WO1999064587A1 - Polypeptides possedant une activite de type beta-secretase - Google Patents
Polypeptides possedant une activite de type beta-secretase Download PDFInfo
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- WO1999064587A1 WO1999064587A1 PCT/FR1999/001326 FR9901326W WO9964587A1 WO 1999064587 A1 WO1999064587 A1 WO 1999064587A1 FR 9901326 W FR9901326 W FR 9901326W WO 9964587 A1 WO9964587 A1 WO 9964587A1
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- 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
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to new polypeptides and their pharmaceutical use. More particularly, the present invention relates to new polypeptides having a ⁇ -secretase activity, characterized in that they are capable of specifically cleaving the natural precursor of the ⁇ -amyloid peptide (APP).
- APP ⁇ -amyloid peptide
- Alzheimer's disease exhibit characteristic symptoms of memory impairment, loss of intellectual capacity and cognitive functions. These pathological changes are accompanied by neuronal atrophy, significant depletion of a certain type of receptor and also a reduction in synaptic connections.
- This syndrome involves the presence of senile plaques and neurofibrillary degeneration in very large quantities, mainly in the cerebral cortex, the hippocampus, the tonsil nucleus and in cortical blood vessels.
- senile plaques are spherical structures which are slowly established over a decade in the extracellular spaces of the hippocampus, the cortex and other brain regions. Their major constituent is the amyloid ⁇ peptide (A ⁇ ) associated with other abnormal proteins. These structures are surrounded by axons and abnormal neurons.
- a ⁇ amyloid ⁇ peptide
- Neurofibrillary degenerations are due to an accumulation of dense bundles of abnormal fibers in the cytoplasm of certain neurons and mainly pyramidal cells of the cortex. These neurofibrillary tangles are made up of a particular form of the protein tau which, associated with other proteins, gives pairs of helical neurofilaments which disturb the conduction of nerve impulses. Familial forms of this disease have been identified and seem to result from various genetic modifications, all of which cause the abnormal accumulation of the A ⁇ peptide. The latter, which are very heterogeneous, have in particular been associated with various mutations on chromosomes 1,14 and 21. The latter has all the more aroused the interest that it carries the gene coding for the precursor protein of A ⁇ . We therefore understand the early onset (55 years) of Alzheimer's disease in subjects with Down Syndrome (Down's syndrome).
- the human A ⁇ peptide is generated by proteolytic cleavages of its precursor (APP) at the Met 596 -Asp 597 and Val 636 - Ile 637 sites.
- the released molecule consists of 39 (to 42) amino acids, the protein sequence of which is as follows:
- the A ⁇ peptide is a natural product secreted by cells and detectable in the blood and cerebrospinal fluid. Although this peptide is neurotoxic, its production is however not sufficient for the formation of amyloid plaques. A altered "processing" or an overexpression of its precursor would predispose to the deposition of A ⁇ in the brain.
- the primary transcript of the ⁇ -amyloid peptide (APP) precursor undergoes alternative splicing to generate mRNAs encoding at least 5 isoforms of 563, 695, 714, 751 and 770 amino acids (aa), expressed ubiquitously in the tissues and the rate of which differs depending on the cell type.
- APP ⁇ -amyloid peptide
- the APP695 and 751 isoforms are however restricted exclusively to the central and peripheral nervous system (in particular at the level of synapses in astrocytes and neurons) where they can play a role in the physiological activity of synapses.
- the isoforms APP751, APP563 and APP770 contain a 56 a "insert" homologous to the protease inhibitor of the "Kunitz” type.
- the secreted form of 1 ⁇ PP751 is identical to nexin IL, a protease inhibitor involved in the regulation of extracellullary serine proteases.
- APP is a glycoprotein of around 120 kDa with the characteristics of an IL-type surface receptor. Although the actual function of APP has not yet been elucidated, studies have shown that this glycoprotein could play a role. role in the regulation of cell growth as well as in adhesion interactions in inflammation, regeneration and immune response.
- APP isoforms are inserted into the endoplasmic reticulum thanks to their "signal" sequence.
- the precursor is then targeted to the Golgi apparatus where it undergoes various post-translational modifications to be then anchored in the membrane.
