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WO1994009364A1 - Procede permettant d'inhiber la liaison de la proteine precurseur d'amyloide a la proteine beta-amyloide - Google Patents

Procede permettant d'inhiber la liaison de la proteine precurseur d'amyloide a la proteine beta-amyloide Download PDF

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Publication number
WO1994009364A1
WO1994009364A1 PCT/US1993/009772 US9309772W WO9409364A1 WO 1994009364 A1 WO1994009364 A1 WO 1994009364A1 US 9309772 W US9309772 W US 9309772W WO 9409364 A1 WO9409364 A1 WO 9409364A1
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Prior art keywords
amyloid
fragment
protein
binding
amyloid protein
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PCT/US1993/009772
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English (en)
Inventor
Warren J. Strittmatter
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Duke University
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Priority to AU53584/94A priority Critical patent/AU5358494A/en
Publication of WO1994009364A1 publication Critical patent/WO1994009364A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present invention relates to Alzheimer's disease, and particularly relates to senile plague formation in Alzheimer's disease.
  • the senile plague and congophilic angiopathy are abnormal extracellular structures found in abundance in brain of patients with Alzheimer's disease.
  • the biochemical composition of these structures has been extensively studied to better understand their possible role in the pathogenesis of this dementing disease.
  • the mature senile plague is a complex structure, consisting of a central core of amyloid fibrils surrounded by dystrophic neurites, axonal terminals and dendrites, microglia and fibrous astrocytes. See D. Selkoe Neuron 6, 487-498 (1991) .
  • the amyloid core of the senile plague and surrounding the blood vessels, producing the congophilic angiopathy is a peptide of 39 to 43 amino acids termed the 0-Amyloid (BA) peptide.
  • BA 0-Amyloid
  • BA peptide is found in brain in Alzheimer's disease, Down's syndrome, hereditary cerebral hemmorhage of the Dutch type, and in old age.
  • /3A is produced by abnormal proteolytic processing of a larger protein, the amyloid precursor protein (APP) . See K. Beyreuther and C. Masters, Brain Path. 1, 241-251 (1991) .
  • the senile plague and congophilic angiopathy contain proteins in addition to BA peptide.
  • APP itself, among others, has been identified in the senile plague by histochemical studies employing antibodies recognizing either the amino- and carboxy-termini of the precursor protein. See, e. g. , F. Tagliavine et al., Neurosci . Lett. 128, 117-120 (1991); C. Joachim et al., Amer. Jour. Path. 138, 373-384 (1991) ; The mechanisms by which these proteins aggregate in the extracellular space to associate with the senile plague and congophilic angiopathy are not known.
  • a first aspect of the present invention is a construct comprising a 3-amyloid protein or fragment thereof (e.g., a fragment which binds to amyloid precursor protein) immobilized on a solid support.
  • a second aspect of the present invention is a method of detecting compounds which bind to 3-amyloid protein.
  • the method comprises providing a construct as given above, contacting an agueous solution (e.g., cerebrospinal fluid) containing a compound suspected of binding to the 3-amyloid protein to the construct; and detecting the presence or absence of target compound bound to said construct.
  • an agueous solution e.g., cerebrospinal fluid
  • a third aspect of the present invention is a method of detecting compounds which inhibit the binding of amyloid precursor protein to 3-amyloid protein.
  • the method comprises providing an agueous solution containing a binding pair, the binding pair comprising (i) the amyloid precursor protein or a fragment thereof which binds to the 0-amyloid protein, and (ii) the 9-amyloid protein or a fragment thereof which binds to the amyloid precursor protein; adding a test compound to the aqueous solution; and then detecting whether or not the test compound inhibits binding between the members of said binding pair.
  • one member of the binding pair may be immobilized on a solid support.
  • a fourth aspect of the present invention is a method of inhibiting the binding of amyloid precursor protein to the ⁇ -amyloid protein.
  • the method comprises contacting to one member of the amyloid precursor protein ⁇ s-amyloid protein binding pair a fragment of the other member of the binding pair, wherein said fragment binds to said one member, and wherein the fragment is provided in an amount sufficient to inhibit binding of amyloid precursor protein to j8-amyloid protein.
  • the one member is ⁇ -amyloid protein and the fragment is an amyloid precursor protein fragment; in another embodiment, the one member is amyloid precursor protein and the fragment is a 0-amyloid protein fragment.
  • a fifth aspect of the present invention is a method of detecting compounds which inhibit the binding of /S-amyloid protein to Apolipoprotein E4.
  • the method comprises, first, providing an aqueous solution containing a binding pair, the binding pair comprising (i) a first compound selected from the group consisting of Apolipoprotein E4 and fragments thereof which bind to the 3-amyloid protein, and (ii) the 0-amyloid protein or a fragment thereof which binds to Apolipoprotein E4, then, adding a test compound to said aqueous solution, and then, detecting whether or not the test compound inhibits binding between the members of the binding pair.
  • a sixth aspect of the present invention is a method of inhibiting the binding of Apolipoprotein E4 to /3-amyloid protein.
