EP1144603A3 - Protease - Google Patents

Protease

Info

Publication number
EP1144603A3
EP1144603A3 EP00903587A EP00903587A EP1144603A3 EP 1144603 A3 EP1144603 A3 EP 1144603A3 EP 00903587 A EP00903587 A EP 00903587A EP 00903587 A EP00903587 A EP 00903587A EP 1144603 A3 EP1144603 A3 EP 1144603A3
Authority
EP
European Patent Office
Prior art keywords
protease
medicament
aspartate
diagnostic agent
proteases
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00903587A
Other languages
German (de)
English (en)
Other versions
EP1144603A2 (fr
Inventor
Kay Hofmann
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
MEMOREC STOFFEL GMBH-MEDIZINISCH-MOLEKULARE ENTWIC
Original Assignee
Memorec Medical Molecular Research Cologne Stoffel GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE19902550A external-priority patent/DE19902550A1/de
Application filed by Memorec Medical Molecular Research Cologne Stoffel GmbH filed Critical Memorec Medical Molecular Research Cologne Stoffel GmbH
Publication of EP1144603A2 publication Critical patent/EP1144603A2/fr
Publication of EP1144603A3 publication Critical patent/EP1144603A3/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6478—Aspartic endopeptidases (3.4.23)
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a protease encoding nucleic acids for the protease and the inhibitors, antibodies, medicaments and diagnostic agents associated therewith.
  • the present invention provides a protease with two aspartate residues in a catalytically active structure, wherein a first aspartate residue is in a motif X1GX2GD and a second aspartate residue is in a motif X 3 X 4 DX 5 , wherein Xi, X 2 , X3 and X 5 are independent are selected from each other from Ala, Val, Leu, Met and Ile and X 4 is an aromatic amino acid, and the motifs X ⁇ GX 2 GD and X 3 X 4 DX 5 are located in a transmembrane region.
  • proteases are very likely to be involved in the cleavage of the amyloid precursor protein (APP).
  • the protease according to the invention represents the hitherto unidentified ⁇ -secretase which is involved in processing the APP to form the amyloid peptides referred to as Aß.
  • Preferred proteases of the present invention additionally have a sequence PALX6YX7V, where X 6 and X 7 independently of one another have the same meaning as Xi. However, it is preferred that X 6 and X 7 are leucine or isoleucine.
  • the proteases are proteases from mammals, in particular from humans.
  • the proteases according to the invention preferably have catalytically active aspartate residues in a region which lies within a transmembrane region.
  • Transmembrane regions can be predicted on the basis of different models if the sequence of a protein is known. They are characterized in that in one area there are predominantly hydrophobic amino acids which are flanked by areas in which there are more hydrophilic amino acids.
  • An aspartate in a transmembrane region can be detected, for example, by using the "GREASE" program, which is part of the FASTA 2.0 program package. With a window width of 17, this program must be used to calculate a hydrophobicity value of at least 80 for the aspartate.
  • the FASTA program package is described in WR Pearson and DJ Lipman, Proc. Natl. Acad. Sci. USA 85 (1988) 2444-2448.
  • the GREASE program uses the Kyte / Doolittle algorithm, described in J. Kyte and RF Doolittle, J. Mol. Biol 157: 105-132 (1982).
  • proteases of the present invention are referred to as psl 1-5 (human psl 1-5: SEQ ID No. 1 to 4 + 19, murine psl 2-4: SEQ ID No. 5-7, sacc. Cerevisiae psl 3: SEQ ID No. 8, human psl2L SEQ ID No. 18).
  • the invention furthermore relates to variants of the proteases according to the invention.
  • Variants are proteins which are derived from the proteases according to the invention by one or more mutations, insertions and deletions, in particular by conservative exchanges. special N- or C-terminal shortened or elongated forms.
  • the invention also relates to nudeic acids which code for the proteases according to the invention.
  • Preferred nucleic acids according to the invention are those with SEQ ID No. 9 - 17 + 20 (human psl 1 - 5: SEQ ID No. 9 - 12 + 20, murine psl 2 - 4: SEQ ID No. 13 - 15, sacc. Cerevisiae psl 3: SEQ ID No. 16, human psl2L SEQ ID No. 17).
  • Complementary nudeic acids are also part of the invention.
  • the proteases according to the invention are involved in the cleavage of the APP to Aß and are thus indirectly involved in the development of, for example, Alzheimer's disease.
  • the invention therefore also relates to inhibitors which inhibit the expression or the activity of the proteases.
  • Such inhibitors can be identified in simple procedures.
