JP2002517239A - Polypeptide having beta-secretase type activity - Google Patents

Abstract

  (57) [Summary] The present invention relates to novel polypeptides and their pharmaceutical uses. More specifically, the present invention relates to a novel polypeptide having β-secretase type activity, which is capable of specifically cleaving natural β-amyloid peptide precursor (APP).

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel polypeptides and their pharmaceutical uses. More specifically, the present invention relates to a novel polypeptide having β-secretase type activity, which is capable of specifically cleaving a natural β-amyloid peptide precursor (APP).

[0002] Individuals suffering from Alzheimer's disease exhibit symptoms characterized by reduced memory, reduced intelligence and reduced cognitive function. These lesions are associated with neuronal atrophy, marked deficiency of certain types of receptors and reduced synaptic connections. This syndrome is mainly associated with the presence of a very large amount of senile plaques and neurofibrillary degeneration in the cerebral cortex, hippocampus, amygdala and cerebral cortical vessels.

The so-called senile plaques are spherical structures that form slowly in the extracellular space of the hippocampus, cortex and other cerebral regions for about a decade. Its main component is β-amyloid (βA) peptide bound to other abnormal proteins. These structures are surrounded by abnormal axons and neurons.

[0004] Neurofibrillary degeneration results from the accumulation of abnormal dense bundles of fibrils in certain neurons, mainly cortical pyramidal cells. These neurofibrillary tangles are composed of specific forms of tau protein and combine with other proteins to form helical neurofilament pairs, disrupting nerve impulse conduction.

[0005] This disease has been reported to be of the familial type, and all are thought to be due to various gene mutations that induce abnormal accumulation of βA peptide. These genetic mutations are very heterogeneous and have been particularly linked to various mutations on chromosomes 1, 14 and 21. Chromosome 21 is of particular interest because it has a gene encoding the precursor protein of βA. Therefore, it is considered that Alzheimer's disease occurs early (55 years old) in a patient with Down syndrome (trisomy 21).

[0006] It should be noted that the proportion of family-type Alzheimer's disease patients to the total number of patients is low.

[0007] Of almost all cases of Alzheimer's disease that are not linked to family type, 70
Individuals older than age show senile plaques in various areas of the cerebrum. However, its distribution depends on the type of dementia in question.

A human βA peptide having a molecular weight of 4 kDa is Met⁵⁹⁶-Asp⁵⁹⁷And Val ⁶³⁶ -Ile⁶³⁷At the site by proteolytic cleavage of its precursor (APP)
Is done. The free molecule is composed of 39 (-42) amino acids with the protein sequence: 10 20 30 40 DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA.

In aqueous solution, this peptide assumes a three-dimensional structure of β-pleated sheet type. This peptide is agglutinating because the COOH terminal moiety is very hydrophobic, and its oligomerization rate depends on pH (maximum formation at pH = 5.4) and peptide concentration. Furthermore,
The sequence between the Gly ²⁵ and Met ³⁵ residues confers on this peptide neurotrophic and neurotoxic properties.

[0010] βA peptides are natural products secreted by cells and detectable in blood and cerebrospinal fluid. Although this peptide is neurotoxic, its production is insufficient for the formation of amyloid plaques. Cerebral deposition of βA is thought to be due to abnormal “processing” or overexpression of its precursor.

The primary transcript of β-amyloid peptide precursor (APP) undergoes alternative splicing and is ubiquitously expressed in tissues at different rates depending on the cell type 563,695
, 714, 751 and 770 amino acids (aa) are generated.

However, the isoforms APP695 and 751 are restricted to the central and peripheral nervous systems (particularly at the level of synapses and neurons in astrocytes) and play a role in synaptic bioactivity. Isoforms APP751, APP563 and APP7
70 contains a 56aa "insert" similar to a "Kunitz" type protease inhibitor. Furthermore, the secreted form of APP751 is consistent with Nexin II, a protease inhibitor involved in the regulation of extracellular serine proteases.

[0013] APP is an approximately 120 kDa glycoprotein with characteristics of the type II surface receptor. Although the actual function of APP has not yet been elucidated, it has been reported that this glycoprotein may play a role in regulating cell proliferation and in adhesive interactions in inflammation, regeneration and immune responses.

[0014] All isoforms of APP are inserted into the endoplasmic reticulum by its "signal" sequence. The precursor is then targeted to the Golgi apparatus, undergoes various post-translational modifications, and binds to the membrane. Under the action of various proteases, APP undergoes various cleavages (see FIG. 1) mainly as shown below.

[0015] A protease activity, called α-secretase activity, activates APP6 inside the βA sequence.
Cut between ^{Lys 612} and ^{Leu 613} residues 95, secreted NH ₂ terminal fragment that contains the first 16aa of [beta] A: generating a (sAPP [alpha] soluble APPα).

A protease activity, called β-secretase activity, cleaves the peptide bond of doublet Met ⁵⁹⁶ -Asp ⁵⁹⁷ inside the precursor, releasing a secreted APPNH _2- terminal fragment (sAPPβ: soluble APPβ) that completely deletes βA. I do.

A third protease activity, called γ-secretase activity, is also associated with the precursor Va
APP acting between l ⁶³⁶ -Ile ⁶³⁷ residues and containing βA
Generate γ.

The main component of senile plaques appearing in both familial and non-familial Alzheimer's disease is β
Amyloid (βA) peptide.