  • the method comprises contacting to one member of the Apolipoprotein E4- / 9-amyloid protein binding pair a fragment of the other member of the binding pair, wherein the fragment binds to the one member, and wherein the fragment is provided in an amount sufficient to inhibit binding of Apolipoprotein E4 to ⁇ -amyloid protein.
  • Figure l illustrates the binding of proteins in human cerebrospinal fluid to BA (1 . 28) peptide (SEQ ID N0:1) or to ethanolamine-immobilized to Immobilon AV membranes.
  • Figure 2 illustrates the binding of proteins in human cerebrospinal fluid proteins to BA (12 . 28) peptide (SEQ ID NO:
  • Figure 3 schematically illustrates the structure of various APP isoforms and APP deletion mutants.
  • Figure 4 illustrates the binding of recombinant- expressed human APP isoforms to BA (1 . 28) peptide or to ethanolamine immobilized to Immobilon AV.
  • Figure 5 illustrates the binding of 770 APP and deletion APP isoforms 770 KX ⁇ and 751 KX ⁇ to BA ⁇ 1 . 28) peptide (BA) and to ethanola ine-control (C) immobilized Immobilon AV membranes.
  • Figure 6 shows the effect of reducing disulfide bonds, and of pH, on the binding of 695-K to BA (1 . 28) peptide.
  • Figure 7 shows the time course of SDS-stable binding of 0A4 peptide by apoE3 and apoE4.
  • One ⁇ l of apoE3 (A) or apoE4 (B) was incubated with 3A4 (1 . 28) (2.5 x 10 _4 M) in a total volume of 20 ⁇ l between 5 minutes and 24 hours at 37°C.
  • the incubation was ended by adding 20 ⁇ l 2X Laemmli buffer (without B-mercaptoethanol) and boiling five minutes. Proteins were electrophoretically separated on a 7.5% polyacrylamide gel, and transferred to Immobilon P membrane.
  • Figure 9 shows the effect of 0 2 and N 2 on the rate of SDS-stable binding of 8A4 by apoE3.
  • Phosphate buffered saline was saturated with 0 2 or N 2 prior to incubating apoE3 with 3A412 (1 . 28) for: Lane 1, 30 min. ; Lane 2, 2 hrs; Lane 3, 4 hrs; Lane 4, 6 hrs. 0A4 peptide was detected with the anti-3A4 peptide antibody.
  • Upper arrow indicates )0A4 (1 . 28) /apoE complex; lower arrow indicates free 9A4.
  • Figure 10 shows SDS-stable binding of various )BA4 peptides to apoE3 and apoE4.
  • ApoE3 and apoE4 were incubated with the indicated concentrations of 0A4 (1 _ 40) (Panel A) . ; 0A4 (1 _ 28) (Panel B) ; or _6A4 (12 . 28) (Panel C) for 5 hrs. The incubation was stopped by the addition of an equal volume of 2X Laemmli buffer (without ⁇ - mercaptoethanol) , and boiled five minutes. 3A4 was detected with the anti-0A4 peptide antibody. Left panels: upper arrows indicate apoE3 dimer//3A4 complex, lower arrows indicate apoE3 monomer//3A4 complex. Right panels: arrows indicate apoEA/ / 8A4 complex.
  • FIG 11 shows the pH dependence of SDS-stable
  • ApoE3 and apoE4 were incubated with 0A4 (1 _ 2g) in citric acid- Na 2 HP0 4 buffer at the indicated pH for 5 hrs.
  • /3A4 peptide was detected with the anti-j8A4 peptide antibody.
  • Figure 12 shows the SDS-stable binding of /8A4 by truncated apoE3.
  • 3A4 (1 . 28) was incubated with truncated apoE3 (1 ⁇ g) for five hrs. and the incubation ended by boiling in Laemmli buffer (without ⁇ -mercaptoethanol) five minutes.
  • Lane 1, apoE3 (1 . 299) (full length);
  • 0A4 peptide was detected with the anti-/3A4 peptide antibody.
  • Amino acid sequences disclosed herein are presented in the amino to carboxy direction, from left to right. The amino and carboxy groups are not presented in the sequence.
  • a first aspect of the instant invention is a construct comprising a ⁇ -amyloid protein or fragment thereof immobilized on a solid support.
  • the fragment is preferably one which binds to amyloid precursor protein binding. Examples of such fragments are the fragments given herein as SEQ ID N0:1 and SEQ ID NO:2, though other fragments, both longer and shorter, may also be employed.
  • the 3-amyloid protein or fragment thereof may be immobilized on the support by any suitable means, either directly or indirectly. Examples include affinity binding and covalent binding, with covalent binding currently preferred. Any suitable solid support may be employed, including, but not limited to, nitrocellulose, silica, and polypropylene.
  • the solid support may be in any suitable configuaration, such as particles, beads, wells, or films.
  • the construct of the present invention may be employed to detect compounds which bind to ⁇ - amyloid protein. Generally this involves contacting an aqueous solution containing a compound suspected of binding to the 9-amyloid protein to the construct, and then detecting the presence or absence of target compound bound to said construct.