  • Corresponding inhibitors can be identified, for example, by measuring the expression or the activity of the proteases in the presence of potential inhibitors.
  • Antibodies directed against the aspartate protesases are particularly suitable for measuring expression and are therefore also part of the invention.
  • the aspartate proteases according to the invention are also involved in the cleavage of other transmembrane proteins, in particular the Notch receptor protein and related proteins which play a role in the development of the nervous system.
  • proteolytic cleavage inside membranes is also involved in other important processes, e.g. :
  • Proteolytic cleavage of the ER stress sensor protein Irel The endoplasmic reticulum has a mechanism that detects the accumulation of unfolded or incorrectly folded proteins and sends a signal to the cell nucleus. This mechanism is called "unfolded protein response" or UPR, and leads to the increased formation of proteins that facilitate folding. In mammals there are two sensor proteins (Irelalpha and Irelbeta) that react to folding defects and are then proteolytically cleaved within the membrane.
  • the processes mentioned can be influenced therapeutically by using the protease or its inhibitors according to the invention.
  • Zeil lines which do not express any of the proteases or nucleic acids according to the invention and preferably also contain no homologous proteases or nucleic acids are particularly suitable for this. These can be used to test the activity of the proteases or to determine inhibitors according to Example 2, preferably in accordance with the method described in Example 1. Saccharomyces cerevisiae is particularly suitable for this.
  • SEQ ID No. 8 and 16 can be used to produce yeast strains by known methods which no longer contain this protein or the nucleic acid. They are therefore preferably suitable as an expression system for characterizing the aspartate proteases according to the invention or for identifying suitable inhibitors.
  • yeast cell lines preferably yeast cell lines and the use of the protein with SEQ ID No. 8 as aspartate protease and the nucleic acid with SEQ ID No. 16 for the expression of a protease are therefore also the subject of the invention.
  • proteases, nudeic acids, inhibitors and antibodies according to the invention can be contained in medicinal and diagnostic agents. They are particularly suitable for the treatment or diagnosis of diseases which are causally linked to the cleavage of the amyloid precursor protein, in particular Alzheimer's disease.
  • the putative ⁇ -secretases are transfected stably or transiently in cos-7 cells, which additionally express SpA4CT (signal peptide fused to ßA4 followed by the APP C-terminus).
  • SpA4CT signal peptide fused to ßA4 followed by the APP C-terminus.
  • ⁇ -Secretase activity is recognizable in this system by the generation of a 4.6 KDa peptide or by the disappearance of the 11 KDa band of the complete SpA4CT. If the pathologically relevant ⁇ -secretase is present, both fragments should be detectable inside the cell.
  • ßA4 which is generated by an endogenous plasma membrane-constant ⁇ -secretase activity, which, however, plays no role in the pathogenesis of Alzheimer's.
  • the transfected cells are washed three times with cold DMEM and then harvested on ice with a cell scraper.
  • the cells (approx. 5X10 6 cells) are collected by centrifugation and in 1 ml Lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 1% NP-40, 1% Triton-x-100, 2 mM EDTA) lysed.
  • the kernels are centrifuged off at 11,000 g.
  • the supernatant is subjected to immunoprecipitation.
  • 1 ⁇ g of the cell lysate is mixed with 2 ⁇ g / ml WO2 immunoglobulin (anti-ßA4 antibody) and shaken overhead at 4 ° C.
  • the Protein-G Sepharose is consecutively two times with the buffers A, B and C (A: 150 mM NaCI, 10 mM Tris-HCl pH 7.5, 0.2% NP-40, 2 mM EDTA; B: 500 mM NaCI, 10 mM Tris-HCI pH 7.5, 0.2% NP-40, 2 mM EDTA; C: 10 mM Tris-HCI pH 7.5), mixed with 20 ⁇ l 3X sample buffer, heated to 95 ° C and the supernatant to a 12% Tris Tricine gel applied. After the gel electrophoretic size fractionation, the proteins are transferred to a PVDF membrane and subsequently detected with an anti-ßA4 antibody.
  • the enzyme is coexpressed according to the above instructions in Cos-7 cells with SpA4CT.
  • the cells are brought into contact with the substance to be investigated in a suitable manner (in the presence or absence of membrane-permeabilizing agents).
  • the intracellularly formed ⁇ A4 is then detected as described above.
  • a reduction in the amount of ⁇ A4 formed suggests that the substance is effective as a ⁇ -secretase inhibitor.

