fragments of a protein with invertase activity. An especially preferred invertase is a yeast invertase or functional fragments thereof. Yet it is obvious for the man skilled in the art that other secretory proteins can be used in a method of the present invention.
Any recognition sequence of a known secretase can be used for the construction of said fusion protein of the present invention. Preferred secretase recognition sites are selected from the β site and the α site of the human amyloid precursor protein (APP) and the S2 site of Notch 1 protein. A much preferred recognition site is the β site of human APP in which the Lys595Asn and Met596Leu changes were introduced (Swedish APP mutant) .
In a much preferred embodiment of the invention said fusion protein comprises amino acid residues 1- 532 of yeast invertase, amino acid residues 590-695 of human APP and optionally an ER retention signal.
In a further preferred embodiments said fusion protein comprises amino acid residues 1-532 of yeast invertase, amino acid residues 1714-1876 of human Notch 1 and optionally an ER retention signal.
Said fusion protein can be expressed from an extrachromosomal gene construct e.g. from an episomal vector enabling expression of the fusion protein in a host cell. Preferably the nucleic acid construct encoding the fusion protein is integrated into the genome of the host cell. The nucleic acid can be introduced into the cell by any transfection method leading to uptake of the nucleic acid sequence into the cell. Such methods are know to the man skilled in the art and are e.g described in Sambrook et al . , Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory, 2001) .
The term secretase activity as used herein encompasses proteolytic enzymes or functional fragments thereof which cleave membrane associated proteins, integral membrane proteins of type I and type II topology.
In a preferred embodiment said protein comprising a secretase activity further comprises an ER sig- nal sequence.
Preferred secretase activities for the present invention are selected from β-secretase and α- secretase of human APP.
In a preferred embodiment of the invention said protein comprising a β-secretase activity further comprises an ER signal sequence and amino acid residues 616-695 of human APP.
The protein comprising said secretase activity can be expressed endogenously by the host cell or it can be encoded by a nucleic acid construct e.g. an episomal expression vector or by a nucleic acid construct that is stably integrated into the genome of the host cell.
Another object of the present invention are compounds identified by a method of the present inven- tion.
Brief Description of the Drawings The invention will be better understood and objects other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such description makes refer- ence to the annexed drawings, wherein:
Fig. 1 shows the primary structure and cellular processing of APP. APP is processed by a sequential cleavage reaction during trafficking through the secretory pathway. The α-secretase pathway is the preferred, constitutive pathway releasing, in combination with the γ-secretase, a non-toxic p3 fragment. In Alzheimer patients, the α-secretase pathway is competed by the amy- loidogenic β-secretase pathway, where, upon γ-secretase cleavage, secretion of the Aβ-peptide leads to formation of extracellular plaques;
Fig. 2A shows the invertase selection system. A portion of APP harboring the α- and the β-site is fused to the enzyme invertase. The β-secretase (BACE) is co- expressed with the invertase-APP fusion protein. Cleavage of the invertase-APP reporter at the β-site liberates the invertase. Secretion of the invertase leads to cleavage of sucrose into glucose and fructose, thus enabling growth on sucrose plates;
Fig. 2B shows a schematic drawing of the in- vertase-APP reporter. The invertase-APP reporter was constructed by fusing the full-length invertase (aa 1-532) to a portion of APP (aa 590-695) harboring the secretase sites, the transmembrane domain and the cytoplasmic tail. Fusion of the ER retention signal DEKKMP (Seq. Id. No. 1) to the C-term further retards growth of clones that do not express an active secretase;
Fig. 2C shows a schematic drawing of the engineered BACE. The signal sequence of BACE was replaced by the SUC2 signal sequence (aa 1-19) to ensure efficient translocation into the yeast ER. To achieve colocaliza- tion of BACE with the invertase-APP fusion protein, the C-term of BACE was substituted by a portion of APP har- boring the transmembrane domain and the cytosolic tail (aa 616-695) ;