  • Any suitable aqueous solution may be employed, such as a biological fluid (blood serum, cerebrospinal fluid, etc.) or an aqueous solution prepared to contain a specific test compound such as a preselected natural or artificial peptide or analog thereof to be tested for the ability to bind 3-amyloid protein. Detecting of the binding event may also be carried out by any suitable means, such as staining, affinity binding, competitive binding assay, etc.
  • the present invention also provides a method of detecting compounds which inhibit the binding of amyloid precursor protein to /3-amyloid protein which involves providing an agueous solution containing a binding pair [the binding pair comprising (i) the amyloid precursor protein or a fragment thereof which binds to the 3-amyloid protein, and (ii) the j8-amyloid protein or a fragment thereof which binds to the amyloid precursor protein] , adding a test compound to the agueous solution; and then detecting whether or not the test compound inhibits binding between the members of said binding pair.
  • This method is useful as an in vitro assay for detecting compounds which may be useful for inhibiting the noted binding event, which is in turn useful in detecting compounds useful for either diagnosing or treating Alzheimer's disease.
  • the detecting step may be carried out by any suitable means, as noted above, and as an aid to detection one member of the binding pair may be immobilized on a solid support.
  • the ⁇ - amyloid 1-28 protein fragment having the sequence given herein as SEQ ID NO:l or the /3-amyloid protein fragment comprises the 3-amyloid protein 12-28 fragment having the sequence given herein as SEQ ID NO:2 may be used in in vitro assays as described, with the fragments optionally bound to a solid support.
  • the solid support thus may, for example, be a construct as discussed above, though other embodiments (e.g., in which APP or a fragment thereof is immobilized on the solid support) are also suitable.
  • the present invention also provides a method of inhibiting the binding of amyloid precursor protein to ⁇ - amyloid protein which comprises contacting to one member of the amyloid precursor protein-3-amyloid protein binding pair a fragment of the other member of said binding pair (also referred to herein as an "active agent") , as noted above.
  • the active agent is the /3-amyloid 1-28 protein fragment having the sequence given herein as SEQ ID NO:l or a fragment thereof which binds to amyloid precursor protein
  • the active agent is the ⁇ -amyloid protein 12-28 fragment having the sequence given herein as SEQ ID NO:2 or a fragment thereof which binds to amyloid precursor protein.
  • the method may be carried out in vitro, as in a diagnostic or screening assay as discussed above, or may be carried out in vivo in a human or animal subject to inhibit plague formation in disorders such as Alzheimer's disease.
  • the present invention also provides a method of detecting compounds which inhibit the binding of 8-amyloid protein to apolipoprotein, including apolipoprotein E3 and apolipoprotein E4.
  • the method comprises, first, providing an aqueous solution containing a binding pair, the binding pair comprising (i) a first compound selected from the group consisting of Apolipoprotein (e.g., ApoE3, ApoE4) and fragments thereof which bind to the / 8-amyloid protein, and (ii) the ⁇ - amyloid protein or a fragment thereof which binds to Apolipoprotein (e.g., ApoE3, ApoE4) , then, adding a test compound to said aqueous solution, and then, detecting whether or not the test compound inhibits binding between the members of the binding pair.
  • Apolipoprotein e.g., ApoE3, ApoE4
  • the present invention also provides a method of inhibiting the binding of apolipoprotein (e.g., ApoE3, ApoE4) to 9-amyloid protein.
  • the method comprises contacting to one member of the apolipoprotein- / S-amyloid protein binding pair a fragment of the other member of the binding pair (also referred to herein as an "active agent") , as also described above.
  • active agent also referred to herein as an "active agent”
  • the method is useful and may be carried out in the same manner as described above (e.g., in vitro as in a diagnostic or screening assay, or in vivo in a human or animal subject to inhibit plaque formation in disorders such as Alzheimer's disease) .
  • Fragments employed in carrying out the present invention are peptides derived from APP, ⁇ h, or Apolipoprotein (e.g., ApoE3, ApoE4) which have N-terminal, C-terminal, or both N-terminal and C-terminal amino acid residues deleted, but retain the biological activity of the parent protein as described herein.
  • active fragments may be prepared by enzymatic digestion of ⁇ h, APP, or ApoE, by direct synthesis, or by genetic engineering procedures.
  • Fragments employed herein include analogs of ⁇ h, APP, ApoE, and the natural sequence active fragments thereof.
  • An "analog" is a chemical compound similar in structure to another which has either a similar or opposite physiological action.
  • Such analogs may be prepared by altering or deleting amino acids.
  • One or more amino acids of a synthetic peptide sequence may be replaced by one or more other amino acids which does not affect the activity of that sequence.
  • Such changes can be guided by known similarities between amino acids in physical features such as charge density, hydrophobicity/hydrophilicity, size and configuration. For example, Thr may be replaced by Ser and vice versa, Asp may be replaced by Glu and vice versa, and Leu may be replaced by lie and vice versa. Further, those skilled in this art will appreciate that minor changes may be made to the naturally occuring amino acids to produce derivatives thereof which retain activity. Fragments of the proteins and peptides described herein include the analogs thereof, as noted above.
