DE19902550A1

DE19902550A1 - A novel aspartate protease, coding sequence, inhibitors and antibodies, useful for treatment and diagnosis of Alzheimer's disease

Info

Publication number: DE19902550A1
Application number: DE19902550A
Authority: DE
Inventors: Kay Hofmann
Original assignee: MEMOREC MEDICAL MOLECULAR RESE
Current assignee: MEMOREC BIOTEC GMBH, 50829 KOELN, DE
Priority date: 1999-01-22
Filing date: 1999-01-22
Publication date: 2000-07-27

Abstract

A novel aspartate protease (AP) has two aspartate residues in a catalytically active structure, where the first aspartate residue is present in motif (I) and a second aspartate residue is present in motif (II), where (I) and (II) are present in a transmembrane region. A novel aspartate protease has two aspartate residues in a catalytically active structure, where the first aspartate residue is present in motif (I) and a second aspartate residue is present in motif (II): where (I) and (II) are present in a transmembrane region. X1, X2, X3, X5 = Ala, Val, Leu, Met, Ile chosen independently from one another; X4 = an aromatic amino acid . Independent claims are also included for the following: (1) a nucleic acid sequence encoding (AP); (2) compounds that inhibit expression or activity of (AP); (3) an antibody targeted against (AP); and (4) a method to identify inhibitors of the aspartate protease activity.

Description

Gegenstand der vorliegenden Erfindung sind eine Aspartatprotease, Nucleinsäuren kodierend für die Aspartatprotease sowie die damit in Verbindung stehenden Inhibitoren, Antikörper, Arznei- und Diagnostikmittel.The present invention relates to an aspartate protease, nucleic acids coding for the aspartate protease and the related Inhibitors, antibodies, medicines and diagnostic agents.

Die vorliegende Erfindung stellt eine Aspartatprotease mit zwei Asparatresten in einer katalytisch aktiven Struktur, wobei ein erster Asparatrest in einem Motiv X₁GX₂GD liegt und ein zweiter Aspartatrest in einem Motiv X₃X₄DX₅ liegt, wobei X₁, X₂, X₃ und X₅ unabhängig voneinander ausgewählt werden aus Ala, ValLeu, Met und Ile und X₄ eine aromatische Aminosäure ist, und die Motive X₁GX₂GD und X₃X₄DX₅ in einer Transmembranregion liegen, zur Verfügung. Für die Motive wurde der Einbuchstabencode der Aminosäuren verwendet, d. h. D = Asp, G = Gly usw.The present invention provides an aspartate protease with two aspartate residues in a catalytically active structure, a first aspartate residue being in a motif X ₁ GX ₂ GD and a second aspartate residue being in a motif X ₃ X ₄ DX ₅ , where X ₁ , X ₂ , X ₃ and X _{5 are} independently selected from Ala, ValLeu, Met and Ile and X _{4 is} an aromatic amino acid, and the motifs X ₁ GX ₂ GD and X ₃ X ₄ DX _{5 are located} in a transmembrane region. The single letter code of the amino acids was used for the motifs, ie D = Asp, G = Gly etc.

Solche Aspartatproteasen sind mit hoher Wahrscheinlichkeit an der Spaltung des Amyloid Precursor Proteins (APP) beteiligt. In einer Ausführungsform der Erfindung stellt die erfindungsgemäße Aspartatprotease die bisher nicht identifizierte γ-Secretase dar, die an der Prozessierung des APP zu den als Aß bezeichneten Amyloidpeptiden beteiligt ist.Such aspartate proteases are very likely to cleave the Amyloid precursor proteins (APP) involved. In one embodiment of the invention represents the aspartate protease according to the invention the previously unidentified γ-secretase represents the processing of the APP to the amyloid peptides referred to as Aß is involved.

