WO1999021995A1 - Traitement et prevention de maladies neuro-degeneratives a l'aide de modulateurs de l'interaction d'app et d'un polypeptide definissant un domaine pi - Google Patents

Traitement et prevention de maladies neuro-degeneratives a l'aide de modulateurs de l'interaction d'app et d'un polypeptide definissant un domaine pi Download PDF

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Publication number
WO1999021995A1
WO1999021995A1 PCT/US1998/022523 US9822523W WO9921995A1 WO 1999021995 A1 WO1999021995 A1 WO 1999021995A1 US 9822523 W US9822523 W US 9822523W WO 9921995 A1 WO9921995 A1 WO 9921995A1
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fragments
amino acid
conserved variants
seq
acid sequence
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PCT/US1998/022523
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English (en)
Inventor
Shasta Sabo
Joseph Buxbaum
Paul Greengard
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The Rockefeller University
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Priority claimed from US08/957,660 external-priority patent/US5928882A/en
Application filed by The Rockefeller University filed Critical The Rockefeller University
Priority to AU11979/99A priority Critical patent/AU1197999A/en
Publication of WO1999021995A1 publication Critical patent/WO1999021995A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Amyloid plaques are a major pathological hallmark of Alzheimer's disease.
  • the principle component of amyloid plaques, A ⁇ , 1 is derived by proteolytic processing of the Alzheimer amyloid protein precursor (APP) 2,3 .
  • APP is a Type I integral membrane protein expressed as three major alternatively spliced isoforms of 695, 751 and 770 amino acids in length. The 695 amino acid isoform is most abundant in neurons.
  • APP is processed in two pathways by at least three unidentified proteases known as the ⁇ - , ⁇ - and ⁇ -secretases. 4'6 A ⁇ is generated from cleavage by both ⁇ - and ⁇ -secretase.
  • ⁇ - secretase cleaves APP within the A ⁇ domain, precluding A ⁇ formation.
  • the majority of processed APP is cleaved by -secretase, releasing the large extracellular domain referred to as APPs.
  • Both APPs and A ⁇ are secreted by normal cells. 4"8 However, the cells in Alzheimer patients abnormally allow the aggregation and accumulation of A ⁇ , and thus facilitate the formation of the amyloid plaques.
  • the present invention relates to applicants' discovery that a polypeptide defining a Phosphotyrosine Interaction (PI) domain, conserved variants thereof, or fragments thereof, interacts with the cytoplasmic domain of APP, and that this binding regulates secretion of APP fragments.
  • PI Phosphotyrosine Interaction
  • the present invention extends to a cell line for screening agents capable of modulating the interaction of a polypeptide defining a PI domain, conserved variants thereof, or fragments thereof with APP, wherein said cell line over-expresses both APP and a polypeptide defining a PI domain, conserved variants thereof, or fragments thereof.
  • a cell line of the present invention can be derived from Madin-Darby canine kidney (MDCK) cells.
  • MDCK Madin-Darby canine kidney
  • cell lines of the present invention can also be derived from other mammalian cell lines presently known, or hereinafter discovered.
  • the cell line over expresses both APP and rat Fe65 (SEQ ID NO: 7), conserved variants thereof, or fragments thereof, as an example of a polypeptide defining a Pl-domain.
  • the present invention further extends to a method for screening for agents capable of modulating the interaction of a polypeptide defining a Pl-domain, conserved variants thereof, or fragments thereof, and the cytoplasmic domain of amyloid precursor protein (APP), wherein the method comprises:
  • step (d) contacting the mammalian cells at the start, or during step (c) with an agent suspected of being capable of modulating the interaction between the optionally detectably labeled polypeptide defining a PI domain, conserved variants thereof or fragments thereof, and the cytoplasmic domain of APP;
  • step (g) comparing the measurements of step (f) with control cells not treated with the suspected agent.
  • Any polypeptide defining a PI domain, conserved variants thereof, or fragments thereof, has applications in the present invention.
  • the PI was initially described by Bork, P. And Margolis, B. , Cell 80:693-694 (March, 1995), which is incorporated by reference herein in its entirety.