  • analogs are those compounds which, while not having amino acid sequences identical to those of the peptides described above, have a similar three-dimensional structure.
  • the interaction between the two binding partners must take place at the surface-accessible sites in a stable three-dimensional molecule.
  • By arranging the critical binding site residues in an appropriate conformation it is possible to synthesize compounds which mimic the essential surface features of the binding partner.
  • a molecule which has a surface region with essentially the same molecular topology to the binding surface of one binding partner will be able to mimic the interaction of that binding partner with its specific binding partner.
  • compositions containing the active agents of the present invention may be prepared in either solid or liquid form.
  • a pharmaceutical carrier according to conventional pharmaceutical compounding techniques, which carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. oral, inhalation or parenteral.
  • any of the usual pharmaceutical media may be employed.
  • suitable carriers and additives include water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like;
  • suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar coated or enteric coated by standard techniques.
  • the carrier will usually comprise sterile water, though other ingredients, for example, for purposes such as aiding solubility or for preservatives, may be included.
  • injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed.
  • Active agents of the present invention may be administered per se or in the form of a pharmaceutically acceptable salt.
  • pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, salicylic, p-toluenesulfonic, tartaric, citric, methanesulphonic, formic, malonic, succinic, naphthalene-2-sulphonic and benzenesulphonic.
  • pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of a carboxylic acid group.
  • BA (1 . 28) peptide (Bachem) (SEQ ID NO:l) or ethanolamine was covalently immobilized to Immobilon AV affinity membrane (Millipore) .
  • the membrane is a chemically activated hydrophilic microporous membrane which covalently immobilizes peptides and proteins through amino and thiol groups.
  • BA peptide was dissolved in distilled water at one mg peptide/100 ⁇ l .
  • Ten microliters (containing one hundred micrograms peptide) were applied to a 13 mm diameter Immobilon disc, and incubated to dryness overnight at room temperature. Peptide was in large excess to the number of functional binding groups on the membrane.
  • Control membranes containing no peptide, were prepared by incubating the membrane in 2.0 M ethanolamine in 1.0 M NaHC0 3 , pH 9.5, to block the reactive groups on the Immobilon AV membrane. Membranes were stored at -20° C in a dessicator. Prior to use the membranes were washed with phosphate buffered saline. EXAMPLE2 Binding of CSF Proteins to Immobilized ⁇ h ( l-28 )
  • the membranes were placed in a Millipore filter holder (Swinnex) and washed with 3.0 ml phosphate-buffered saline (Bracket 1) , and then washed with 700 ⁇ l 10% sodium dodecyl sulfate (SDS) (Bracket 2) , 700 ⁇ l 4 M urea (Bracket 3) , or 700 ⁇ l 6 M guanidine hydrochloride (Bracket 4) .
  • the membranes were removed from the filter holder, cut in half and placed in 150 ⁇ l of Laemmli buffer (2% sodium dodecylsulfate, 5% beta-mercaptoethanol, pH 6.8) and boiled five minutes to solubilize retained proteins.
  • the membrane was next incubated in the Anti-Alzheimer amyloid precursor antibody (mouse monoclonal : clone 22C11) from Boehringer Mannheim (at 1:200 dilution) in Blotto overnight at 4° C, then washed five times in Blotto.
  • the membrane was exposed to secondary antibody conjugated with horseradish peroxidase for one hour at room temperature, then washed seven times in Blotto. Horseradish peroxidase was then visualized with Enhanced Chemiluminesce Detection kit (Amersham) , and exposed to Hyperfilm ECL (Amersham) .
  • APP retained by immobilized BA peptide (1-28) is only partially eluted by 10% SDS ( Figure IB; Bracket 2) and 6 M guanidine hydrochloride (Bracket 4) , and is not eluted by 4 M urea (Bracket 3).
  • Membranes were then washed and prepared identically to those shown in Figure 1: Bracket 1, Phosphate-buffered saline; Bracket 2, 10% SDS; Bracket 3, 4M urea; Bracket 4, 6M guanidine hydrochloride. Proteins were electrophoresed and then visualized by silver staining (Figure 2A) or immunodetection with Anti- Alzheimer precursor protein antibody following Western transfer to Immobilon P ( Figure 2B) . The data collected showed that the shorter BA peptide (amino acids 12-28) given in SEQ ID NO:2 also bound APP.
  • APP Binding of APP to immobilized BA (12 . 28) peptide or to immobilized H.M. control peptide is shown in Figure 2B.
  • APP was retained by both the BA (12 . 28) peptide and the control peptide following wash with phosphate buffered saline (Bracket 1) .
  • 10% SDS eluted little APP from BA ⁇ 12 . 28) peptide, but eluted most of the APP from the control peptide (Bracket 2) .
  • guanidine hydrochloride eluted little APP from BA (12 . 28) peptide, but eluted virtually all detectable APP from the H.M. control peptide (Bracket 4) .