Ein Überblick über die Rolle der y-Secretasen bei der Entstehung der Alzheimerschen Erkrankung geben S. L. Ross et al. in J. of Biol. Chem. 273 (1998), 15309-15312. An overview of the role of y-secretases in the development of Alzheimer's Disease give S. L. Ross et al. in J. of Biol. Chem. 273 (1998), 15309-15312.

Bevorzugte Aspartatproteasen der vorliegenden Erfindung weisen zusätzlich eine Sequenz PALX₆YX₇VP auf, wobei X₆ und X₇ die gleiche Bedeutung haben wie X1. Es wird jedoch bevorzugt, daß X₆ und X₇ Leucin oder Isoleucin sind.Preferred aspartate proteases of the present invention additionally have a sequence PALX ₆ YX ₇ VP, where X ₆ and X _{7 have} the same meaning as X1. However, it is preferred that X ₆ and X _{7 are} leucine or isoleucine.

Insbesondere handelt es sich bei den Aspartatproteasen um Aspartatproteasen von Säugetieren, insbesondere von Menschen.In particular, the aspartate proteases are aspartate proteases from Mammals, especially humans.

Die erfindungsgemäßen Aspartatproteasen weisen die katalytisch aktiven Aspartat reste in einer Region auf, die innerhalb eines Transmembranbereichs liegt. Transmembranbereiche lassen sich bei Kenntnis der Sequenz eines Proteins aufgrund verschiedener Modelle vorhersagen. Sie sind dadurch gekennzeichnet, daß in einem Bereich überwiegend hydrophobe Aminosäuren liegen, die von Bereichen flankiert werden, in denen eher hydrophile Aminosäuren liegen.The aspartate proteases according to the invention have the catalytically active aspartate remains in a region that lies within a transmembrane region. Transmembrane regions can be based on knowledge of the sequence of a protein predict different models. They are characterized in that in one The range is predominantly hydrophobic amino acids flanked by areas which contain more hydrophilic amino acids.

Besonders bevorzugte Aspartatproteasen der vorliegenden Erfindung werden als psnlike 1 bis 4 bezeichnet. Die humanen und murinen Aminosäuresequenzen sind in der Fig. 1 gezeigt.Particularly preferred aspartate proteases of the present invention are referred to as psnlike 1 to 4. The human and murine amino acid sequences are shown in FIG. 1.

Weiterhin sind Varianten der erfindungsgemäßen Aspartatproteasen Gegenstand der Erfindung. Varianten sind Proteine, die durch einen oder mehrere Mutationen, Insertionen und Deletionen, insbesondere durch konservative Austausche, von den erfindungsgemäßen Aspartatproteasen abgeleitet sind. Auch Nucleinsäuren, die für die erfindungsgemäßen Aspartatproteasen kodieren, sind Gegenstand der Erfindung. Bevorzugte erfindungsgemäße Nucleinsäuren sind solche, die in der Fig. 2 gezeigt sind. Auch komplementäre Nucleinsäuren sind Bestandteil der Erfindung.The invention furthermore relates to variants of the aspartate proteases according to the invention. Variants are proteins which are derived from the aspartate proteases according to the invention by one or more mutations, insertions and deletions, in particular by conservative exchanges. The invention also relates to nucleic acids which code for the aspartate proteases according to the invention. Preferred nucleic acids according to the invention are those shown in FIG. 2. Complementary nucleic acids are also part of the invention.