Fig. 2D shows that BACE enhances growth on sucrose plates. The invertase-APP fusion protein harbor- ing the Swedish mutation was co-expressed with the indicated constructs [BACE, empty vector or Yap3p (a yeast secretase that specifically cleaves APP at the α-site) ] in a yeast strain deficient for the suc2 gene and the endogenous α-secretases mkc7 and yap3. Growth on sucrose plates was monitored after 2.5 days and 3 days, respectively;
Fig. 3 shows principles of a cellular selection system to identify modulators of secretases. Fig. 3A-C show schematic drawings of yeast cells. The re- porter gene is either the LacZ or different selectable marker genes, that can act either positively or negatively on growth. These genes are under the control of the Gall/10 promoter (denoted by the GAL4 transcription factor) ; Fig. 3A shows default readout (without BACE) :
The reporter gene is induced. Biscrel cells harboring the invertase-APP fusion protein in the absence of any secretase activity express the reporter gene when grown on 5% sucrose/2% galactose. Galactose activates transcription of the reporter gene, whereas sucrose is inert to the system in the absence of secreted invertase;
Fig. 3B shows that in the presence of an active BACE the reporter gene is repressed. Cleavage of the invertase-APP (Sw) by the β-secretase leads to liberation and subsequent secretion of invertase. In the periplasm, invertase hydrolyses sucrose into glucose and fructose. The yielded glucose is taken up by the cell where it dominantly represses transcription of the reporter system; Fig.3C shows that in the presence of an inhibited BACE the reporter gene is induced. If the action of BACE is blocked by a potent inhibitor the readout of the system is the same as in the absence of BACE (described in 3A) ;
Fig. 4A shows a schematic representation of the reporter gene. The HIS3 open reading frame (ORF) was fused to the 47 N-terminal amino acids of GAL10. The expression of the GAL10-HIS3 fusion protein is under the control of the GAL10 promoter, the expression of the LacZ ORF is controlled by the GAL1 promoter. The arrows indicate the transcription start points. The boxes labeled I, II, III and IV represent GAL4 binding sites;
Fig. 4B shows LacZ assays. Biscrel cells harboring the indicated constructs were cultivated in 5% sucrose 2% galactose -ura -trp drop out medium. Expression of the LacZ reporter gene was quantified using the β- galactosidase substrate ONPG. The columns represent the average β-galactosidase units of three samples each. Error bars are indicated. Invertase: soluble and secreted invertase; Invertase-APP: displayed in figure 2B; Invertase-APP (Sw) : the same as invertase-APP, but harboring the Swedish mutation at the β-site; BACE: displayed in figure 2C; BACEi : the same as BACE, but harboring a point mutation in the active center, which renders the enzyme inactive. The constructs named "empty" only carry the yeast marker genes necessary to grow in the drop out me- dium,
Fig. 4C shows liquid growth assays. Biscrel cells harboring the indicated constructs were cultivated in non-selective -ura -trp 2% glucose medium as well as in selective -his -ura -trp 5% sucrose 2% galactose me- dium. After 24 h, the cell density of the cultures was determined by measuring the optical density (OD600) . The cell density in the selective medium is an indicator for the expression of the HIS3 gene. For the explanation of the different constructs, see legend of figure 4C. For the selective cultures, the average OD600 of three cultures is displayed. The standard deviation is indicated by an error bar . Fig. 5 shows that cleavage of invertase-APP by the yeast secretase Yap3p can be monitored by the cellular system to identify modulators of secretases. Induction of reporter gene expression (LacZ) was measured in a LacZ assay.
Fig. 6 shows a schematic drawing of the in- vertase-Notch 1 reporter. The invertase-Notch 1 reporter was constructed by fusing the full-length invertase (aa 1-532) to a portion of Notch 1 (aa 1714-1876) harboring the S2 site of Notch 1 protein, the transmembrane domain and the cytoplasmic tail. Fusion of the ER retention signal DEKKMP (Seq. Id. No. 1) to the C-term further retards growth of clones that do not express an active secretase and Fig. 7 shows that cleavage of invertase-Notch by the yeast α-secretase Yap3p can be monitored by the cellular system to identify modulators of secretases. Induction of reporter gene expression (LacZ) was measured in a LacZ assay.
Modes for Carrying Out the Invention
A reporter gene for the use in the present invention is under control of a transcriptional activation system. A preferred transcriptional activation system is a GAL gene regulatory system, more preferably a yeast GAL gene regulatory system.
In a preferred embodiment of the invention said reporter gene is under control of the yeast GALl-10 gene regulatory region.