  • FIG. 3 Various APP isoforms and deletion mutants produced in baculovirus-transformed insect cells are schematically illustrated in Figure 3. These cells synthesize and release into the culture media human recombinant APP after proteolytically processing the precursor protein at the same peptide bond hydrolyzed by mammalian cells. See R. Bhasin et al., Proc. Natl . Acad. Sci . USA. 88, 10307-10311 (1991); D. Lowery et al., J. Biol . Chem. 266, 19842-19850, (1991).
  • APP-695, APP-751, and APP-770 were produced by known techniques. See R. Bhasin et al., supra .
  • RIS were created by digestion of full length clones of APP with EcoRl, filling in the 5' - overhangs with Klenow, and self ligation, in accordance with known technigues. See generally T. Maniatis et al., in Molecular Cloning: A Laboratory Manual , (Cold Spring Harbor Laboratory, N.Y. 1989) .
  • This frameshift results in a stop codon seven amino acids after the start of the BA domain. The last three amino acids before the stop condon were altered in the process.
  • ⁇ were created by digestion of full length clones of APP with Xhol and Bglll followed by filling in of the overhangs by Klenow. See generally T. Maniatis et al., supra.
  • the 5 Kb top fragment was gel purified and self ligated, creating a deletion of 295 amino acids between the Kunitz protease inhibitor domain and the BA domain.
  • K mutants were created by digestion of the corresponding APP ⁇ plasmids with Xhol followed by filling in of the 5' - overhangs with Klenow and self ligation.
  • the resultant plasmids contained a stop condon after 308 amino acids in APP695-K, and 383 amino acids in APP770-K.
  • KX ⁇ was created by digestion of full length APP with Kpnl and Xcml, followed by digestion of the 3' - overhangs with T4 DNA polymerase.
  • the " 5 Kb bands were gel purified and self ligated.
  • the resultant plasmids had 263 amino acids deleted after amino acid 19 of APP, thereby deleting the cysteine rich and negatively charged domains of APP. All deleted APP clones were subsequently transferred to the baculovirus expression plasmid pJVPIO in accordance with known techniques. See R. Bhasin et al., Proc. Natl . Acad. Sci . USA. 88, 10307-10311 (1991).
  • Baculovirus expression plasmids containing the deleted APPs were co-transfected with either AcNPV DNA or linearized RP6 baculovirus DNA (for APP ⁇ and APP-K clones) as previously described. See R. Bhasin et al., supra ; P. Kitts et al., Nucl . Acid Res. 18, 5667-5672, (1990) .
  • conditioned media (20 ⁇ l) from baculovirus-infected insect cells transfected with the genes for APP-695, APP-751 or APP-770 (in 130 ⁇ l phosphate-buffered saline) prepared as described in Example 4 above were incubated with Immobilon AV membranes previously prepared with BA (1 . 28) peptide (BA) , or with ethanolamine (C) , then washed with 3.0 ml phosphate buffered saline and 700 ⁇ l 10% SDS. The membranes were then boiled five minutes in Laemmli buffer and the eluted proteins electrophoresed.
  • Lanes marked STD are 4 ⁇ l of conditioned media in Laemmli buffer loaded directly on the gel. Following electrophoresis, proteins were transferred to Immobilon P by Western technique, and the APP isoforms detected by the Anti-Alzheimer precursor protein antibody, and visualized as described previously.
  • APP-695, APP-751, and APP-770 was retained by membranes with immobilized BA (1 . 28) peptide, but was eluted from control membranes following 10% SDS. Since the APP-695 isoform bound to the BA (1 . 28) peptide, the Kunitz domain and a unigue 19 amino acid region in APP-770 do not appear to be required for APP binding.
  • deletion mutants of APP were tested. Mutants of APP containing only the amino terminus of APP (695-K and 770-K) were examined for BA binding, to determine whether the amino terminus of APP is capable of binding BA peptide. As shown in Figure 4B, these amino- terminus fragments of APP bound to the immobilized BA peptide, but not to the control membrane.
  • the RIS mutant terminates after the seventh amino acid of the amyloid domain, with substitution of three unrelated amino acids, and also bound to immobilized BA (1 . 28) peptide.
  • the ⁇ deletion mutant lacks the peptide region between the Kunitz domain and BA amyloid, and similarly bound to BA (1 . 28) peptide.
  • the KX ⁇ deletion mutants lack the amino terminus of APP between the signal sequence and the Kunitz domain.
  • the membranes were washed with phosphate buffered saline and 10% SDS, as described previously.
  • Gel lanes marked STD were loaded with conditioned media in Laemmli (4 ⁇ l of 770-APP and 770-KX ⁇ and 0.8 ⁇ l of 751-KX ⁇ ) .
  • the eluted proteins were electrophoresed, transferred by Western technique and visualized by the Alpha-5-anti-Alzheimer amyloid precursor protein antibody, a rabbit polyclonal antibody directed against amino acids 444-595 of APP-695, produced and felicitly supplied by Dr.Ivan Lieberburg.
  • Increasing the exposure time of the Hyperfilm with the i munoblot by five times as shown in Figure 5B, demonstrates small amounts of the KX ⁇ mutants bound to BA peptide, not detected on the ethanolamine-control membranes.