[0019] βA peptide ^Met 596 ^{-Asp 597} sites of APP in the β-secretase-type protease activity, in the γ-secretase-type protease activity ^Val 636 -I
It is produced by cleavage of its precursor APP at site le ⁶³⁷ .

In familial Alzheimer's disease, mutations at the β-secretase cleavage site have been identified. This is ^{(Lys 595 -Met 596 → Asn-} Leu in APP695) "Swedish" double mutation of APP, indicating increased production of βA peptide (hence maturation increased advantageous APP to amyloid generation path).

However, in most cases of Alzheimer's disease, APP is in its natural form, with a non-mutated β-secretase cleavage site.

Certain proteases from human, rat or monkey have been studied by various investigators and are expected to be involved in the maturation of precursor APP. Among these enzymes, serine proteases 1 and 2 (Abraham et al. (1991), Bi)
ochem. Biophys. Res. Commun. , 174, 790-79
6; Matsumoto et al. (1994), Biochemistry, 33, 3;
941-3948; Matsumoto et al. (1994), Neuroscien.
ces Letters, 195, 171-174) and cathepsin G-like protease (Razzaboni et al. (1992), Brain Research 5).
89, 207-216). These human or monkey enzyme cleaves at the level of ^Met 596 -Asp ^{59 7} site of APP by β-secretase-type protease activity, but is detected or partially purified from Alzheimer's disease patients.

Since the formation of β-amyloid peptide is caused by the action of β-secretase-type enzyme on APP, Met ⁵⁹⁶ − in human cells not derived from Alzheimer's disease patients.
It would be important to identify and characterize the β-secretase-type enzyme system that is selectively involved in the post-translational maturation of the β-amyloid peptide precursor at the level of the Asp ⁵⁹⁷ site. By recognizing these novel enzyme systems in this way, it is possible to anticipate the production of novel molecules that can be used as medicaments and that can mediate the metabolism of β-amyloid peptide in non-familial Alzheimer's disease.

Therefore, the present inventors have proposed the β-secretase type β-amyloid peptide precursor (APP
The present inventors have completed the present invention by identifying and characterizing a polypeptide having a catalytic activity against). Unlike other proteins that have been identified, the polypeptides of the present invention act specifically on the native form of APP. The invention is based in particular on the discovery of a 70 kDa polypeptide capable of cleaving the non-mutated form of APP.

Accordingly, a first object of the present invention is to provide a natural β-amyloid peptide precursor (APP)
A polypeptide having β-secretase type activity or a mutant thereof, which is capable of specifically cleaving.

The term mutant in the sense of the present invention has the same activity as the polypeptide of the present invention,
It means any molecule obtained by gene and / or chemical modification of the peptide sequence. Genetic and / or chemical modification means any mutation, substitution, deletion, addition and / or modification of one or more residues. Such variants can be made for a variety of purposes, such as to improve their production levels, increase their protease resistance, increase and / or alter their activity, or confer novel biological properties. it can. Additional variants include, for example, chimeric polypeptides containing an additional heterologous moiety attached to the terminus. The term variant also includes polypeptides similar to the polypeptides described in the present invention, which are derived from other cell sources, especially cells of other organisms.

The substrate cleaved by the polypeptide of the invention does not have a mutation in its peptide sequence, in particular the β-amyloid peptide precursor has no Swedish double mutation. Polypeptide or variants thereof of the present invention can selectively cleave peptide bonds of ^Met 596 ^{-Asp 597} doublet inside the wild or native form of APP. In particular, the polypeptide Swedish mutation ^(Lys 595 ^-Met being detected from patients with Alzheimer's disease cerebral sample ⁵⁹⁶ → As the present invention
Does not cut APP forms with n-Leu).

The polypeptides of the present invention can be purified from human cells of a subject not afflicted with Alzheimer's disease and can cleave only its native form of APP at the level of the peptide bond of Met ⁵⁹⁶ -Asp ⁵⁹⁷ doublet.

The polypeptide of the present invention is characterized in that its activity does not depend on the second substrate and / or ligand. By way of example, mention may be made of ions, especially calcium or magnesium cations. In fact, other proteins, such as serine proteases 1 and 2 and cathepsin G-like proteases with protease activity, require the presence of calcium to be active.

The polypeptide of the present invention has a molecular weight of 65 to 75 kDa, preferably about 70 kDa.
With a molecular weight of Its isoelectric point is 6.0-7.0, preferably 6.0.

[0031] These polypeptides are endopeptidases of the serine protease family.
These endopeptidases are preferably of the chymotrypsin-sensitive type.
In fact, according to the inhibition profile, these endopeptidases are completely inhibited by PMSF (phenylmethanesulfonyl fluoride),
TPCK (L-1-chloro-3- [4-tosylamido] -4-phenyl-2-butanone), partially inhibited by benzamidine. Furthermore, it is completely resistant to inhibition by antipapain.

The polypeptide of the present invention or a mutant thereof is characterized by having a maximum β-secretase activity at pH 7-8.

The present invention further relates to non-peptide or non-exclusive peptide compounds capable of cleaving the β-amyloid peptide precursor at the Met ⁵⁹⁶ -Asp ⁵⁹⁷ site.
Such compounds are obtained by reproducing the active motif of the polypeptide of the invention with a non-peptide or non-exclusive peptide structure compatible with use as a medicament. Thus, the present invention provides for the production of pharmacologically active non-peptide or non-exclusive peptide molecules by determining the structural components of these polypeptides that are important for their activity,
And the use of said polypeptides to reproduce said components with non-peptide or non-exclusive peptide structures. The invention also relates to a pharmaceutical composition comprising one or more molecules thus produced.