- the APP Under the action of different proteases, the APP can then undergo various cleavages (see Figure 1), some of which are predominant: The protease activity, called ⁇ -secretase, cleaves within the sequence
- Protease activity called ⁇ -secretase. cleaves the peptide bond of the 96 5 97 doublet Met -Asp within the precursor to release an APP NH fragment - secreted terminal (designated sAPP ⁇ : soluble APP ⁇ ) completely deleted from A ⁇ .
- the 3rd protease activity could also act between residues Val 6 to Ile 637 of the precursor to generate a secreted APP ⁇ proform containing the ⁇ A.
- a ⁇ ⁇ -amyloid peptide
- proteases from humans, rats and monkeys have been studied by various authors and are believed to be involved in the maturation of the APP precursor.
- these enzymes mention may be made most particularly of serine proteases 1 and 2 (Abraham et al. (1991), Biochem. Biophys. Res. Commun., 174, 790-796; Matsumoto et al. (1994), Biochemistry, 33, 3941-3948; Matsumoto et al. (1994), Neurosciences Letters, 195, 171-174) as well as G-like Cathepsin (Razzaboni et al. (1992), Brain Research, 589, 207-216).
- the present invention therefore results from the identification and characterization by the applicant of polypeptides having a catalytic activity vis-à-vis the precursor of the ⁇ -amyloid peptide (APP) of the ⁇ -secretase type. Unlike the other proteases identified, the polypeptides of the present invention have a specificity of action towards the natural form of APP.
- the present invention derives in particular from the discovery of a 70 kDa polypeptide capable of cleaving the non-mutated forms of APP.
- a first object of the invention therefore relates to polypeptides or their variants having a ⁇ -secretase type activity characterized in that they are capable of specifically cleaving the natural precursor of the ⁇ -amyloid peptide (APP).
- APP ⁇ -amyloid peptide
- variant designates any molecule having the same activity as the polypeptides of the invention, obtained by modification of a genetic and / or chemical nature of the peptide sequence.
- modification of a genetic and / or chemical nature one can hear any mutation, substitution, deletion, addition, and / or modification of one or more residues.
- Such variants can be generated for different purposes, such as that of improving its production levels, that of increasing its resistance to proteases, that of increasing and / or modifying its activity, or that of imparting new ones to it.
- biological properties may, for example, be made of chimeric polypeptides comprising an additional heterologous part linked to the end.
- the term variant also includes polypeptides homologous to the polypeptides described in the present invention, derived from other cellular sources and in particular from cells of other organisms.
- the substrate which cleaves the polypeptides of the invention does not have a mutation in its peptide sequence and in particular the precursor of the ⁇ -amyloid peptide does not carry the Swedish double mutation.
- the polypeptides of the invention or their variants are capable of selectively cleaving the peptide bond of the Met 596 -Asp 597 doublet within the native or natural form of APP.
- polypeptides according to the invention have been purified from human cells of subjects not suffering from Alzheimer's disease and are capable of exclusively cleaving APP in its natural form at the peptide bond of the Met 596 -Asp 597 doublet. .
- the polypeptides of the invention are characterized in that their activity is not dependent on a second substrate and / or on a ligand.
- ions and more particularly cations such as magnesium or calcium cations.
- other proteins such as serine proteases 1 and 2 or Cathepsin G-like having protease activity, require the presence of calcium to be active.
- the polypeptides according to the invention have a molecular mass of between 65 and 75 kDa and preferably their molecular mass is around 70 kDa.
- Their isoelectric point is between 6.0 and 7.0 and preferably is equal to 6.0.
- polypeptides are endopeptidases of the serine protease family.
- these endopeptidases are of the sensitive chymotrypsin type.
- the inhibition profile shows that these endopeptidases are totally inhibited by PMSF (Phenylmethane-sulfonil fluoride) and partially inhibited by pefablock, TPCK (Ll-Chloro-3- [4tosylamido] -4-phenyl-2- butanone), benzamidine.
- PMSF Phhenylmethane-sulfonil fluoride
- pefablock Phenylmethane-sulfonil fluoride
- TPCK Ll-Chloro-3- [4tosylamido] -4-phenyl-2- butanone
- benzamidine are completely resistant to inhibition by antipapain.
- polypeptides of the invention or their variants are characterized by a maximum ⁇ -secretase activity at a pH of between 7 and 8.
- a subject of the invention is also non-peptide or non-exclusively peptide compounds capable of cleaving at the Met 5 -Asp 5 7 site the precursor of the ⁇ -amyloid peptide.