  • the domain is characterized by: having at least 150 amino acid residues; having a putative secondary structure generally comprising seven ⁇ -sheets and, in most PI domains, at least two ⁇ -helices in the order cd ' (when present), ⁇ l, ⁇ l, ⁇ 2- 7, ⁇ 2; and having a putative globular tertiary structure composed of a ⁇ -sandwich with two nearly orthogonal antiparallel ⁇ -sheets and at least two ⁇ -helices.
  • polypeptides defimng a Pl-domain, conserved variants thereof, or fragments thereof which have applications in the present invention include, but are not limited to: human She comprising an amino acid sequence of Figure 3 (SEQ ID NO:l), conserved variants thereof or fragments thereof; murine She comprising an amino acid sequence of Figure 4 (SEQ ID NO: 2) conserved variants thereof or fragments thereof; human Sck comprising an amino acid sequence of Figure 5 (SEQ ID NO: 3) conserved variants thereof or fragments thereof; murine xl 1 comprising an amino acid sequence of Figure 6 (SEQ ID NO:4) conserved variants thereof or fragments thereof; human xl 1 comprising an amino acid sequence of Figure 7 (SEQ ID NO:5) conserved variants thereof or fragments thereof;
  • Numb comprising an amino acid sequence of Figure 8 (SEQ ID NO:6), conserved variants thereof, or fragments thereof;
  • rat Fe65 comprising an amino acid sequence of Figure 9 (SEQ ID NO:7), conserved variants thereof, or fragments thereof;
  • Disabled comprising an amino acid sequence of Figure 10 (SEQ ID NO:8), conserved variants thereof or fragments thereof human DOC-2 comprising an amino acid sequence of Figure 11 (SEQ ID NO:9), conserved variants thereof, or fragments thereof; murine p96 comprising an amino acid sequence of Figure 12 (SEQ ID NO:10), conserved variants thereof or a fragment thereof; human Fe65 comprising an amino acid sequence of Figure 13 (SEQ ID NO:l 1), conserved variants thereof or fragments thereof; human FE65-like protein comprising an amino acid of Figure 14 (SEQ ID NO: 12), conserved variants thereof, or a fragment thereof; rat Fe65L2 protein comprising an amino acid sequence of figure 15 (SEQ ID NO: 13), conserved variants thereof, or fragments thereof; or human IRS-1 protein comprising an amino acid sequence of Figure 16 (SEQ ID NO: 14), conserved variants thereof, or fragments thereof.
  • a polypeptide defining a PI domain, conserved variants thereof or fragments thereof is human Fe65, comprising an amino acid sequence of Figure 14 (SEQ ID NO:12), conserved variants thereof or fragments thereof.
  • the present invention extends to a mammalian cell line which over expresses a polypeptide defining a PI domain, conserved variants thereof or fragments thereof, and APP.
  • the mammalian cell line comprises MDCK cells, which over-express APP and rat Fe65 (SEQ ID NO:7), conserved variants thereof, or fragments thereof.
  • Methods which can be used to measure optionally labeled APP and polypeptide defining a PI domain conserved variants thereof or fragments thereof include, but are not limited to, immunoprecipitation, or immunoassays, such as sandwich ELISA.
  • a further aspect of the present invention involves a therapeutic composition
  • a therapeutic composition comprising an agent which modulates the interaction of a polypeptide defining a PI domain conserved variants thereof or fragments thereof, and the cytoplasmic domain of an amyloid precursor protein (APP), and a pharmaceutically acceptable carrier thereof.
  • APP amyloid precursor protein
  • the present invention further extends to a therapeutic composition as described above, wherein the agent is a non-peptidyl agent.
  • the present invention extends to a method for treating and/or preventing neurodegenerative disease in a mammal in need of such therapy, which comprises administering a therapeutically effective amount of a therapeutic composition comprising an agent capable of modulating the interaction of a polypeptide defining a PI domain, conserved variants thereof or fragments thereof, and the cytoplasmic domain of amyloid precursor protein (APP), and a pharmaceutically acceptable carrier thereof.
  • a therapeutic composition comprising an agent capable of modulating the interaction of a polypeptide defining a PI domain, conserved variants thereof or fragments thereof, and the cytoplasmic domain of amyloid precursor protein (APP), and a pharmaceutically acceptable carrier thereof.
  • the agent can be a non-peptidyl agent.
  • Examples of a neurodegenerative disease which can be treated or prevented with the present invention include, but are not limited to, Alzheimer's disease or dementia.