The use of said system in a preferred embodiment of the present invention has the following theoretical background: The preferred carbohydrate of yeast cells is glucose since it can be metabolized directly. Other carbohydrates are converted to the glycolytic substrate glu- cose-1-phosphate in several steps. Addition of galactose to yeast cultures grown on glycerol induces expression of the GAL genes at least 1000-fold. If glucose is added to the galactose-containing media, the GAL genes are induced to only 1% of the levels obtained with galactose alone. This phenomenon is known as glucose repression. GA 2 , GAL1-GAL7-GAL10, and MEL1 are the GAL structural genes. Their products transport galactose into the cell and convert it to glucose-1-phosphate. The major regulatory pro- teins of this system are the products of the genes GAL3 , GAL4, and GAL80. Gal4p, the product of the GAL4 gene, is a transcriptional activator that binds specific regulatory DNA elements called UASG, which are present near the promoter regions of the galactose inducible genes. Its activity is required to stimulate expression of the GAL genes in the presence of galactose. GalδOp is a direct repressor of Gal4p, while Gal3p mediates the galactose- dependent release of GalδOp inhibition of Gal4p (for review see [5] . The glucose repression is exerted through several mechanisms [5] . Here, we focus on the regulation of the divergently oriented GALl-10 genes, whose regulatory region was used for the experiments described below. The GALl-10 regulatory region contains four UASG elements located between the divergent GALl and GAL10 promoters. These UASG elements are bound by Gal4p in a cooperative manner. Due to this cooperativity, the system is rather sensitive towards changes in the concentration of Gal4p, which is reduced 3- to 5-fold in the presence of glucose. GalδOp significantly contributes to glucose repression of the GALl-10 genes by binding to Gal4p to mask its activation domain, thereby preventing expression of these GAL genes [6]. The GALl promoter contains an additional regulatory element, which is the binding site for the re- pressor Miglp. The activity of this repressor is also regulated by glucose. (A Miglp site is also present in the GAL4 promoter . ) The construction of suitable host cells and the other molecular biological reagents for the use in the present invention e.g. fusion protein constructs can be done using standard molecular biology techniques as described e.g. in Sambrook et al . , Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory, 2001) .
The man skilled in the art is as well able to determine suitable culturing conditions allowing the de- tection and/or survival of the used cells. Said conditions are dependent on the used genetic constructs and the host cells.
There are at least three different categories of compounds that can be screened by a screening method of the present invention: chemical libraries, natural product libraries and combinatorial libraries. Chemical libraries consist of structural analogues of known compounds. Natural product libraries are collections of microorganism, animals, plants or marine organisms which are used to create mixtures for screening by for example fermentation and extraction of broths from soil, plant or marine microorganisms or extraction of plants or marine organisms. Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic com- pounds as a mixture. They are relatively easy to prepare e.g. by traditional synthesis methods, PCR or cloning.
In a screening test of the present invention a test compound can e.g. be added to the culture medium of the host cells or it can be expressed by the host cells e.g. from an expression construct. The expression of test substances within the host cells is e.g. suitable for the screening of peptide libraries.
The present invention is now further illustrated by means of examples. Reconstitution of secretase activities in yeast
To monitor BACE activity within yeast cells, we made use of the invertase reporter system described in our patent application WO 01/75088, Title: "Method for identification of polypeptides with protease activity" .
Secretion of invertase enables yeast to use sucrose as a carbon source. The invertase hydrolyses su- crose to yield fructose and glucose. For the reporter system described in our patent application WO 01/75086, the endogenous gene encoding the invertase (the SUC2 gene) was knocked out. A recombinant invertase was fused to the N-terminus of a portion of APP harboring the transmembrane domain as well as the α- and β-sites (residues 590-695) . In addition, an ER retention signal was added to the C-terminus of this fusion construct (Fig. 2A, B) . These modifications prevent invertase from being secreted and, consequently, the yeast cells are unable to efficiently grow on plates containing sucrose as the only carbohydrate. The system described here is based on the observation that, upon cleavage of the APP portion at the α- or β-site, the invertase is liberated and secreted in the extracellular environment, where it hydrolyzes su- crose into fructose and glucose.
There are two yeast endogenous secretases described in the literature which can cleave APP at the α- site: Yap3p and Mkc7 [1 2] . Since these proteins have α- secretase activity that constitutively cleaves APP, their respective genes had to be knocked out in order to investigate β-secretase activity in yeast using the invertase reporter system described in WO 01/75068.
To detect APP-specific BACE activity within yeast cells, the protein had to be modified. Since trans- membrane and cytosolic sequences contribute to the sub- cellular localization of transmembrane proteins, the transmembrane and cytosolic portion of BACE was substi- tuted by the transmembrane and cytosolic portion of APP. In this way, co-localization of the enzyme with its substrate was facilitated. To ensure efficient translocation into the yeast ER, the signal sequence of BACE was sub- stituted with the SUC2-signal peptide at the N-terminus of the protein (Fig. 2C) . Since BACE cleaves the APP wild type less efficiently than the so-called Swedish APP mutant [3], the β-site of the invertase-APP (590-695) reporter construct was altered such as to introduce the Lys595Asn and Met596Leu changes indicated as invertase- APP (Sw) . Figure 2D shows the growth effect on sucrose plates due to cleavage of the invertase-APP (Sw) reporter protein by BACE or Yap3p.