Die erfindungsgemäßen Aspartatproteasen sind an der Spaltung des APP zum Aβ beteiligt und sind damit indirekt an der Entstehung beispielsweise der Alzheimerschen Erkrankung beteiligt. Daher sind auch Inhibitoren, die die Expression oder die Aktivität der Aspartatproteasen hemmen, Gegenstand der Erfindung. Solche Inhibitoren können in einfachen Verfahren identifiziert werden. Entsprechende Inhibitoren können beispielsweise durch Messung der Expression oder der Aktivität der Aspartatproteasen in Gegenwart von potentiellen Inhibitoren identifiziert werden. Insbesondere zur Messung der Expression eignen sich gegen die Aspartatprotesasen gerichtete Antikörper, die somit ebenfalls Bestandteil der Erfindung sind.The aspartate proteases according to the invention are at the cleavage of the APP to Aβ and are indirectly involved in the development of Alzheimer's, for example Disease involved. Therefore are also inhibitors that the expression or the Inhibit the activity of aspartate proteases, object of the invention. Such Inhibitors can be identified in simple procedures. Appropriate Inhibitors can be, for example, by measuring expression or activity of the aspartate proteases can be identified in the presence of potential inhibitors. The aspartate protesases are particularly suitable for measuring expression directed antibodies, which are therefore also part of the invention.

Die erfindungsgemäßen Aspartatproteasen, Nucleinsäuren, Inhibitoren und Antikörper können in Arznei- und Diagnostikmitteln enthalten sein. Sie eignen sich insbesondere zur Behandlung oder Diagnose von Erkrankungen, die mit der Spaltung des Amyloid IPrecursor Proteins ursächlich verbunden sind, insbesondere der Alzheimerschen Erkrankung.The aspartate proteases, nucleic 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 associated with the cleavage of the amyloid IPrecursor proteins are causally linked, especially to Alzheimer's Illness.

Claims

1. Aspartate protease with two residues of asparagus in a catalytically active structure, with a first aspartate residue in a motif X ₁ GX ₂ GD and a second aspartate residue in a motif X ₃ X ₄ DX ₅ , where X ₁ , X ₂ , X ₃ and X _{5 is} independently selected from Ala, Val, Leu, Met and Ile and X _{4 is} an aromatic amino acid, and the motifs X ₁ GX ₂ GD and X ₃ X ₄ DX ₅ lie in a transmembrane region.

2. Aspartate protease according to claim 1, characterized in that the aspartate protease has the sequence PALX ₆ YX ₇ VP, where X ₆ and X _{7 have} the same meaning as X ₁ .

3. Aspartate protease according to claim 2, characterized in that X ₆ and X _{7 are} selected from leucine and isoleucine.

4. aspartate protease according to any one of claims 1 to 3, characterized in that the transmembrane region is a region in which the amino acids are predominantly hydrophobic.

5. aspartate protease according to any one of claims 1 to 4, characterized in that the aspartate protease is one of the sequences psnlike 1, psnlike 2, psnlike 3 or psnlike 4.

6. Nucleic acids coding for an aspartate protease according to at least one of claims 1 to 5.

7. Nucleic acids according to claim 6, characterized in that they are the Sequences psnlike 1, psnlike 2, psnlike 3 or psnlike 4 have.

8. Inhibitors, characterized in that they express the expression of Inhibit aspartate protease according to one of claims 1 to 5.

9. Inhibitors, characterized in that they have the activity of Inhibit aspartate protease according to one of claims 1 to 5.

10. Antibody directed against aspartate proteases according to one of claims 1 until 5.

11. A method for identifying inhibitors, characterized in that the activity of the aspartate proteases according to any one of claims 1 to 5 in Presence of potential inhibitors is measured.

12. Medicament containing an aspartate protease according to one of claims 1 to 5, a nucleic acid according to any one of claims 6 to. 7, an inhibitor according to claim 8 or 9 or an antibody according to claim 10.

13. Use of a medicament according to claim 12 for the treatment of Diseases associated with the cleavage of the amyloid precursor protein are causally linked, especially Alzheimer's disease.

14. Diagnostic agent containing an aspartate protease according to one of the claims 1 to 5, a nucleic acid according to any one of claims 6 to 7, an inhibitor according to claim 8 or 9 or an antibody according to claim 10.

15. Use of the diagnostic agent according to claim 14 for the diagnosis of Diseases associated with the cleavage of the amyloid precursor protein are causally linked, especially Alzheimer's disease.