[0034] According to one aspect of the invention, the polypeptide or variant thereof further comprises a signal sequence enabling a precise cellular localization. Examples of usable sequences include IgkB signal peptide sequence, APP signal peptide, signal peptide of muscle and central nicotinic acetylcholine receptor subunits, and the like.

Another object of the present invention is a method for purifying the polypeptide of the present invention, which has a β-secretase type activity and is capable of specifically cleaving a natural precursor of APP. The method comprises the steps of: firstly concentrating the cell culture supernatant with a membrane; and subsequently subjecting the concentrate to various purification steps, in particular centrifugation through a tangential membrane, followed by exclusion chromatography and ion exchange chromatography. And then to hydrophobic interaction chromatography and finally to exclusion chromatography again.

[0036] The present invention also relates to the use of cell lines. This cell line was selected from a number of other human lines derived from various sources other than Alzheimer's disease patients (see Materials and Methods).
These cell lines were used to test polypeptides of the invention and variants thereof. For example, these human cell lines are representative of the central or peripheral nervous system and the immune system and are capable of normal metabolism of the beta amyloid peptide precursor and inducing the production of said peptide. The selected cell line was the THP1 line derived from monocytes (ATCCTIB20
2) is preferred.

The above cell lines are used in particular as hosts for detecting compounds (ligands, antagonists, agonists) capable of inhibiting the interaction between the polypeptide of the invention and its substrate.

Another object of the invention is a polyclonal or monoclonal antibody or antibody fragment against a polypeptide as described above. Such an antibody can be produced by a method known to those skilled in the art. In particular, these antibodies can be prepared by immunizing an animal against one of the polyclonal or a variant thereof of the present invention, collecting blood, and isolating the antibody. These antibodies can also be produced by producing hybridomas according to techniques known to those skilled in the art. The antibody or antibody fragment of the invention particularly inhibits at least partially the interaction of said polypeptide with a β-amyloid peptide precursor and / or reduces the β-secretase activity of the polypeptide of the invention on a native β-amyloid peptide precursor. Can be used with the ability to at least partially inhibit. In particular, these antibodies are used in particular as medicaments for treating neurodegenerative diseases such as Alzheimer's disease.

Another object of the invention is to at least partially inhibit the interaction of the polypeptide with the β-amyloid peptide precursor and / or at least partially modulate or inhibit the β-secretase activity of the polypeptide of the invention. The present invention relates to a method for identifying a compound capable of performing the following.

The detection and / or identification of such compounds may include:-if the molecule has an affinity for the polypeptide of the invention, under conditions that allow the polypeptide to interact with the molecule. Contacting a recombinant cell expressing the peptide with a mixture comprising said molecule or various molecules that have not been identified, and detecting and / or isolating the molecule bound to the polypeptide of the invention. Is done.

In certain embodiments, the methods of the invention are suitable for the detection and / or isolation of agonists and antagonists of β-secretase activity of a polypeptide of the invention. The corresponding nucleotide sequence can be obtained from these agonist or antagonist molecules by conventional techniques known to those skilled in the art, in particular by sequencing.

Thus, according to one aspect of the invention, it is particularly advantageous to express in situ an agonist or antagonist molecule of the polypeptide of the invention from its nucleotide sequence. In order to produce these molecules and express them in vivo, ex vivo and / or in vitro, their nucleotide sequences need to be carried on a viral or plasmid vector and transfected into an appropriate host cell by this vector.

The invention further provides that the interaction of the polypeptide with the β-amyloid peptide precursor is at least partially inhibited and / or that the β-secretase activity of the polypeptide of the invention is inhibited and / or It also relates to the use of the above polypeptides or variants thereof for detecting ligands and compounds which mediate the metabolism of amyloid peptide precursors and / or can alleviate the production of β-amyloid peptide.

Indeed, the invention also relates to a method for detecting a molecule capable of affecting the activity of a polypeptide according to the invention.

The “screening” method comprises the steps of: contacting a polypeptide of the present invention having β-secretase type activity with a molecule or a mixture containing various molecules, as the case may be, which have not been identified; Contacting the mixture with a substrate of a polypeptide of the invention, preferably native APP; measuring β-secretase activity on APP; detecting molecules that act on β-secretase activity of the polypeptide of the invention. And / or isolating.

Another object of the present invention relates to the use of a ligand or modulator identified and / or obtained by the above method as a medicament. Such ligands or modulators can modulate the level of β-secretase activity of the polypeptides of the present invention relative to the native β-amyloid peptide precursor, and can treat certain neurological disorders, especially Alzheimer's disease.

The present invention further relates to any pharmaceutical composition comprising a polypeptide as described above or an agonist, antagonist or ligand molecule as an active ingredient.

The present invention also relates to any pharmaceutical composition comprising as an active ingredient at least one of the antibodies or antibody fragments and / or antisense oligonucleotides as described above.

The present invention further relates to pharmaceutical compositions wherein the peptides, antibodies, ligands and / or nucleotide sequences as described above are linked to one another or to other active ingredients.

The pharmaceutical composition of the invention may at least partially inhibit the interaction of a polypeptide of the invention or a variant thereof with a natural β-amyloid peptide precursor, and / or
It can be used to at least partially inhibit secretase activity and / or to mediate the metabolism of β-amyloid peptide precursor and inhibit or reduce the production of β-amyloid peptide. As the pharmaceutical composition, a pharmaceutical composition for treating a neurodegenerative disease such as Alzheimer's disease is more preferable.