Cellular system to screen for secretase inhibitors
In order to establish the described cellular system to screen for BACE inhibitors, we combined the BACE-dependent invertase secretion system with the endogenous GAL gene regulatory network.
A reporter construct was cloned which expresses the LacZ gene under the control of the GALl promoter and the divergently oriented HIS3 gene under the control of the GAL10 promoter. As in the case of the endogenous GALl and GAL10 genes, four UASG elements are located between these two promoters. This reporter construct was integrated in yeast cells deficient for suc2 , mkc7 and yap3 to create the strain Biscrel. If this strain is grown in the presence of galactose, expression of the LacZ gene and the HIS3 gene is induced by the UASG-binding Gal4p activator. As in the case of the endogenous GALl and GAL10 genes, these reporter genes become dominantly repressed upon addition of glucose to the medium. The product of the HIS3 gene is needed for growth on histidine-depleted (-his) medium. Consequently, Biscrel can only grow on -his media that contain galactose but no glucose. The HIS3 reporter gene of the strain Biscrel, which lacks the endogenous invertase activity, is also induced by Gal4p when these cells grow in the presence of sucrose together with galactose (Fig 3A) . How- ever, if Biscrel is allowed to secrete the invertase, glucose is generated by the hydrolysis of sucrose. As consequences, the newly generated glucose dominantly represses the HIS3 gene expressed from the GAL10 promoter and growth on -his medium is inhibited. Biscrel cultured on sucrose and galactose and expressing the invertase-APP (Sw) fusion protein provides a tool to select for inhibited BACE. Indeed, if BACE expressed in Biscrel is active, the membrane-anchored invertase-APP (Sw) fusion protein is cleaved at the β site and the invertase moiety is secreted. The secreted invertase hydrolyses sucrose into fructose and glucose, the latter of which represses the HIS3 and the LacZ reporter genes (Fig 3B) . If BACE is inhibited, either by mutations or by an inhibiting compound, the invertase moiety re- mains anchored to the ER-membrane via the APP domain, the sucrose in the medium is not hydrolyzed, and expression of the HIS3 and LacZ genes is induced by galactose (Fig 3C) .
Results
Expression of the product of the LacZ gene, a β-galactosidase, can be measured by using the substrate analogue o-nitro-phenyl-β-galactopyranosid (ONPG) . A cleavage product of ONPG has a yellow colour. OD measure- ment at 420 nm allows quantification of the reaction catalyzed by β-galactosidase, and indirectly of the LacZ- expression. This assay allowed us to investigate the effect of invertase-APP cleavage by BACE in the context of Biscrel . Biscrel was transformed with plasmids expressing different β-secretase activities together with the invertase-APP (Sw) construct, or its wildtype variant (invertase-APP) . Empty plasmids and an invertase- expressing plasmid were used as negative and positive controls, respectively. Liquid cultures were grown in 5 % sucrose, 2 % galactose, 0.1 % glucose drop-out medium (The medium contained histidine for this assay as the cultures should grow equally) . The maximal level of LacZ expression in this assay was obtained with transformants harboring two empty plasmids. In these cells there was no invertase activity, and consequently no glucose was pro- duced in the presence of sucrose. In this way, values of >0.6 β-galactosidase units were measured (Fig. 4B, column 1) . Expression of soluble and secreted invertase resulted in a strong repression of the reporter gene (Fig. 4B, column 2) . Expression of the invertase-APP (Sw) fusion protein alone resulted in a β-galactosidase activity of 0.25 units (Fig. 4B, column 3). Expression of the invertase-APP wild-type protein resulted in a comparable level of LacZ gene activation (Fig. 4B, column 4) . The 2-3-fold reduction in LacZ expression observed in the presence of these invertase fusion proteins is most likely due to the residual appearance of the invertase moiety at the surface of the cell. This could be due to a small portion of the fusion protein that reaches the cell surface, or to some residual endogenous secretase activity present in the cell despite the fact that the two major endogenous cc-secretases Yap3p and Mkc7p were knocked out.