- Such compounds are obtained by reproduction of the active motifs of the polypeptide according to the invention by non-peptide or non-exclusively peptide structures and which are compatible with pharmaceutical use.
- the invention relates to the use of polypeptides as described above for the preparation of non-peptide, or not exclusively peptide, pharmacologically active molecules, by determining the structural elements of these polypeptides which are important for their activity and the reproduction of these elements by non-peptide or non-exclusively peptide structures.
- the invention also relates to pharmaceutical compositions comprising one or more molecules thus prepared.
- the polypeptides or their variants further comprise a signal sequence allowing precise cell localization.
- a signal sequence allowing precise cell localization.
- sequences which can be used mention may be made preferably of the sequence of the signal peptide of IgkB, the signal peptide of APP, the signal peptides of nicotinic muscle and central acetylcholine receptor subunits etc.
- Another object of the invention consists in a method of enzymatic purification of the polypeptides of the invention, having an activity of ⁇ -secretase type and capable of specifically cleaving the natural precursor of APP. This process includes the following steps:
- the concentration product then undergoes the various stages of purification with in particular centrifugation on a tangential membrane followed by exclusion chromatography and ion exchange chromatography then by hydrophobic interaction chromatography and finally by a new exclusion chromatography.
- the present invention also relates to the use of a cell line.
- This line was selected from many other human lines (see Materials and Methods) coming from various origins but from subjects not affected by Alzheimer's disease. These lines were used to search for the polypeptides of the invention or their variants.
- these human cell lines are representative of the Central or Peripheral Nervous System and of the Immune System and are capable of carrying out the normal metabolism of the precursor of the amyloid ⁇ peptide leading to its production.
- the cell line selected is the THP1 line (ATCC TIB 202) derived from a monocyte.
- the cell lines described above are used in particular as a host for the detection of compounds (ligands, antagonists, agonists) capable of inhibiting the interaction between the polypeptides of the invention and their substrate.
- Another object of the invention resides in antibodies or fragment of polyclonal or monoclonal antibodies directed against a polypeptide as defined above.
- Such antibodies can be generated by methods known to those skilled in the art.
- these antibodies can be prepared by immunizing an animal against a polypeptide of the invention or one of its variants, then by drawing blood and isolating the antibodies.
- These antibodies can also be generated by preparing hybridomas according to techniques known to those skilled in the art.
- the antibodies or antibody fragments according to the invention can in particular be used for their ability to inhibit, at least in part, the interaction between said polypeptide and the precursor of the ⁇ -amyloid peptide and / or to inhibit at least in part the ⁇ -secretase activity of the polypeptides of the invention vis-à-vis the natural precursor of the ⁇ -amyloid peptide.
- these antibodies are used as a medicament, in particular for the treatment of neurodegenerative diseases such as Alzheimer's disease.
- Another object of the present invention relates to a method for identifying compounds capable of inhibiting, at least in part, the interaction of the polypeptide and the precursor of the ⁇ -amyloid peptide and / or of modulating or inhibiting at least in part of the ⁇ -secretase activity of the polypeptides of the invention.
- a molecule or a mixture containing different molecules, possibly unidentified, is brought into contact with a recombinant cell such as expressing a polypeptide of the invention under conditions allowing the interaction between said polypeptide and said molecule in the case where that -this would have an affinity for said polypeptide, and,
- this method of the invention is suitable for the detection and / or isolation of agonists and antagonists of the ⁇ -secretase activity of the polypeptides of the invention. From these agonist or antagonist molecules, it is possible by conventional techniques known to those skilled in the art and in particular by sequencing to obtain their corresponding nucleotide sequences.
- agonist or antagonist molecules of the polypeptides of the invention expressed in situ from their nucleotide sequences.
- the preparation of these molecules and their expression in vivo, ex-vivo and / or in vitro, require that their nucleotide sequences are carried by a viral or plasmid vector and are transfected by means of said vector into appropriate host cells.
- the present invention also relates to the use of the polypeptides defined above or of their variants for the detection of ligands as well as of compounds capable of inhibiting, at least in part, the interaction between the polypeptide and the precursor of the ⁇ - peptide.
- the present invention also relates to a method for demonstrating molecules which can influence the activity of the polypeptides of the invention.
- This screening method involves the following steps:
- polypeptides of the invention which have a ⁇ -secretase type activity are brought into contact with a molecule or a mixture containing different molecules, possibly not identified.