Another object of the present invention is to at least partially inhibit the interaction of a polypeptide of the invention or a variant thereof with a natural β-amyloid peptide precursor, and / or a polypeptide of the invention or a variant thereof. The above-mentioned molecule (ligand, antibody or antibody fragment, which inhibits or alleviates the production of β-amyloid peptide by at least partially inhibiting the β-secretase activity of β-amyloid peptide and / or mediating the metabolism of the precursor of β-amyloid peptide. Antagonists, agonists). These molecules are preferably used as medicaments, in particular for the treatment of neurodegenerative diseases, in particular for the treatment of Alzheimer's disease.

According to one aspect of the invention, the polypeptide of the invention or a variant thereof is used to mediate the metabolism of β-amyloid peptide.

According to another embodiment of the present invention, said polypeptide or a variant thereof at least partially inhibits the interaction of the polypeptide of the present invention or a variant thereof with a natural β-amyloid peptide precursor; and And / or at least partially inhibit the β-secretase activity of the polypeptide of the present invention or a variant thereof, and / or mediate the metabolism of the β-amyloid peptide precursor to inhibit or alleviate β-amyloid peptide production. Used to detect possible ligands or compounds.

For use in accordance with the present invention, the polypeptides of the present invention, particularly antagonists, agonists, antibodies and ligands thereof, are topically, orally, parenterally, intranasally, intravenously, intramuscularly, subcutaneously, intraocularly, transdermally. It is preferred to combine with one or more pharmaceutically acceptable carriers to formulate such a route of administration. It is preferably used as an injection. In particular, sterile isotonic saline (monosodium phosphate, disodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc., or a mixture of such salts), or sterile water or physiological serum as the case may be. (Particularly freeze-dried) compositions which can be added to form injectable solutes.

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are merely illustrative and do not limit the present invention.

Materials and Methods Cell Line Origins Thirteen human cell lines of various origins were used for mature enzyme testing.

Central Nervous System SW1088 ATCCHTB12 Astrocytoma SW1788 ATCCHTB13 Astrocytoma U-138MG ATCCHTB16 Glioblastoma, U-373MG ATCCHTB17 Glioblastoma, Astrocytoma II degree Peripheral nervous system Foreskin MRC5 ATCC CCL171 Lung, Diploid Immune system DAKIKI ATCC TIB206 B cells, IgA secretion H9 ATCCCHTB176 T cell lymphoma IM-9 ATCC CCL 159 lymphoblast, Ig secretion K-562 ATCC CCLAT CC IAT CTB1 CT lymphocyte leukemia CTCC IAT CTB1 CT lymphocyte leukemia CT16 ball.

Cell Culture After thawing, cells were plated in the presence of 10% fetal calf serum, depending on the cell origin, in “DMEM”
Culture in medium or "RPMI1640" medium. These cultures were carried out at 37 ° C. in 1-liter flasks while changing the medium every 2-3 days. Depending on the test cell line, it takes 2-5 months to obtain a volume of 18 liters of medium. The final culture step is performed for 48 hours in the absence of fetal calf serum and phenol red. Thereafter, these cell cultures are centrifuged to recover the supernatant, which is used for enzyme purification.

The cell lines HMCB, U-373MG, U-138MG, MRC5 and Hs27 were cultured in “DMEM” medium, and cell lines SW1088, SW1783, K-562,
H9, DAKIKI, THP1, RPMI1788 and IM-9 are "RPMI1
640 "culture.

Enzyme purification ULTRASETTE® (FI) with a cut-off threshold of 10 kDa
(LTRON) After concentrating 18 liters of the supernatant of each cell culture with a membrane, the proteolytic activity was purified using the obtained concentrate according to the following protocol.

In the first stage, centrifugation at 7000 rpm with a tangential membrane. Specifically, it is concentrated on an ULTRAFREE® (MILLIPORE) membrane with a cut-off threshold of 10 kDa.

Next, exclusion chromatography is performed. According to a particular aspect of the present invention, Sephacryl S-100 column exclusion limit ¹⁰ 3 ^{Da~10 5} Da (Pharmac
Exclusion chromatography was performed in ia).

The third step of the method is ion exchange chromatography. In particular, a Q-Sepharose column (Pharmacia) composed of a gel from strong anions was used.
Solvent A (25 mM Tris, pH 7.5) and solvent B (25 mM Tris, 1
The column is eluted with a 0 → 1M salt gradient using M NaCl, pH 7.5).

The penultimate step is to carry out hydrophobic interaction chromatography on a phenyl sepharose-6 column (Pharmacia) with a particularly high degree of substitution (40 μmol / g gel). 1 → 0 using solvent A (25 mM Tris, 1 M (NH ₄ ) ₂ SO ₄ , pH 7.5) and solvent B (25 mM Tris, pH 7.5)
The column was eluted with a M ammonium sulfate gradient.

Finally, as a final step, exclusion chromatography is carried out on a TSK gel G2000SW column (Interchim), in which the gel is composed of a hard silica support, especially grafted with hydrophilic groups. The eluent was 25 mM with 250 mM NaCl
Tris, pH 7.5.

Enzyme Tests β-secretase activity was tested by tests using various peptides similar or identical to the amino acid sequence of the precursor APP at the level of the β-secretase enzyme cleavage site (Table 1).