Co-expression of BACE with the invertase-APP (Sw) fusion- protein led to a significant reduction of β- galactosidase activity (0.026 β-galactosidase units, Fig. 4B, column 5), whereas co-expression with the construct harboring the APP wild-type sequence (invertase-APP) resulted in an only slight reduction (Fig. 4B, column 6). A catalytically inactive version of BACE (BACEi) behaved similarly as an empty plasmid when expressed together with either one of these fusion constructs (Fig. 4B, columns 7 , 8) . These results show that the expression level of the reporter gene system is dependent on the presence of free invertase. Free invertase was generated either by direct expression of the natural SUC2 gene or by libera- tion from a membrane-bound fusion protein. The liberation and subsequent secretion of the invertase moiety only took place efficiently when the active BACE secretase was co-expressed with the invertase fusion protein harboring the Swedish mutation, thus underlying the specificity of the system.
The expression of the HIS3 gene was quantified by a growth assay in liquid medium. For this assay, Biscrel cells were cultivated in liquid -his medium containing 5% sucrose and 2% galactose (selective condi- tions) . Two-ml cultures were inoculated with equal amounts of cells transformed with the constructs of interest. Cell density was measured after 24 h. The same amounts of cells were used to inoculate cultures in non- selective medium. This control experiment showed that none of the constructs per se had an effect on cell growth under non-selective conditions (Fig. 4C, columns 1-4) . The cell densities measured after growth in selective medium are displayed in figure 4C, columns 5-8. Biscrel harboring two empty plasmids fully induced the HIS3 gene and could grow under selective conditions (Fig. 4C, column 5) . The cell density of this culture was comparable to those of all the cultures grown in non-selective medium. The cells transformed with the invertase-APP (Sw) construct together with an empty plasmid or with the in- activated BACE grew to a density of about 75% of the one reached with two empty plasmids (Fig. 4C, column 6, 7) . Expression of active BACE together with the invertase-APP (Sw) fusion protein reduced cell growth to about 10% of the values obtained with inactivated BACE (Fig. 4C, col- umn 8) . Therefore, this assay provides a tool to screen compound libraries to identify BACE inhibitors that are active in a eukaryotic cellular environment and that do not negatively affect basic cellular functions.
The screening system is as well suitable to screen for modulators of other secretases . Figure 5 shows the results of such an experiment. Yap3p is a yeast secretase which cleaves APP at the α-site. The experimental procedures were the same as described for fig. 4B. The mutated Yap3p as well as the wildtype version were provided on a yeast expression vector. Yap3p cleaves the in- vertase-APP fusion protein at the α-site thereby liberating the invertase. The observed effect on LacZ expression is comparable to the effect observed when soluble and secreted invertase was expressed (last column) .
To further investigate the general applica- bility of our system, a different target, namely the human Notch 1 protein, was fused to invertase (Fig. 6) . The features of this new construct are identical to those of the invertase-APP fusion protein, except that the APP portion is replaced by the amino acids 1714-1876 of the Notch 1 protein (NCBI Protein Databank accession number AAG33848) , which include the transmembrane domain. The invertase-Notch construct was co-expressed with Yap3p (α- secretase) or with an empty vector in the Biscrel strain and a β-gal assay was performed. The results of this ex- periment are displayed in figure 7. The first column indicates the value obtained with the empty vector (0.4532 β-gal units) ; the second column indicates the value obtained for Yap3p (0.0724 β-gal units). The cleavage of the invertase-Notch fusion protein was confirmed by de- tecting the cleavage products in a Western blot (data not shown) .
While there are shown and described presently preferred embodiments of the invention, it is to be distinctly understood that the invention is not limited thereto but may be otherwise variously embodied and practiced within the scope of the following claims. References
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2. Komano, H. , et al . , Involvement of cell surface glycosyl-phosphatidylinosi tol -linked aspartyl proteases in alpha- secretase- type cleavage and ectodomain solubilization of human Alzheimer beta-amyloid precursor protein in yeast . J Biol Chem, 1998. 273(48): p. 31648- 51.
3. Citron, M. , et al . , Mutation of the beta- amyloid precursor protein in familial Alzheimer ' s disease increases beta-protein production . Nature, 1992. 360(6405) : p. 672-4.
4. Lohr, D. , P. Venkov, and J. Zlatanova, Transcriptional regulation in the yeast GAL gene family: a complex genetic network. The FASEB Journal, 1995. 9: p. 777-786.