Abstract

L'invention concerne une protéase comportant deux restes aspartate dans une structure catalytiquement active, un premier reste aspartate se situant dans un motif X1GX2GD et un deuxième reste aspartate se situant dans un motif X3X4DX5. La protéase selon l'invention est caractérisée en ce que X1, X2, X3 et X5 pouvent être choisis indépendamment les uns des autres parmi Ala, Val, Leu, Met et Ile, en ce que X4 est un acide aminé aromatique et en ce que les motifs X1GX2GD et X3X4DX5 se situent dans une région transmembranaire.
EP00903587A 1999-01-22 2000-01-19 Protease Withdrawn EP1144603A3 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
DE19902550 1999-01-22
DE19902550A DE19902550A1 (de) 1999-01-22 1999-01-22 Aspartatprotease
DE19925946 1999-06-08
DE19925946 1999-06-08
DE19929115 1999-06-24
DE19929115 1999-06-24
PCT/EP2000/000390 WO2000043505A2 (fr) 1999-01-22 2000-01-19 Protease

Publications (2)

Publication Number Publication Date
EP1144603A2 EP1144603A2 (fr) 2001-10-17
EP1144603A3 true EP1144603A3 (fr) 2002-02-06

Family

ID=27218935

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00903587A Withdrawn EP1144603A3 (fr) 1999-01-22 2000-01-19 Protease

Country Status (4)

Country Link
EP (1) EP1144603A3 (fr)
AU (1) AU2542100A (fr)
CA (1) CA2360585A1 (fr)
WO (1) WO2000043505A2 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8609087B2 (en) 1999-07-12 2013-12-17 Trustees Of Dartmouth College Compounds and methods for identifying compounds which inhibit a new class of aspartyl proteases
US6887677B1 (en) 1999-07-12 2005-05-03 Trustees Of Dartmouth College Compounds and methods for identifying compounds which inhibit a new class of aspartyl proteases
GB0126782D0 (en) * 2001-11-07 2002-01-02 Medical Res Council Assay

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5965397A (en) * 1997-01-31 1999-10-12 Genetics Institute, Inc. Secreted proteins and polynucleotides encoding them
WO1999043693A1 (fr) * 1998-02-26 1999-09-02 Human Genome Sciences, Inc. Proteines secretees par l'homme
JP2001514520A (ja) * 1997-03-11 2001-09-11 ジェネティックス・インスチチュート・インコーポレーテッド 分泌蛋白およびそれらをコードするポリヌクレオチド
EP1027430A4 (fr) * 1997-07-16 2001-09-19 Human Genome Sciences Serie de 64 proteines humaines secretees
JPH11187882A (ja) * 1997-12-26 1999-07-13 Ono Pharmaceut Co Ltd 新規なポリペプチド、その製造方法、そのポリペプチドをコードするｃＤＮＡ、そのｃＤＮＡからなるベクター、そのベクターで形質転換された宿主細胞、そのポリペプチドの抗体、およびそのペプチドまたは抗体を含有する薬学的組成物
AU5928899A (en) * 1998-09-23 2000-04-10 Human Genome Sciences, Inc. 31 human secreted proteins

Also Published As

Publication number Publication date
WO2000043505A2 (fr) 2000-07-27
CA2360585A1 (fr) 2000-07-27
EP1144603A2 (fr) 2001-10-17
WO2000043505A3 (fr) 2001-11-29
AU2542100A (en) 2000-08-07

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