To make the chromophore peptide, the peptide Z-
Incubate 5 μl of Val-Lys-Met-AMC (7-amino-6-methylcoumarin) with 5 μl of supernatant at 37 ° C. for 6 hours. 0.1N HCl 3μ
The reaction is stopped by adding 1 and the enzyme activity is measured by measuring the fluorescence of the free AMC chromophore at 460 nm.

Synthetic peptides of various sizes similar or identical to the β-secretase enzyme cleavage site were synthesized and used as substrates in characterization and enzyme specificity studies (Table 1).

Incubate 5 μl of supernatant with 5 μl of peptide at 37 ° C. for 6 hours. 0.
The reaction is stopped by adding 3 μl of 1N HCl, and the enzymatic activity is determined by measuring the 215 nm optical density of the cleavage fragment previously separated by HPLC.

The cleavage site is estimated by sequencing the fragment obtained by cleavage.

Under the same experimental conditions (incubation time, pH and concentration) in the absence of the enzyme (
215 nm absorbance (A) of substrate incubated in A ₀ ) and in the presence (A _j )
By measuring the percentage of cleavage of each peptide substrate [% = 100 (A ₀ −A _j )]
/ A ₀ ].

Under the same experimental conditions, the 215 nm absorbance of the peptide substrate or 460 nm of the substrate Z-Val-Lys-Met-MCA in the absence (I ₀ ) and presence (I _j ) of the inhibitor
The percent inhibition [% = 100 (I ₀ −I _j ) / I ₀ ] of each substrate incubated in the presence of the enzyme was determined by measuring the fluorescence.

Baculovirus-infected insect cell membrane extraction containing a human gene encoding a normal APP (APP-K ⁵⁹⁵ M ⁵⁹⁶ ) and a precursor with an “Swedish” double mutation APP (APP-N ⁵⁹⁵ L ⁵⁹⁶ ) These precursors were obtained from the product.
These membrane extracts are incubated with the polypeptides of the invention having purified β-secretase activity and the antibody WO- against the first amino acid of the β-amyloid peptide
2 (Ida N. et al. (1996) J. Biol Chem 271, 22908).
Monoclonal antibodies to the NH ₂ -terminal motifs -22914) and precursor 22
C11 (Boehringer Mannheim; Hilbich C. (1
993) Journal of Biochemical Chemistry
, 268, 26571-26577).

Embodiments Embodiment 1 Detection of Enzyme Activity This example is aimed at detecting enzyme activity in a human cell line of a subject not suffering from Alzheimer's disease.

[0075] Two approaches were used to detect proteases likely to be involved in the maturation of human APP.

Immunological Approach: Monoclonal antibody 22C from 13 human cell lines as described in Materials and Methods
The enzyme activity test was carried out according to No. 11, and a measurable amount of AP in the membrane level and in the cell culture medium.
A cell line capable of producing P was selected. The various cleavage sites of APP were detected and identified using the monoclonal antibody WO-2.

The results are as follows.

The monoclonal antibody 22C11 has a total of 13 to 8 cell lines tested (
HMCB, MRC5, Hs27, SW1088, SW1783, H9, THP1
And IM-9) could be selected. Immunoblot analysis of selected cells revealed molecular weight differences between membrane APP (120 kDa) and soluble APP (110-100 kDa). This indicates that the precursor has undergone more than one enzymatic cleavage at the level of the COOH terminal sequence.

As a result of immunoblot analysis of molecular entities produced by the eight selection systems, various cleavage sites of APP can be detected and identified by the monoclonal antibody WO-2, and the precursor of the βA peptide is differentially detected. I was able to prove that I was undergoing sexual maturity.

Thus, this approach involves a measurable amount of APP at the membrane level and in the cell culture medium.
A cell line capable of producing was selected, and enzymatic cleavage at the precursor of the βA peptide could be demonstrated.

Peptide Substrate: Peptide [KMD] APP (−5) derived from APP and having a similar cleavage site
+5) and Z-Val-Lys-Met-MCA (see Materials and Methods) were used as substrates to detect various enzyme activities present in eight cell lines.

The fragments generated by cleavage of the substrate [KMD] APP (−5, + 5) were combined analysis (HPLC, amino acid composition and sequencing) in a selection system. Indeed, after incubation of the substrate [KMD] APP (−5, + 5) with the supernatant,
The resulting fragments were first separated by HPLC on a reverse phase RPC ₁₈ column (VYDAC) eluting with a gradient of 5 → 40% acetonitrile / 0.05% TFA. The sequence and / or amino acid composition of these fragments was determined by conventional techniques.

As a result of this analysis, Met was detected in each of the eight selection systems.^-1-Asp⁺¹(Complete APP
Met in⁵⁹⁶-Asp⁵⁹⁷At the level of peptide bonds)
Main cutting and Ser^-5-Glu^-4(Ser in complete APP⁵⁹²-Gl
u⁵⁹³And Ala⁺²-Glu⁺³(Ala in complete APP ⁵⁹⁸ -Glu⁵⁹⁹Identifies two types of secondary cleavage at the binding level
We were able to.

An inhibition profile analysis of the enzyme activity of the eight cell lines against the fluorescent substrate Z-Val-Lys-Met-MCA was also performed (Table 2).

As a result of this analysis, the presence of serine (inhibition by aprotinin and pefablock) and metalloprotease (inhibition by EDTA and phosphoramidone) types could be detected in each of the eight selection systems. (Table 2).