- the reaction mixture described in the previous step is brought into contact with the substrate of the polypeptides of the invention which is preferably APP in its natural form - the ⁇ -secretase activity is measured on the APP
- the molecules which have had an effect on the ⁇ -secretase activity of the polypeptides of the invention are detected and / or isolated.
- Another object of the invention relates to the use of a ligand or of a modulator identified and / or obtained according to the process described above as a medicament.
- ligands or modulators by their capacity to interfere at the level of the ⁇ -secretase activity of the polypeptides of the invention vis-à-vis the natural precursor of the ⁇ -amyloid peptide may, in fact, make it possible to treat certain neurological conditions and in particular Alzheimer's disease.
- the subject of the invention is also any pharmaceutical composition comprising as active ingredient either a polypeptide as defined above or the agonist, antagonist or ligand molecules defined above.
- composition comprising as active ingredient at least one antibody or an antibody fragment as defined above, and / or an antisense oligonucleotide.
- compositions in which the peptides, antibodies, ligands and / or nucleotide sequences defined above are associated with each other or with other active ingredients.
- compositions according to the invention can be used to at least partially inhibit the interaction of the polypeptides of the invention or their variants with the natural precursor of the ⁇ -amyloid peptide and / or to at least partially inhibit the activity ⁇ -secretase and / or intervene on the metabolism of the precursor of the ⁇ -amyloid peptide to inhibit or slow down the production of the ⁇ -amyloid peptide. It is more preferably pharmaceutical compositions intended for the treatment of neurodegenerative diseases such as for example Alzheimer's disease.
- Another object of the present invention is the use of the molecules described above (ligands, antibodies or antibody fragments, antagonists, agonists) to at least partially inhibit the interaction of the polypeptides of the invention or their variants and the natural precursor of the ⁇ -amyloid peptide and / or to inhibit, at least in part, the ⁇ -secretase activity of the polypeptides of the invention or their variants and / or intervene in the metabolism of the precursor of the ⁇ -amyloid peptide to inhibit or slow the production of the ⁇ -amyloid peptide.
- the use of these molecules is envisaged as a medicament, in particular for the treatment of neurodegenerative diseases and in particular for the treatment of Alzheimer's disease.
- the polypeptides of the invention or their variants are used to intervene in the metabolism of the ⁇ -amyloid peptide.
- the polypeptides or their variants defined above are used to demonstrate ligands or compounds capable of at least partially inhibiting the interaction between the polypeptides of the invention or their variants and the natural precursor of the ⁇ -amyloid peptide and / or to inhibit, at least in part, the ⁇ -secretase activity of the polypeptides of the invention or their variants and / or intervene in the metabolism of the precursor of the ⁇ peptide - amyloid to inhibit or slow down the production of the ⁇ -amyloid peptide.
- polypeptides of the invention and especially their antagonists, agonists, antibodies and ligands are preferably associated with one or more pharmaceutically acceptable vehicles to be formulated for administration by topical, oral, parenteral route, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. They are preferably used in an injectable form.
- saline solutions monosodium phosphate, disodium, sodium chloride, potassium, calcium or magnesium, etc., or mixtures of such salts
- sterile, isotonic, or dry compositions in particular lyophilized, which, through addition according to the case of sterilized water or physiological saline, allow the constitution of injectable solutes.
- Figure 1 APP topography and cleavage sites.
- FIG. 1 description of the process for purifying the polypeptides of the invention.
- Figure 3 analysis by immunoblotting of the cleavage by the polypeptides of the invention of complete precursors of membrane origin of the ⁇ -amyloid peptide "normal” (APP-K 595 M 596 ) and “double mutated” (APP-N 595 L 596 ). Demonstration of the specificity of cleavage of the polypeptides of the invention towards the precursor of membrane origin of the "normal” ⁇ -amyloid peptide (APP-KM).
- column 1 represents the membranes not incubated without enzyme
- column 2 represents the membranes incubated at 37 ° C without enzyme
- column 3 corresponds to the membranes incubated at 37 ° C with the polypeptides of the invention having a ⁇ -secretase activity.