  • Disulfide bonds of the 695-K deletion mutant were reduced by boiling 100 ⁇ l of conditioned media in 10 ⁇ l of 300 mM dithiothreitol for five minutes. After cooling, the conditioned media was incubated in the dark with 10 ⁇ l iodoacetic acid (20 mg in 100 ⁇ l distilled water) at 37° C for thirty minutes. 30 ⁇ l of this material (Reduced) or 20 ⁇ l untreated material (Control) in 140 ⁇ l phosphate buffered saline were incubated with immobilized BA ⁇ 1 _ 28) (BA) , or ethanolamine (C) and the membranes were washed with phosphate buffered saline and 10% SDS.
  • BA immobilized BA ⁇ 1 _ 28
  • C ethanolamine
  • the retained proteins were then eluted in Laemmli buffer as described previously.
  • the lanes marked STD standards
  • the proteins were loaded directly with 6 ⁇ l of treated or 4 ⁇ l of control conditioned media.
  • the proteins were transferred to Immobilon P, and detected with the Anti- Alzheimer precursor protein antibody.
  • BOTTOM To determine the effects of pH on binding, 20 ⁇ l of conditioned media containing the 695-K deletion mutant in 130 ⁇ l of 0.1 M citric acid , 0.2 M Na 2 HP0 4 at the indicated pH, were incubated with immobilized BA (1 . 28) peptide thirty minutes.
  • the membranes were washed and eluted with 10 % SDS, and the 695-K APP was detected on the immunoblot following electrophoresis and transfer, as described above.
  • the 695-K mutant APP which binds BA peptide, is the amino terminus domain of APP containing twelve cysteines and the negatively-charged region rich in glutamic (28 residues) and aspartic (17 residues) acids. To determine whether the tertiary structure of this peptide established by disulfide bridges was necessary for binding BA peptide, the disulfide bonds were reduced. As shown in Figure 6A, reduction of disulfide bonds did not prevent binding of the 695-K mutant to BA peptide, suggesting that this binding does not require large domains of the protein with native tertiary structure. To determine whether charge interactions between the 695-K mutant and BA peptide were important in binding, binding was assayed over the pH range 2.5 to 7.6. The calculated pl of the 695-K mutant is 3.98. As shown in Figure 6B, binding between 695-K mutant and BA (1 . 28) was dependent on pH, with increased binding observed at pH values lower than 7.0.
  • the apoE3 and apoE4 isoforms were isolated from the plasma of fasting subjects with the E3/3 and E4/4 homozygous phenotypes, using techniques previously described in S. Rail, Methods Enzymol . 128, 273 (1986).
  • Synthetic 0A4 peptides were obtained from Bachem. One mg
  • 0A4 peptide was dissolved in 60 ⁇ l distilled water, then diluted with phosphate buffered saline, pH 7.30 as needed.
  • the Immobilon membrane was washed and incubated in primary antibody overnight, as described in W.J. Strittmatter, Proc. Natl . Acad. Sci . USA, 90:1977 (1993). Rabbit-anti human apoE antibody was used at 1:5000. Rabbit-anti 3A4 amyloid peptide antibody was used at 1:80. The Immobilon membrane was incubated with horseradish peroxidase-conjugated secondary antibody, and chemiluminescence was visualized by exposure to Hyperfiled obtained from Amersham. Quantitative scanning densitometry was on a Hoeffer gel scanner, and was analyzed with the included GS370 software.
  • V/V ⁇ -mercaptoethanol was added during incubation.
  • 30 mM dithiothreitol was added during the incubation.
  • the incubation was stopped by the addition of two aliquots of an equal volume of Laemmle buffer (without ⁇ -mercaptoethanol) , and boiled five minutes.
  • ⁇ - mercaptoethanol was added to a third sample after the incubation.
  • Dithiothreitol was added to a fourth sample after incubation. Proteins were electrophoresed on a 10% polyacrylamide gel, transferred to Immobilon P membrane, and the /SA4 peptide/apoE complexes were detected by anti- jSA4 peptide antibody.
  • apoE3 and apoE4 bound 3A4 (1 _ 40) , jSA4 (1 _ 28) , and 3A4 (12 _ 28) and was maximal at 10 '4 molar peptide in all three cases and half- maximal binding was approximately 10 "5 molar.
  • 0A4 (1 _ 28) was incubated with l ⁇ g truncated apoE3 for five hours and the incubation ended by boiling in Laemmli buffer (without ⁇ - mercaptoethanol) five minutes.
  • ⁇ h4 peptide was detected with the anti-SA4 peptide antibody.
  • ⁇ h4 peptide did not bind to the 22-kDa apoE3 fragment. See Figure 12. Binding of ⁇ h4 peptide to apoE3 (1 _ 244) was very low or minimal. In contrast, apoE3 (1 . 266) did form SDS-stable ⁇ h4 peptide complex, which was further increased with apoE (1 . 272) . See Figure 12.