Embodiment 2 Purification and Characterization of β-Secretase Activity This example describes the purification of a polypeptide of the invention having β-secretase activity,
The purpose is to elucidate the characteristics.

Based on the selection of the eight human cell lines described in Example 1 and the results obtained, the THP-1 cell line was selected for the reason that the cell cycle was rapid and a large amount of protein was obtained, And a method for purification of the desired β-secretase activity according to the purification protocol described in the section “Method”.

For tracking the purification of the polypeptide of the invention, the substrate Z-Val-Lys-Me
The residual activity of the fraction having proteolytic activity on t-MCA and [KMD] APP (-5, +5) was analyzed. In each purification step, each fraction was first contacted with the peptide Z-Val-Lys-Met-MCA and the fraction with endoprotease activity was isolated. These fractions are then combined with the peptide [KM
D] tested against APP (−5, + 5), Met ⁻¹ −Asp ⁺¹ (complete AP
The fraction that preferentially cleaves this peptide substrate at the level of peptide binding was isolated (corresponding to the Met ⁵⁹⁶ -Asp ⁵⁹⁷ site in P). As a result of these tests, it was possible to detect various fractions having endoprotease activity isolated from the supernatants of eight types of selected cell cultures by using these two substrates simultaneously.

Several protein fractions were obtained at the end of the purification process, which is an exclusion chromatography using a TSK2000 column (see Materials and Methods for properties).

The residual activity of these fractions on the peptide [KMD] APP (−5, + 5) was measured, and only one fraction having β-secretase type activity was obtained. Molecular weight measurement by polyacrylamide gel electrophoresis, isoelectric point measurement, pH
-Dependent maximum activity tests and inhibition profiles with conventional inhibitors ("Materials and Methods")
) To determine its characteristics.

Electrophoresis analysis was performed on a 4-20% polyacrylamide gel on a Past system (Pharmacia) under denaturing or normal conditions, and a molecular weight band of approximately 70 kDa was detected.

Maximum activity on the peptide [KMD] APP (−5, + 5) was observed at pH 7-8.

According to the inhibition profile of this fraction for the peptide [KMD] APP (−5, + 5), this fraction is a serine protease and the percentage inhibition was calculated to be 100% PMSF, 75% Pefabloc, 25% TPCK, Benzamidine 10% and antipapain 0%.

Therefore, this example relates to a method for purifying a polypeptide of the present invention having β-secretase activity and measurement of various properties.

Example 3 Specificity of β-Secretase Activity This example relates to the analysis of β-secretase activity of a polypeptide of the invention.

This specificity was analyzed by using various substrates as described below.

Peptides similar or identical to the precursor amino acid sequence at the level of the cleavage site (Table 1).

-The native and mutant (Swedish mutant) forms of the beta amyloid peptide precursor.

The polypeptides of the invention were contacted with various substrates, and the percent cleavage of these substrates was calculated. Table 3 shows the results.

Analysis of the data summarized in Table 3 for the synthetic peptides revealed features relating to the importance of several subsites involved in this β-secretase substrate recognition,
We can conclude as follows.

1) Regardless of the size of the substrate, subsites P ₁ and P ₂ are essential (A in Table 3)
). The double mutation (Lys-Met → Asn-Leu) was found to completely suppress enzymatic cleavage.

2) Subsites P ₂ and P ₁ are interactive or cooperative (Table 3B). In fact, a simple substitution of P ₂ (Lys → Asn) or P ₁ (Met → Leu) only reduces the cleavage rate, whereas the so-called “Swedish” double mutation suppresses substrate recognition.

The substitution of the P ₂ residue (Lys → Arg) can be performed by using a peptide having Leu at P ₁ ([KLD
] -APP (-5, + 5) and [RLD] -APP (-5, + 5) Substrate ([KMD] -APP (-5, + 5) for cutting index difference has a Met on _{P 1)} of the [RMD] -A
It is larger than the difference in the cutting rate observed for PP (−5, +5)).

[0104] 3) the size and / or amount of the P ₁ residue is large (C in Table 3): the subsite restrictions to be added to the peptide backbone by a side chain of P ₁ increases, the enzymatic cleavage rate decreases. Indeed, experiments can rank the cut rates as a function of the replacement performed.

[KMD] APP (−5, + 5)> [KLD] APP (−5, + 5)> [KI
D] APP (−5, + 5)> [KVD] APP (−5, + 5).

[0106] 4) residues of the subsites P _'1 is always Asp or Glu (D in Table 3). In fact, as is evident from the results, mutating Asp to Asn or Gln does not eliminate substrate cleavage, further results in cleavage at the equivalent Ala-Glu site of the Met-Asp site, and further under the same experimental conditions APP Fragmentation rate of fragment (1,5) is 35%
The pseudo site Ala-Glu is only available on natural substrates.

Thus, based on the results previously obtained with the polypeptides of the present invention in terms of β-secretase type cleavage specificity, the peptide, pseudopeptide or non-peptide species of Met-As
It is believed that a competitive inhibitor is obtained at the p-cleavage site.

For the membrane-originated precursor of β-amyloid peptide, “normal” (APP-K ⁵⁹⁵ M ⁵⁹⁶ ) and “double mutation” (APP-K ⁵⁹⁵ M ⁵⁹⁶ ) by the polypeptides found to have β-secretase activity in Examples 1 and 2 APP-N ⁵⁹⁵ L ⁵⁹⁶ ) full (full length)
The precursor was cleaved and the cleavage products were immunoblot analyzed using antibodies 22C11 and WO02 (see Materials and Methods).