- the cells After thawing, the cells, depending on their origin, are cultured either in “DMEM” medium or in “RPMI 1640” medium in the presence of 10% fetal calf serum. These cultures were carried out in 1 liter flasks at 37 ° C. with a renewal of the culture media every 2 to 3 days. Depending on the cell line studied, a period of 2 to 5 months is necessary to obtain a volume of 18 liters of culture medium. The last culture step is done for 48 hours in the absence of fetal calf serum and phenol red. These cell cultures are then centrifuged to recover the supernatant which will be used for the purification of the enzymes.
- the HMCB, U-373 MG, U-138 MG, MRC5 and Hs27 cell lines were cultured in “DMEM” medium while the lines SW 1088, SW 1783, K-562, H9, DAKIKI, THP1, RPMI 1788 and IM-9 were in the “RPMI 1640” environment.
- Enzyme purification The 18 liters of supernatant from each cell culture are concentrated on ULTRASETTE TM membranes (FILTRON) having a cut-off threshold of lOkDa, then the concentration product obtained was used for the purification of the proteolytic activities according to the following protocol: -
- the first step consists in a tangential membrane centrifugation at 7000 rpm. More particularly, the concentration is carried out on an ULTRAFREE® membrane (MILLIPORE) having a cut-off threshold of lOkDa
- an exclusion chromatography is carried out.
- the exclusion chromatography was carried out on a Sephacryl column S-100 (Pharmacia), the exclusion limits of which are 10 3 Da and 10 5 Da.
- - Ion exchange chromatography represents the third step of the process. It was used in particular a Q-Sepharose column (Pharmacia) whose gel consists of strong anions. The column is eluted by a salt gradient from 0 to 1 M using solvent A (25 mM Tris, pH 7.5) and solvent B (25 mM Tris, 1 M NaCl, pH 7.5).
- the penultimate step consists of chromatography of hydrophobic interactions, in particular on a phenyl-sepharose-6 column (Pharmacia) having a high degree of substitution (40 ⁇ mol / g of gel).
- This column was eluted with a 1 to 0 M ammonium sulfate gradient using solvent A (25 mM Tris, (NH 4 ) 1 M SO 4 , pH 7.5) and solvent B (25 mM Tris, pH 7.5) .
- the last step is an exclusion chromatography, carried out in particular on a TSKgel G2000SW column (Interchim), the gel of which consists of rigid supports of silica, grafted with a hydrophilic group.
- the eluent is a 25 mM Tris buffer, pH 7.5 containing 250 mM NaCl.
- the enzymatic activity is determined by measuring the fluorescence of the free AMC chromophore at 460 nm.
- the reaction is stopped by adding 3 ⁇ l of 0.1N HCl, and the enzymatic activity is determined by measuring the optical density at 215 nm of the cleavage fragments previously separated by HPLC.
- the cleavage sites are deduced by determining the sequence of the fragments resulting from the cleavage.
- the percentage inhibition [% 100 (Io-Ij) / Io] of each substrate incubated in the presence of the enzyme was evaluated by measuring the absorbance at 215 nm for a peptide substrate or the fluorescence at 460 nm for the substrate Z-Val-Lys-Met-MCA in the absence (Io) and in the presence (L) of the inhibitor under the same experimental conditions.
- the precursors normal APP (APP-K 595 M 596 ) and APP having the double "Swedish” mutation (APP-N 595 L 596 ) were obtained from membrane extracts of cells of insects infected with baculovirus containing the genes humans coding for these precursors. These membrane extracts, incubated with the polypeptides of the invention having purified ⁇ -secretase activity, are analyzed by immunoblot using the antibody WO-2 (Ida N. et al. (1996) J. Biol Chem 271, 22908- 22914) directed against the first amino acids of the amyloid ⁇ peptide and the monoclonal antibody 22C11 (Boehringer Mannhein; Hilbich C. (1993) Journal of Biochemical Chemistry, 268, 26571-26577) directed against the NH2-terminal motif of the precursor.
- WO-2 Ida N. et al. (1996) J. Biol Chem 271, 22908- 22914
- the purpose of this example is to demonstrate enzymatic activities in human cell lines of subjects not affected by Alzheimer's disease.
- the search for enzymatic activities was carried out using the 22C11 monoclonal antibody to select the cell lines having the capacity to produce measurable amounts of APP at the level of the membrane and in the cell culture medium.
- the monoclonal antibody WO-2 was used to reveal and identify the different APP cleavage sites.
- the 22C11 monoclonal antibody made it possible to select 8 cell lines (HMCB, MRC5, Hs27, SW1088, SW1783, H9, THP1 and IM-9) out of the 13 tested in total.