5. Lamphier, M.S. and M. Ptashne, Mul tiple mechanisms mediate glucose repression of the yeast GALl gene . Proc . Natl . Acad. Sci . USA, 1992. 89: p. 5922-5926. 6. Melcher, K. and H.E. Xu, Gal80-Gal80 interaction on adjacent Gal4p binding si tes is required for complete GAL gene repression . Embo J, 2001. 20(4): p. 841-δ51.
7. Hooper, N.M. , Karran, E.H., Turner, A.J. Membrane protein secretases Biochem. J, 1997. 321: 265- 279.

Claims

Claims
1. A method for the identification/isolation of modulators of a secretase activity wherein suitable eukaryotic host cells are contacted with a test substance wherein said suitable host cells comprise: a) a fusion protein comprising a secretory protein, a membrane anchor domain and a secretase cleavage se- quence, b) a protein comprising a secretase activity recognising said cleavage sequence of said fusion protein and c) at least one reporter gene under control of a tran- scriptional activation system wherein said transcriptional activation system is regulated by the release of said secretory protein from said fusion protein by said secretase activity and its subsequent secretion then culturing said cells under suitable conditions such that said reporter gene allowing detection and/or survival of cells is only expressed or repressed in a manner that is dependent on an altered secretase activity due to said test substance. 2. Method for the isolation of a secretase inhibitor according to claim 1, wherein a reduced or no release of said secretory protein due to a reduced/ inhibited secretase activity leads to expression of said at least one reporter gene thereby allowing detection and/or survival of cells under suitable culturing conditions.
3. Method according to claim 1 or 2 , wherein said at least one reporter gene is selected from genes conferring antibiotic resistance, genes encoding reporter molecules with an activity that can be detected by col- orimetric or fluorescent methods and genes complementing auxotrophies, preferably a His3 gene.
4. Method for the identification of agonists of a secretase activity according to claim 1, wherein the release of said secretory protein due to an enhanced secretase activity leads to a reduced expression of said at least one reporter gene thereby allowing detection and/or survival of cells under suitable conditions.
5. Method according to claim 4, wherein said at least one reporter gene is selected from genes conferring sensitivity to a chemical. 6. Method according to anyone of the preceding claims, wherein said cells comprise a second reporter gene selected from the group consisting of: a) genes encoding reporter molecules with an activity that can be detected by colorimetric or fluorescent methods, b) genes conferring antibiotic resistance and genes conferring sensitivity to a chemical and c) genes complementing auxotrophies .
7. Method according to anyone of the preced- ing claims, wherein said cell is a yeast cell. δ . Method according to anyone of the preceding claims, wherein said secretory protein has an enzymatic activity, preferably a protein with invertase activity or functional fragments of a protein with inver- tase activity.
9. Method according to anyone of the preceding claims, wherein said secretory protein is a yeast invertase or functional fragments of a yeast invertase.
10. Method according to anyone of the preced- ing claims, wherein said secretase cleavage sequence is selected from the β site and the α site of the human amyloid precursor protein and the S2 site of Notch 1 protein.
11. Method according to claim 10, wherein into said β site the Lys595Asn and Met596Leu changes were introduced.
12. Method according to anyone of the preceding claims, wherein said fusion protein comprises an ER retention signal.
13. Method according to anyone of the preced- ing claims, wherein said fusion protein comprises amino acid residues 1-532 of yeast invertase, amino acid residues 590-695 of human APP and an ER retention signal.
14. Method according to anyone of the preceding claims, wherein said fusion protein comprises amino acid residues 1-532 of yeast invertase, amino acid residues 1714-lδ76 of human Notch 1 and an ER retention signal .
15. Method according to anyone of the preceding claims, wherein a nucleic acid construct encoding said fusion protein is integrated into the genome of said host cell.
16. Method according to anyone of the preceding claims, wherein said protein comprising a secretase activity recognising said cleavage sequence of said fu- sion protein further comprises an ER signal sequence.
17. Method according to anyone of the preceding claims, wherein said secretase activity is selected from β-secretase and α-secretase. lδ . Method according to claim 16, wherein said protein comprising a β-secretase activity further comprises an ER signal sequence and amino acid residues 616-695 of human APP.
19. Method according to anyone of the preceding claims, wherein said transcriptional activation sys- tem comprises at least part of the yeast GAL gene regulatory system.
20. Method according to anyone of the preceding claims, wherein said reporter gene is under control of the yeast GALl-10 regulatory region.
PCT/IB2002/001342 2002-04-18 2002-04-18 Method for the identification of modulators of a secretase activity WO2003087842A1 (en)

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