Analysis of the molecules with these antibodies increases the percentage of cleavage of the precursor APP-KM, irrespective of the length of the enzyme contact incubation time, but increases the percentage of precursor APP-KM.
At NL, it remains almost zero. Actually, the following points are apparent from the results shown in FIG.

-Bac. When the NL membrane, ie, the membrane containing the precursor APP-NL, is incubated, the same band is observed regardless of the experimental conditions (columns 1, 2 and 3). Therefore, cleavage by the polypeptide of the present invention is not observed.

-Bac. When a WT membrane, that is, a membrane containing the natural precursor APP-KM, is incubated, a new band appears at around 12 kDa in column 3 as compared to the other two columns. Column 1 shows the membrane incubated in the absence of enzyme, column 2 shows the membrane incubated in the absence of enzyme at 37 ° C., and column 3 incubates at 37 ° C. with the polypeptide of the invention having β-secretase type activity. Corresponding to the film. bac. Note that the intensity difference between the bands of columns 1 and 2 of the WT membrane is due to the amount of starting material loaded on the gel.

As a result of analysis with the antibody WO-2, it was possible to detect this novel band having a molecular weight of about 12 kDa corresponding to the COOH-terminal fragment obtained by cleavage of the precursor with β-secretase at the Met-Asp binding level. As a result of this analysis, it can be concluded that the native precursor APP-KM is cleaved by the polypeptide of the present invention.

Thus, this result indicates that the precursor APP-NL was not cleaved, but the precursor APP-NL
KM has been shown to be selectively cleaved, supporting the data obtained with the 10, 20, or 40 amino acid substrate peptide APP.

Further, this example demonstrates that the polypeptide of the invention isolated above has specific β-secretase activity of a natural precursor of β-amyloid peptide.

[0115]

[Table 1]

[0116]

[Table 2]

[0117]

[Table 3]

[0118]

[Table 4]

[Brief description of the drawings]

FIG. 1 shows a schematic diagram of APP and a cleavage site.

FIG. 2 is an explanatory diagram of a method for purifying the polypeptide of the present invention.

[3] "normal" immuno the ^{(APP-K 595 M 596)} and "double mutant" ^{(APP-N 5 95 L 596} ) cut by the polypeptide of the present invention the β-amyloid peptide of the membrane origin complete precursor It is a blot analysis figure. The polypeptide of the invention is "normal"
It was found that it specifically cleaves the membrane-originated precursor of β-amyloid peptide (APP-KM). For each of the precursors (APP-NL and APP-KM), column 1 shows the membrane incubated in the absence of the enzyme, column 2 shows the membrane incubated at 37 ° C. in the absence of the enzyme, and column 3 shows the β This corresponds to a membrane incubated at 37 ° C. with a polypeptide of the invention having secretase type activity.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 39/395 A61P 25/00 4C086 45/00 25/28 4H045 48/00 C07K 16/40 A61P 25/00 C12N 9/64 25/28 C12Q 1/37 C07K 16/40 C12N 15/00 A C12N 9/64 A61K 37/02 C12Q 1/37 37/54 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG) KZ, MD, RU, TJ, TM), AE, AL, AU, BA, BB, BG, BR, CA, CN, CU, CZ, EE, GD, GE, HR, HU, ID, IL, IN, IS , JP, KP, KR, LC, LK, LR, LT, LV, MG, MK, MN, MX, NO, NZ, PL, RO, RU, SG, SI, SK, SL, TR, TT, UA, US, UZ, VN, YU, ZA (72) Inventor Miyunoz-Zimene, Noeri France, F-94190 Villeneuve-Saint-Georgieu, Lille-dou-Ciuman-dou-Fouère, 53 (72) Inventor Mt. , Philippe franc F-94240 ・ Ray-Rose, Liu Douyu Commandin Lermini, 16F Term (Reference) 4B024 AA01 BA07 BA44 CA01 DA03 FA18 GA11 HA03 HA15 4B050 CC03 DD11 FF05C FF11C FF20C LL01 4B063 QA18 QR16 QS28 QX01 4C084 AA02 AA07 AA13 AA17 DC22 DC50 NA14 ZA152 ZA162 ZC202 4C085 AA13 AA14 CC02 CC04 CC13 4C086 AA01 AA02 AA04 EA16 MA01 MA04 NA14 ZA15 ZA16 ZC20 4H045 AA10 AA11 GA10 GA10

Claims (29)