  • a method for treating and/or preventing neurodegenerative disease, as disclosed herein extends to any agent that modulates the binding of any polypeptide defining a PI domain, conserved variants thereof or fragments thereof, to the cytoplasmic domain of an amyloid precursor protein.
  • the PI domain is characterized by: having at least 150 amino acid residues; having a putative secondary structure generally comprising seven ⁇ -sheets and, in most PI domains, at least two ⁇ -helices in the order ⁇ l ' (when present), ⁇ l, ⁇ l, ⁇ 2- 7, ⁇ 2; and having a putative globular tertiary structure composed of a ⁇ -sandwich with two nearly orthogonal antiparallel ⁇ -sheets and at least two ⁇ -helices.
  • polypeptides defining a PI domain conserved variants thereof, or fragments thereof, comprises: human She comprising an amino acid sequence of Figure 3 (SEQ ID NO:l), conserved variants thereof or fragments thereof; murine She comprising an amino acid sequence of Figure 4 (SEQ ID NO: 2) conserved variants thereof or fragments thereof; human Sck comprising an amino acid sequence of Figure 5 (SEQ ID NO: 3) conserved variants thereof or fragments thereof; murine xl 1 comprising an amino acid sequence of Figure 6 (SEQ ID NO:4) conserved variants thereof or fragments thereof; human xl 1 comprising an amino acid sequence of Figure 7 (SEQ ID NO:5) conserved variants thereof or fragments thereof;
  • Numb comprising an amino acid sequence of Figure 8 (SEQ ID NO:6), conserved variants thereof, or fragments thereof; rat Fe65 comprising an amino acid sequence of Figure 9 (SEQ ID NO:7), conserved variants thereof, or fragments thereof; Disabled comprising an amino acid sequence of Figure 10 (SEQ ID NO:8), conserved variants thereof or fragments thereof human DOC-2 comprising an amino acid sequence of Figure 11 (SEQ ID NO:9), conserved variants thereof, or fragments thereof; murine p96 comprising an amino acid sequence of Figure 12 (SEQ ID NO: 10), conserved variants thereof or a fragment thereof; human Fe65 comprising an amino acid sequence of Figure 13 (SEQ ID NO:l 1), conserved variants thereof or fragments thereof; human FE65-like protein comprising an amino acid of Figure 14 (SEQ ID NO : 12), conserved variants thereof, or a fragment thereof; rat Fe65L2 protein comprising an amino acid sequence of figure 15 (SEQ ID NO: 13), conserved variants thereof, or
  • one object of the present invention is to exploit Applicants' discovery that intervening in the binding of a polypeptide defining a PI domain, conserved variants thereof or fragments thereof with the cytoplasmic domain of APP decreases APP processing, and hence decreases the amount of A ⁇ produced so that the progression and/or onset of Alzheimer's disease can be inhibited or prevented.
  • Another object of the present invention is to provide a reliable method for screening agents which can interfere with the protein-protein interaction of a polypeptide defining a PI domain, conserved variants thereof or fragments thereof and APP.
  • agents have applications in the prevention and/or inhibition of a neurodegenerative disease, such as Alzheimer's Disease or dementia.
  • Yet another object of the present invention is to provide a mammalian cell line which over-expresses APP and a polypeptide defining a PI domain, conserved variants thereof or fragments thereof so as to provide a facile and convenient method for identifying agents which modulate the interaction between APP and a polypeptide defining a PI domain, conserved variants thereof or fragments thereof.
  • Yet still another object is to provide a therapeutic composition for treating and/or preventing Alzheimer's disease, comprising an agent which can modulate the protein- protein interaction of a polypeptide defining a PI domain, conserved variants thereof or fragments thereof, with APP, along with a pharmaceutically acceptable carrier thereof.
  • Still yet another object of the present invention is to provide a method for treating and/or preventing a neurodegenerative disease, such as Alzheimer's Disease, comprising administering a therapeutically effective amount of a therapeutic composition of the present invention.
  • Figure 1 is a graph showing that over-expression of Fe65 causes an increase in secretion of APPs from MDCK cells.
  • cells were pulsed with [ 35 S] -methionine for two hours, followed by a two-hour chase.
  • APPs was immunoprecipitated from the chase medium with the monoclonal antibody, 6E10.
  • Figure 2A is a graph showing that over-expression of Fe65 causes an increase in A ⁇ secretion from MDCK cells.