- the immunoblot analysis also showed a difference in molecular mass between the membrane APP (120 kDa) and the soluble APP (110-100 kDa). This indicates that the precursor has undergone, at its COOH-terminal sequence, one or more enzymatic cleavage (s).
- this approach made it possible to select cell lines having the capacity to produce measurable amounts of APP at the membrane level and in the cell culture medium and to show enzymatic cleavages in the precursor of the A ⁇ peptide.
- a combined analysis (HPLC, amino acid composition and determination of the sequences) of the fragments generated by cleavage of the substrate [KMD] APP (-5, + 5) was carried out in the selected lines. Indeed, after incubation of the substrate [KMD] APP (-5, + 5) with the supernatants, the fragments generated were first separated by HPLC on a reverse phase RPCig column (VYDAC) eluted by a guard of 5-40 % acetonetrile / 0.05% TFA. The sequence and / or the amino acid composition of these fragments have been determined by conventional techniques.
- the purpose of this example is to describe the purification and to highlight the characteristics of the polypeptides of the invention having a ⁇ -secretase activity.
- the THP-1 cell line was chosen, because of its rapid cell cycle making it possible to obtain large quantities of proteins, to be used as a model for the purification of the ⁇ -secretase activity sought after according to the purification protocol described in "Materials and Methods".
- [KMD] APP (-5, + 5) made it possible to obtain a single fraction having a ⁇ -secretase activity. Its characterization was carried out by measuring the molecular weight by electrophoresis on polyacrilamide gel, measuring the isoelectric point, finding the maximum activity as a function of pH as well as the inhibition profile by conventional inhibitors ("Materials and Methods").
- the electrophoresis analysis was carried out on a 4- 20% polyacrylamide gel on Phast-system (Pharmacia) under denaturing or normal conditions and shows a band of molecular mass close to 70 kDa.
- the inhibition profile of this fraction shows that it is a serine protease: the percentages of inhibition calculated being respectively 100% for PMSF, 75% for pefablock, 25% for TPCK, 10% o for benzamidine and 0% for antipapain.
- This example therefore describes the purification process as well as the research and the demonstration of the various characteristics of the polypeptides of the invention having a ⁇ -secretase activity.
- Example 3 Specificity of the ⁇ -secretase activity This example describes the analysis of the ⁇ -secretase activity of the polypeptides of the invention.
- polypeptides of the invention were contacted with the various substrates and the percentage of cleavage of these substrates was calculated. The results are presented in Table 3.
- the residue of the P'i subsite is necessarily Asp or Glu (Part D Tab.3): In fact, the results have shown that the mutation of Asp by Asn or Gln does not eliminate the cleavage of the substrate; moreover, the cleavage occurs at the Ala-Glu site equivalent to the Met-Asp site; in addition, the Ala-Glu pseudo site is only accessible in the natural substrate because, under the same experimental conditions, the rate of cleavage of the APP fragment (1.5) is only 35%.
- this example demonstrates that the polypeptides of the invention isolated previously have a ⁇ -secretase type activity specific for the natural precursor of the amyloid ⁇ peptide.
- Table 1 table of peptides of different sizes mimicking or reproducing the ⁇ -secretase cleavage site, synthesized for use as a substrate in the characterization and enzymatic specificity of the polypeptide according to the invention.
- Table 2 Inhibition profile of the enzymatic activities of the 8 selected cell lines with respect to the Z-Val-Lys-Met-MCA peptide, expressed as a percentage of activity.
- Table 3 results of the analysis of the enzymatic specificity of the polypeptide of the invention using peptides mimicking or reproducing the amino acid sequence of the APP precursor, at the cleavage site.