[Claims] 1. A polypeptide having β-secretase type activity, which is capable of specifically cleaving a natural β-amyloid peptide precursor (APP). 2. The polypeptide according to claim 1, wherein the β-amyloid peptide precursor (APP) has no mutation in its protein sequence. 3. The polypeptide according to claim 1, which is a polypeptide purified from a human cell of a subject not suffering from Alzheimer's disease. 4. A molecular weight of about 70 kDa, an isoelectric point of about 6.0, a serine protease group endopeptidase, a chymotrypsin-sensitive endopeptidase, which reaches a maximum activity at pH 7 to 8. 4. The polypeptide according to any one of claims 1 to 3. 5. The polypeptide according to claim 4, wherein the activity is independent of the second substrate and / or ligand. 6. Polypeptide according to claim 5, characterized in that its activity does not depend on ions, preferably calcium or magnesium cations. 7. A β-amyloid peptide precursor can be cleaved at a β-secretase site, and an active motif of the polypeptide according to any one of claims 1 to 6 is reproduced by a non-peptide or non-exclusive peptide structure. A non-peptide or non-exclusive peptide compound obtained by the following method. 8. The polypeptide according to claim 1, further comprising a signal sequence. 9. A signal peptide sequence having an IgkB signal sequence, APP
9. The polypeptide according to claim 8, wherein the polypeptide is selected from the following signal peptides: muscle and central signal peptides of subunits of the nicotinic acetylcholine receptor. 10. A method for purifying a polypeptide according to any one of claims 1 to 9 from cells derived from a subject not suffering from Alzheimer's disease, wherein a supernatant of a cell culture solution is collected. Followed by the steps of concentrating, reconcentrating the concentrate with a tangential membrane and subsequently purifying the resulting product by continuous chromatography, in particular exclusion, ion exchange and hydrophobic interaction chromatography. The method as described above. 11. Representative of the central or peripheral nervous system and the immune system capable of effecting normal metabolism of the β-amyloid peptide precursor for producing the polypeptide according to any one of claims 1 to 9. TH from a human cell line, preferably a monocyte
Use of a selected cell line that is line P1 (ATCCTIB202). 12. Normal metabolism of a β-amyloid peptide precursor for detecting a compound capable of inhibiting the interaction between the polypeptide according to any one of claims 1 to 9 and its substrate. THP1 line derived from representative human cell lines of the central or peripheral nervous system and immune system, preferably monocytes (ATCCTIB20
Use of a selected cell line that is 2). 13. An antibody or an antibody fragment against the polypeptide according to any one of claims 1 to 9, which at least partially inhibits the interaction between the polypeptide and the β-amyloid peptide precursor, and 2. An antibody, which has the ability to inhibit the activity of the polypeptide according to claim 1 and / or mediate the metabolism of β-amyloid peptide. 14. A polypeptide according to any one of claims 1 to 9 and β
At least partially inhibit the interaction of the amyloid peptide precursor, and / or
Or a method for detecting or isolating a compound capable of inhibiting the activity of the polypeptide, wherein the a-molecule has an affinity for the polypeptide according to any one of claims 1 to 9. Contacting a recombinant cell expressing said polypeptide with a mixture comprising said molecule or various unidentified molecules, optionally under conditions that allow said polypeptide to interact with said molecule; b. The method comprising the step of detecting and / or isolating a molecule bound to the polypeptide. 15. A method according to claim 14, wherein the method is obtainable by a method according to claim 14.
10. A ligand of the polypeptide according to any one of claims 1 to 9. 16. The ligand according to claim 15, which is an antagonist, agonist or inhibitor of the polypeptide according to any one of claims 1 to 9. 17. A pharmaceutical composition comprising at least one inhibitor of the polypeptide according to any one of claims 1 to 9 as an active ingredient. 18. A pharmaceutical composition comprising at least one of the antibody or antibody fragment according to claim 13 and / or the ligand according to claim 15 as an active ingredient. 19. A pharmaceutical composition comprising the peptide, antibody or antibody fragment according to claim 13 and the ligand according to claim 15 and / or the corresponding nucleotide sequence linked to each other or to another active ingredient. 20. The method according to any one of claims 17 to 19 for at least partially inhibiting the interaction between the polypeptide and the β-amyloid peptide precursor and / or inhibiting the activity of the polypeptide. Composition. 21. The composition according to any one of claims 17 to 20, which mediates the metabolism of β-amyloid peptide, and preferably suppresses or delays the production of β-amyloid peptide. 22. A composition according to any one of claims 17 to 21 for the treatment of a neurodegenerative disease. 23. The composition according to claim 22, for treating Alzheimer's disease. 24. at least partially inhibiting the interaction of the polypeptide with the β-amyloid peptide precursor and / or inhibiting the activity of the polypeptide;
Use of an antibody or antibody fragment according to claim 13 and / or a ligand according to claim 15 for mediating the metabolism of β-amyloid peptide. 25. Use of an antibody or antibody fragment according to claim 13 and / or a ligand according to claim 15, especially as a medicament for the treatment of neurodegenerative diseases, in particular Alzheimer's disease. 26. Use of a polypeptide according to any one of claims 1 to 9 for the manufacture of a medicament for the treatment of neurodegenerative diseases, in particular Alzheimer's disease. 27. at least partially inhibiting the interaction of the polypeptide with the beta-amyloid peptide precursor and / or inhibiting the activity of the polypeptide;
And / or a compound capable of mediating the metabolism of β-amyloid peptide and / or a ligand of the polypeptide according to any one of claims 1 to 9 for detecting a ligand of the polypeptide according to any one of claims 1 to 9. Use of the polypeptide according to the above item. 28. A method for detecting a molecule that alters the activity of a polypeptide of the present invention, comprising: a mixture of a polypeptide of the present invention having a β-secretase type activity or various unidentified molecules. Contacting the reaction mixture described in the preceding step with a substrate of the polypeptide of the present invention, which is preferably native APP; measuring β-secretase activity on APP; Detecting and / or isolating a molecule that alters the β-secretase activity of the polypeptide. 29. A virus or plasmid vector comprising the nucleotide sequence of an agonist or antagonist molecule of the polypeptide of the invention, wherein said sequence is transfected into a suitable host cell and said agonist or antagonist of said polypeptide of the invention. Such a viral or plasmid vector for expressing a molecule in vivo, ex vivo and / or in vitro.