  • ELISA of conditioned medium from MDCK cells reveals an increase in A ⁇ secretion upon Fe65 expression.
  • cells were incubated in serum-free medium for four hours at 37C. Then, the medium was collected and subjected to a sandwich ELISA for A ⁇ using 6E10 as the capture antibody and 4G8 as the detection antibody.
  • the values obtained are normalized to total cellular holoAPP determined by immunoblotting with 369.
  • Figure 2B is a gel showing the effect of Fe65 on A ⁇ secretion in the MDCK cells was confirmed by immunoprecipitation. These experiments were performed essentially as described above for APPs except A ⁇ was immunoprecipitated with both 6E10 and 4G8. The data shown are from a representative experiment.
  • Figure 3 is the amino acid sequence of human She.
  • Figure 4 is the amino acid sequence of murine She.
  • Figure 5 is the amino acid sequence of human Sck.
  • Figure 6 is the amino acid sequence of murine xll.
  • Figure 7 is the amino acid sequence of human xl l.
  • Figure 8 is the amino acid sequence of Numb.
  • Figure 9 is the amino acid sequence of rat Fe65.
  • Figure 10 is the amino acid sequence of Disabled.
  • Figure 11 is the amino acid sequence of human DOC-2.
  • Figure 12 is the amino acid sequence of murine p96.
  • Figure 13 is the amino acid sequence of human Fe65.
  • Figure 14 is the amino acid sequence of human Fe65-like protein.
  • Figure 15 is the amino acid sequence of rat Fe65L2 protein.
  • Figure 16 is the amino acid sequence of human IRS-1 protein. DETAILED DESCRIPTION OF THE INVENTION
  • phrases “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • terapéuticaally effective amount is used herein to mean an amount sufficient to prevent, and preferably reduced by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, the occurrence of amyloid formation.
  • domain is used herein to describe any amino acid sequence which represents a fragment of a larger polypeptide, which fragment has a characteristic sequence, structure, and/or activity.
  • amino acid residues described herein are preferred to be in the "L" isomeric form.
  • residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property of immunoglobulin-binding is retained by the polypeptide.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.
  • amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino- terminus to carboxy -terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues.
  • the above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein.
  • Mutations can be made in a DNA sequence encoding a polypeptide defining a PI domain such that a particular codon is changed to a codon which codes for a different amino acid. Such a mutation is generally made by making the fewest nucleotide changes possible.
  • a substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping).
  • Such a conservative change generally leads to less change in the structure and function of the resulting protein.
  • a non-conservative change is more likely to alter the structure, activity or function of the resulting protein.
  • the present invention should be considered to include sequences containing conservative changes which do not significantly alter the activity or binding characteristics of the resulting protein.
  • Another grouping may be those amino acids with phenyl groups:
  • Phenylalanine Tryptophan Tyrosine Another grouping may be according to molecular weight (i.e., size of R groups):
  • Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property.
  • a Cys may be introduced a potential site for disulfide bridges with another Cys.
  • a His may be introduced as a particularly "catalytic" site (i.e., His can act as an acid or base and is the most common amino acid in biochemical catalysis).
  • Pro may be introduced because of its particularly planar structure, which induces ⁇ -turns in the protein's structure.
  • Two amino acid sequences are "substantially homologous" when at least about 70% of the amino acid residues (preferably at least about 80% , and most preferably at least about 90 or 95%) are identical, or represent conservative substitutions.
  • the screening assays of the present invention are based on cell lines which over-express both APP and a polypeptide defining a PI domain, conserved variants thereof, or fragments thereof derived from a mammalian cell line. Any mammalian cell line presently known or subsequently discovered can be used as the basis for a cell line of the present invention.
  • a cell line of the present invention is derived from Madin-Darby canine kidney (MDCK) cells.
  • cell lines of the present invention can be used to examine the effects of a polypeptide defining a PI domain, conserved variants thereof or fragments thereof on processing of APP.
  • the mammalian cell line comprises Madin-Darby canine kidney (MDCK) cells stably expressing the 695 amino acid isoform of APP 12 (MDCK-APP) which are transfected with cDNA encoding a polypeptide defining a PI domain, conserved variants thereof, or fragments thereof and selected for puromycin resistance.