  • ⁇ h4 binding by apoE appears to require the domain of apoE between amino acids 244 to 272 within the region previously demonstrated to mediate binding to lipoprotein particles as suggested in J.A. Westerlund, et al., J " . Biol . Chem. 263:6249 (1988).

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Abstract

Procédés permettant d'inhiber la liaison de la protéine β-amyloïde à la protéine précurseur d'amyloïde. Des produits d'assemblage comportant une protéine β-amyloïde ou un fragment de ladite protéine, immobilisés sur un support solide, des procédés de détection de composés qui se lient à la protéine β-amyloïde et des procédés de détection des composés qui inhibent la liaison de la protéine précurseur d'amyloïde à la protéine β-amyloïde sont décrits.
PCT/US1993/009772 1992-10-13 1993-10-13 Procede permettant d'inhiber la liaison de la proteine precurseur d'amyloide a la proteine beta-amyloide WO1994009364A1 (fr)

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Cited By (23)

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WO1996012736A3 (fr) * 1994-10-25 1996-08-01 Harvard College COMPOSES ET PROCEDES D'INHIBITION DE LA FORMATION ET DE LA NEUROTOXICITE DES FILAMENTS DE β-PROTEINES
WO1998037421A1 (fr) * 1997-02-19 1998-08-27 Regents Of The University Of Minnesota Criblage pour la recherche d'inhibiteur de formation de depot a-beta, au moyen d'amyloide synthetique
US5955472A (en) * 1995-11-02 1999-09-21 Warner-Lambert Company Naphthylazo inhibition of amyloidosis
US6017913A (en) * 1999-05-03 2000-01-25 Warner-Lambert Company Naphthylazo inhibition of amyloidosis
WO2000043791A2 (fr) 1999-01-25 2000-07-27 Minerva Biotechnologies Corporation Detection rapide et efficace d'une agregation de proteines aberrantes dans des maladies neurodegeneratives
US7575880B1 (en) 2000-05-26 2009-08-18 Elan Pharma International Limited Method of screening an antibody for activity in clearing an amyloid deposit
US7582733B2 (en) 1998-11-30 2009-09-01 Elan Pharma International Limited Humanized antibodies that recognize beta amyloid peptide
US7588766B1 (en) 2000-05-26 2009-09-15 Elan Pharma International Limited Treatment of amyloidogenic disease
US7625560B2 (en) 2004-12-15 2009-12-01 Janssen Alzheimer Immunotherapy Humanized antibodies that recognize beta amyloid peptide
US7700751B2 (en) 2000-12-06 2010-04-20 Janssen Alzheimer Immunotherapy Humanized antibodies that recognize β-amyloid peptide
US7790856B2 (en) 1998-04-07 2010-09-07 Janssen Alzheimer Immunotherapy Humanized antibodies that recognize beta amyloid peptide
US7871615B2 (en) 2003-05-30 2011-01-18 Janssen Alzheimer Immunotherapy Humanized antibodies that recognize beta amyloid peptide
US7893214B2 (en) 1997-12-02 2011-02-22 Janssen Alzheimer Immunotherapy Humanized antibodies that recognize beta amyloid peptide
US7964192B1 (en) 1997-12-02 2011-06-21 Janssen Alzheimer Immunotherapy Prevention and treatment of amyloidgenic disease
US8003097B2 (en) 2007-04-18 2011-08-23 Janssen Alzheimer Immunotherapy Treatment of cerebral amyloid angiopathy
US8034339B2 (en) 1997-12-02 2011-10-11 Janssen Alzheimer Immunotherapy Prevention and treatment of amyloidogenic disease
US8128928B2 (en) 2002-03-12 2012-03-06 Wyeth Llc Humanized antibodies that recognize beta amyloid peptide
US8613920B2 (en) 2007-07-27 2013-12-24 Janssen Alzheimer Immunotherapy Treatment of amyloidogenic diseases
US8784810B2 (en) 2006-04-18 2014-07-22 Janssen Alzheimer Immunotherapy Treatment of amyloidogenic diseases
US8916165B2 (en) 2004-12-15 2014-12-23 Janssen Alzheimer Immunotherapy Humanized Aβ antibodies for use in improving cognition
US9067981B1 (en) 2008-10-30 2015-06-30 Janssen Sciences Ireland Uc Hybrid amyloid-beta antibodies
US9644025B2 (en) 2007-10-17 2017-05-09 Wyeth Llc Immunotherapy regimes dependent on ApoE status
WO2023175225A1 (fr) * 2022-03-17 2023-09-21 Universidad Miguel Hernández De Elche Méthode et trousse de diagnostic de la maladie d'alzheimer reposant sur la détection de l'apolipotrotéine e

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US6214569B1 (en) 1992-01-13 2001-04-10 President And Fellows Of Harvard College Methods for screening for inhibitors of Alzheimer β-peptide filament formation