Abstract
Description
Claims
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
HU0104022A HUP0104022A2 (hu) | 1998-06-05 | 1999-06-04 | Béta-szekretáz típusú aktivitással rendelkező polipeptidek |
AU40455/99A AU4045599A (en) | 1998-06-05 | 1999-06-04 | Polypeptides with beta-secretase type activity |
CA002330242A CA2330242A1 (fr) | 1998-06-05 | 1999-06-04 | Polypeptides possedant une activite de type beta-secretase |
JP2000553577A JP2002517239A (ja) | 1998-06-05 | 1999-06-04 | ベータセクレターゼ型活性をもつポリペプチド |
EP99923674A EP1084240A1 (fr) | 1998-06-05 | 1999-06-04 | Polypeptides possedant une activite de type beta-secretase |
KR1020007013630A KR20010052499A (ko) | 1998-06-05 | 1999-06-04 | 베타-시크리타제형 활성을 지닌 폴리펩타이드 |
IL13973999A IL139739A0 (en) | 1998-06-05 | 1999-06-04 | Polypeptides with beta-secretase type activity |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
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FR98/07068 | 1998-06-05 | ||
FR9807068A FR2779444A1 (fr) | 1998-06-05 | 1998-06-05 | Polypeptides possedant une activite de type beta-secretase et capables de cliver le precurseur naturel du peptide beta-amyloide (app) |
US12259999P | 1999-03-03 | 1999-03-03 | |
US60/122,599 | 1999-03-03 |
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WO1999064587A1 true WO1999064587A1 (fr) | 1999-12-16 |
WO1999064587A8 WO1999064587A8 (fr) | 2001-08-02 |
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PCT/FR1999/001326 WO1999064587A1 (fr) | 1998-06-05 | 1999-06-04 | Polypeptides possedant une activite de type beta-secretase |
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Country | Link |
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EP (1) | EP1084240A1 (fr) |
KR (1) | KR20010052499A (fr) |
AU (1) | AU4045599A (fr) |
CA (1) | CA2330242A1 (fr) |
HU (1) | HUP0104022A2 (fr) |
IL (1) | IL139739A0 (fr) |
WO (1) | WO1999064587A1 (fr) |
Cited By (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000058479A1 (fr) * | 1999-03-26 | 2000-10-05 | Amgen Inc. | Genes et polypeptides beta secretase |
WO2002006306A2 (fr) * | 2000-07-19 | 2002-01-24 | Pharmacia & Upjohn Company | SUBSTRATS ET DOSAGES PERMETTANT DE SURVEILLER L'ACTIVITE DE LA β-SECRETASE |
US6420534B1 (en) | 1998-09-24 | 2002-07-16 | Pharmacia & Upjohn Company | Alzheimer's disease secretase, APP substrates therefor, and uses thereof |
US6545127B1 (en) | 1999-06-28 | 2003-04-08 | Oklahoma Medical Research Foundation | Catalytically active recombinant memapsin and methods of use thereof |
US6627739B1 (en) | 1999-02-10 | 2003-09-30 | Elan Pharmaceuticals, Inc. | β-secretase enzyme compositions and methods |
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US7456007B1 (en) | 1998-12-31 | 2008-11-25 | Elan Pharmaceuticals, Inc. | β-secretase enzyme compositions and methods |
US7514408B1 (en) | 1999-12-02 | 2009-04-07 | Elan Pharmaceuticals, Inc. | β-secretase enzyme compositions and methods |
JP2011236246A (ja) * | 2000-08-18 | 2011-11-24 | Shire Human Genetic Therapies Inc | 高マンノースタンパク質および高マンノースタンパク質の製造方法 |
US9453847B2 (en) | 2010-07-19 | 2016-09-27 | Shire Human Genetic Therapies, Inc. | Mannose receptor C type 1 (MRC1) codon optimized cell line and uses thereof |
US9623090B2 (en) | 2012-03-02 | 2017-04-18 | Shire Human Genetic Therapies, Inc. | Compositions and methods for treating type III gaucher disease |
US9631196B2 (en) | 2011-06-10 | 2017-04-25 | Temasek Life Sciences Laboratory Limited | Genetic manipulation and expression systems for Pucciniomycotina and Ustilaginomycotina subphyla |
US9694057B2 (en) | 2006-02-07 | 2017-07-04 | Shire Huma Genetic Therapies, Inc. | Stabilized compositions of proteins having a free thiol moiety |
US11571466B2 (en) | 2009-07-28 | 2023-02-07 | Takeda Pharmaceutical Company Limited | Compositions and methods for treating Gaucher disease |
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Also Published As
Publication number | Publication date |
---|---|
WO1999064587A8 (fr) | 2001-08-02 |
EP1084240A1 (fr) | 2001-03-21 |
AU4045599A (en) | 1999-12-30 |
IL139739A0 (en) | 2002-02-10 |
KR20010052499A (ko) | 2001-06-25 |
CA2330242A1 (fr) | 1999-12-16 |
HUP0104022A2 (hu) | 2002-03-28 |
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