  • MDCK Madin-Darby canine kidney
  • MDCK-APP 695 amino acid isoform of APP 12
  • a polypeptide defining a PI domain, conserved variants thereof, or fragments thereof, whose effect on processing of APP was studied in this embodiment of the present invention, and is used in a method to screen for agents that modulate the interaction of a polypeptide defining a PI domain and the APP protein of the present invention is Fe65, a brain enriched protein of unknown function which binds to the NPTY sequence in the cytoplasmic tail of APP.
  • Fe65 contains two types of protein-protein interaction domains, a WW domain, and a PI domain. Binding of Fe65 to APP occurs through the second PI domain of Fe65.
  • the amino acid sequence of Fe65 is set forth in Figure 10 (SEQ ID NO:8).
  • any polypeptide defining a PI domain, conserved variants thereof, or fragments thereof, has applications in the present invention.
  • doubly stable clonal cell lines (MDCK- APP/Fe65) were isolated using cloning rings, and cells expressing high levels of Fe65 were identified by immunoblotting with antibodies raised against the WW domain of human Fe65.
  • a polypeptide defining a PI domain conserved variants thereof or fragments thereof such as Fe65, may either increase the amount of APP transported from the Golgi to the plasma membrane or the amount of APP recycled back to the cell surface after endocytosis.
  • agents or drugs that inhibit the interaction of a polypeptide defining a PI domain can be used to inhibit A ⁇ secretion in the brain, preventing and/or retarding the formation of plaques.
  • agents suspected of having the desired activity can be screened for both effects on the interaction of APP with polypeptide defining a PI domain, such as Fe65, conserved variants thereof or fragments thereof, and effects on the Fe65- dependent increase in secretion of A ⁇ .
  • agents or drugs are typically formulated in compositions for administration to mammalian patients in need of such therapy.
  • unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e. , carrier, or vehicle.
  • the therapeutic compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • the quantity to be administered depends on the subject to be treated, and the severity of the condition under treatment. Precise amount of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosages may range from about 0.1 to 20, preferably about 0.5 to about 10, and more preferably 1 to 5 milligrams of active ingredient per kilogram body weight of individual per day, and depend on the route of administration.
  • the unit dosage form contains from about 0.5 mg to about 750 mg depending on the activity of the particular Fe65 modulator being utilized as the active ingredient.
  • rat Fe65 SEQ ID NO: 7
  • pcDNA3 Invitrogen
  • Rat Fe65 was subcloned into the mammalian expression vector, pcDNA3 (Invitrogen) by PCR using rat Fe65 in pGEX as a template.
  • the 5' PCR primer encoded an amino terminal FLAG epitope tag.
  • MDCK-695 cells 12 were transfected in 10 cm diameter plates using the calcium phosphate transfection system (Gibco) essentially following the manufacturers instructions. Each plate was transfected with 15 g of rat Fe65 in pcDNA3 and 5 ⁇ g of pPUR (Clontech), a selection vector containing a puromycin resistance gene. After selection with 2.5 ⁇ g/ml puromycin (Clontech), individual clones were isolated using cloning rings. Cell lines expressing high levels of rat Fe65 were identified by immunoblotting with polyclonal antibodies raised against a GST- fusion protein of amino acids 201-240 of rat Fe65. These cells are maintained in DMEM containing 200 ⁇ g/ml of G418 (Gibco) and 1 ⁇ g/ml of puromycin.
  • DMEM containing 200 ⁇ g/ml of G418 (Gibco) and 1 ⁇ g/ml of puromycin.
  • Immunoprecipitation was performed essentially as described previously. 13 Briefly, cells were plated at a density of 5 X 10* cells/cm 2 . After washing, they were incubated with [ 35 S]-methionine (NEN) in methionine-free
  • DMEM Dulbeco's Modified Eagle Medium
  • APPs generated by ⁇ -cleavage and A ⁇ were immunoprecipitated from the chase medium with monoclonal antibodies 6E10 and 4G8 and agarose-linked goat anti-mouse IgG (American Qualex). Immunoprecipitates were separated by SDS-PAGE and quantified by Phosphorlmager (Molecular Dynamics). Sister cultures were lysed with 1 % NP40 in phosphate- buffered saline (PBS) immediately after labeling. Values obtained for APPs and A ⁇ were normalized to total labeled cellular holo/APP immunoprecipitated from these lysates with the polyclonal antibody, 369, raised against the cytoplasmic domain of APP. 14
  • ELISA ELISA
  • the cell medium was changed to fresh serum-free DMEM and incubated for four hours at 37 °C. Then the conditioned medium was collected and subjected to sandwich ELISA for A ⁇ using 6E10 as the capture antibody and 4G8 as the detection antibody. A more detailed description of the ELISA can be found elsewhere. 15 Samples were incubated with biotinylated antibody and anti-biotin alkaline phosphatase conjugate. After 1 hour, alkaline phosphatase substrate was added and A ⁇ was quantified using the Dynatech Plate Reader. The values obtained were normalized to total cellular holoAPP determined by immunoblotting with 369 and [ 125 I] -protein A (Amersham) followed by Phosphorlmager quantification.