WO1996012736A3 (fr) * 1994-10-25 1996-08-01 Harvard College COMPOSES ET PROCEDES D'INHIBITION DE LA FORMATION ET DE LA NEUROTOXICITE DES FILAMENTS DE β-PROTEINES
EP1172377A1 (fr) * 1994-10-25 2002-01-16 The President And Fellows Of Harvard College Composés et procédés d'inhibition de la formation et de la neurotoxicité des filaments d' amyloid BETA-protéines
US5955472A (en) * 1995-11-02 1999-09-21 Warner-Lambert Company Naphthylazo inhibition of amyloidosis
WO1998037421A1 (fr) * 1997-02-19 1998-08-27 Regents Of The University Of Minnesota Criblage pour la recherche d'inhibiteur de formation de depot a-beta, au moyen d'amyloide synthetique
US8535673B2 (en) 1997-12-02 2013-09-17 Janssen Alzheimer Immunotherapy Prevention and treatment of amyloidogenic disease
US8034348B2 (en) 1997-12-02 2011-10-11 Janssen Alzheimer Immunotherapy Prevention and treatment of amyloidogenic disease
US7964192B1 (en) 1997-12-02 2011-06-21 Janssen Alzheimer Immunotherapy Prevention and treatment of amyloidgenic disease
US7893214B2 (en) 1997-12-02 2011-02-22 Janssen Alzheimer Immunotherapy Humanized antibodies that recognize beta amyloid peptide
US8642044B2 (en) 1997-12-02 2014-02-04 Janssen Alzheimer Immunotherapy Prevention and treatment of amyloidogenic disease
US8034339B2 (en) 1997-12-02 2011-10-11 Janssen Alzheimer Immunotherapy Prevention and treatment of amyloidogenic disease
US9051363B2 (en) 1997-12-02 2015-06-09 Janssen Sciences Ireland Uc Humanized antibodies that recognize beta amyloid peptide
US7790856B2 (en) 1998-04-07 2010-09-07 Janssen Alzheimer Immunotherapy Humanized antibodies that recognize beta amyloid peptide
US7582733B2 (en) 1998-11-30 2009-09-01 Elan Pharma International Limited Humanized antibodies that recognize beta amyloid peptide
JP2012230124A (ja) * 1999-01-25 2012-11-22 Minerva Biotechnologies Corp 神経変性疾患における異常型タンパク質凝集の迅速かつ高感度の検出
JP2002540383A (ja) * 1999-01-25 2002-11-26 ミナーヴァ・バイオテクノロジーズ・コーポレーション 神経変性疾患における異常型タンパク質凝集の迅速かつ高感度の検出
WO2000043791A2 (fr) 1999-01-25 2000-07-27 Minerva Biotechnologies Corporation Detection rapide et efficace d'une agregation de proteines aberrantes dans des maladies neurodegeneratives
WO2000043791A3 (fr) * 1999-01-25 2001-07-26 Minerva Biotechnologies Corp Detection rapide et efficace d'une agregation de proteines aberrantes dans des maladies neurodegeneratives
US6017913A (en) * 1999-05-03 2000-01-25 Warner-Lambert Company Naphthylazo inhibition of amyloidosis
US7588766B1 (en) 2000-05-26 2009-09-15 Elan Pharma International Limited Treatment of amyloidogenic disease
US7575880B1 (en) 2000-05-26 2009-08-18 Elan Pharma International Limited Method of screening an antibody for activity in clearing an amyloid deposit
US7700751B2 (en) 2000-12-06 2010-04-20 Janssen Alzheimer Immunotherapy Humanized antibodies that recognize β-amyloid peptide
US8128928B2 (en) 2002-03-12 2012-03-06 Wyeth Llc Humanized antibodies that recognize beta amyloid peptide
US7871615B2 (en) 2003-05-30 2011-01-18 Janssen Alzheimer Immunotherapy Humanized antibodies that recognize beta amyloid peptide
US7625560B2 (en) 2004-12-15 2009-12-01 Janssen Alzheimer Immunotherapy Humanized antibodies that recognize beta amyloid peptide
US8916165B2 (en) 2004-12-15 2014-12-23 Janssen Alzheimer Immunotherapy Humanized Aβ antibodies for use in improving cognition
US8784810B2 (en) 2006-04-18 2014-07-22 Janssen Alzheimer Immunotherapy Treatment of amyloidogenic diseases
US8003097B2 (en) 2007-04-18 2011-08-23 Janssen Alzheimer Immunotherapy Treatment of cerebral amyloid angiopathy
US8613920B2 (en) 2007-07-27 2013-12-24 Janssen Alzheimer Immunotherapy Treatment of amyloidogenic diseases
US9644025B2 (en) 2007-10-17 2017-05-09 Wyeth Llc Immunotherapy regimes dependent on ApoE status
US9067981B1 (en) 2008-10-30 2015-06-30 Janssen Sciences Ireland Uc Hybrid amyloid-beta antibodies
WO2023175225A1 (fr) * 2022-03-17 2023-09-21 Universidad Miguel Hernández De Elche Méthode et trousse de diagnostic de la maladie d'alzheimer reposant sur la détection de l'apolipotrotéine e
ES2951901A1 (es) * 2022-03-17 2023-10-25 Univ Miguel Hernandez De Elche Metodo y kit de diagnostico de la enfermedad de alzheimer basado en la deteccion de apolipoproteina e

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