Abstract

Cette invention concerne des modulateurs qui modifient l'interaction entre un polypeptide définissant un domaine PI et le domaine cytoplasmique d'une protéine précurseur d'amyloïde. Ces modulateurs vont réduire la production globale de bêta amyloïde (Aβ) dans une cellule, et permettent ainsi d'obtenir des agents thérapeutiques que l'on utilise dans le traitement et/ou la prévention de maladies neuro-dégénératives, telles que la maladie d'Alzheimer et la démence.
PCT/US1998/022523 1997-10-24 1998-10-23 Traitement et prevention de maladies neuro-degeneratives a l'aide de modulateurs de l'interaction d'app et d'un polypeptide definissant un domaine pi WO1999021995A1 (fr)

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AU11979/99A AU1197999A (en) 1997-10-24 1998-10-23 Treatment and prevention of neurodegenerative diseases using modulators of the interaction of app and a polypeptide defining a pi domain

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US08/957,660 1997-10-24
US08/957,660 US5928882A (en) 1997-10-24 1997-10-24 Treatment and prevention of neurodegenerative diseases using modulators of Fe65
US6208598A 1998-04-17 1998-04-17
US09/062,085 1998-04-17

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WO2007098417A2 (fr) * 2006-02-21 2007-08-30 Oklahoma Medical Research Foundation TRAITEMENT DE LA MALADIE D'ALZHEIMER AVEC DES INHIBITEURS DE LA FIXATION DE L'ApoE AU RÉCEPTEUR DE L'ApoE

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WO1996017866A1 (fr) * 1994-12-09 1996-06-13 The Regents Of The University Of California Nouveau domaine proteinique fixant des proteines tyrosine-phosphorylees
WO1997012039A2 (fr) * 1995-09-27 1997-04-03 Gerald Krystal Inositol-phosphatase contenant le site sh2
FR2740454A1 (fr) * 1995-10-26 1997-04-30 Rhone Poulenc Rorer Sa Peptides capables d'inhiber l'endocytose de l'app et sequences nucleotidiques correspondantes
WO1997032020A2 (fr) * 1996-02-29 1997-09-04 Mount Sinai Hospital Corporation Proteines shc

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BORK P ET AL: "A phosphotyrosine interaction domain [letter]", CELL, MAR 10 1995, 80 (5) P693-4, UNITED STATES, XP002093478 *
GUENETTE SY ET AL: "Association of a novel human FE65-like protein with the cytoplasmic domain of the beta-amyloid precursor protein.", PROC NATL ACAD SCI U S A, OCT 1 1996, 93 (20) P10832-7, UNITED STATES, XP002093476 *
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ZHANG Z ET AL: "Sequence-specific recognition of the internalization motif of the Alzheimer's amyloid precursor protein by the X11 PTB domain.", EMBO J, OCT 15 1997, 16 (20) P6141-50, ENGLAND, XP002093477 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007098417A2 (fr) * 2006-02-21 2007-08-30 Oklahoma Medical Research Foundation TRAITEMENT DE LA MALADIE D'ALZHEIMER AVEC DES INHIBITEURS DE LA FIXATION DE L'ApoE AU RÉCEPTEUR DE L'ApoE
WO2007098417A3 (fr) * 2006-02-21 2007-10-18 Oklahoma Med Res Found TRAITEMENT DE LA MALADIE D'ALZHEIMER AVEC DES INHIBITEURS DE LA FIXATION DE L'ApoE AU RÉCEPTEUR DE L'ApoE

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