Classifications

• • 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
  • C12N9/6472—Cysteine endopeptidases (3.4.22)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/55—Protease inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • 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

Definitions

• This invention relates to treatment of Alzheimer's disease.
• Amyloid deposits are thought to be trophic or toxic to their surroundings. Amyloid deposits are found in the center of senile plaques and in the blood vessels in the brains of Alzheimer's disease ("AD") patients.
• the major component of brain amyloid is the ⁇ -protein, a 4 Kd (39-42 a ino acids) fragment (see, e.g., G.G. Glenner et al. (1984), Biochem. Biophys. Res. Comm. , Vol. 12, pp. 1131-35; C.L.
• brain amyloid In addition to and tightly associated with the ⁇ -protein, brain amyloid also contains a serine protease inhibitor, ⁇ l-antichymotrypsin ("ACT").
• ACT serine protease inhibitor
• Certain ⁇ -PP transcripts include a domain homologous to the Kunitz- type protease inhibitors (described, for example, in N. Kitaguchi et al.
• DSS disuccinimidyl suberate
• AD proteolytic factors from the brain of AD patients here termed "AD proteolytic factors”.
• accumulation of the ⁇ -protein is a consequence of an alternative degradati pathway that results in abnormal ⁇ -PP processing, and one or more of the AD proteolytic factors participates in this abnormal pathway.
• the invention features an AD proteolytic factor capable of cleaving ⁇ -protein precursor at a site near the ⁇ -protein N-terminus.
• the AD proteolytic factor is capabl of cleaving ⁇ -PP at a site outside the ⁇ -protein domain and near the ⁇ -protein N-terminus, more preferably at a site following lysine or at a sit following methionine;
• a first AD proteolytic factor includes a calcium- activated protease, preferably a serine protease; activity of the serine AD proteolytic factor is inhibited by PN2 and by ACT;
• a second AD proteolytic factor includes a cysteine protease; the cysteine protease is a metalloprotease, Ca 2+ - or Mg 2+ -dependent (and possibly Zn 2+ -dependent) having a molecular weight about 43-68 kDa.
• the invention features a method for treating Alzheimer's disease in a patient, by reducing ⁇ -protein precursor proteolysis at a site near the ⁇ -protein N-terminus.
• the method includes administering to the patient an inhibit that inhibits proteolysis at a site outside the ⁇ -protein domain of ⁇ -PP and at or near the ⁇ -protein N-terminus, and preferably inhibits proteolysis in the vicinity of the ⁇ -protein N-terminus, preferably by inhibiting the proteolytic activity of a proteolytic factor that acts at such a site; the inhibitor is capable of passing the blood-brain barrier, and the inhibitor ca be administered, for example, parenterally (intravascularly or intramuscularly) or orally.
• the invention features a method for diagnosis in a subject of a disease characterized by accumulation of amyloi and particularly of Alzheimer's Disease, by determining the level, in a sample from the subject, such as a tissue or fluid sample, of an AD proteolytic factor.
• the invention features a method for screening for an agent useful in treatment of a disease characterized by accumulation of amyloid, by incubating an AD protease with a peptide having an amino acid sequence corresponding to the sequence spanning the ⁇ -protein N-terminus in the presence of the candidate agent, and determining degradation of the peptide.
• a candidate agent may be useful i treating such a disease where peptide degradation by the AD protease is less in the presence of the candidate agent than would have been expected under the same or similar reaction conditions in the absence of the candidate agent.
• the peptide has an amino acid sequence that includes a 10-amino acid sequence spanning the ⁇ -protein N-terminus,. and more preferably beginning five or six amino acids upstream from the N-terminus.
• the invention features a method for purifying an enzyme from a sample, and particularly a proteolytic enzyme, by incubating the sample with a substrate of the enzyme or with a fragment of the substrate to which the enzyme binds, treating the sample with DSS to crosslink any enzyme-substrate complexes in the sample, and recovering the complexes.
• the substrate or substrate fragment is labelled (more preferably radiolabelled). Description of Preferred Embodiments
• Fig. 1 is a series of prints showing serine protease activity in fractions initially purified from brain homogenates from Alzheimer's disea (“AD") patients.
• Panel A is a photograph of a coomassie blue stained SDS PAGE gel showing cleavage products of the iodinated peptide 125 I- HSEVKMDAEF (peptide "PI") following reaction with brain homogenates fractions and cross-linking with disuccinimidyl suberate (“DSS”).
• Panel B an X-radiograph of a gel prepared as in Panel A.
• Panel C is an autoradiograph of a cellulose microcrystalline thin layer chromatography (“TLC”) plate showing cleavage products of 126 I-P1 following reaction with brain homogenate fractions.
• TLC thin layer chromatography
• Fig. 2 is a series of prints showing serine protease activity in fractions from brain homogenates from AD patients, further purified by si exclusion chromatography.
• the respective panels A, B, C are as describe in Fig. 1.
• Fig. 3 is a print showing inhibition by various agents of serine protease activity in fractions from brain homogenates from AD patients.
• Fig. 4 is a sequence map showing cleavage of PI by cathepsin G (upper) and by Ca 2+ activated specific serine protease ("CASP") according the invention (lower).
• H histidine
• S serine
• E glutamic acid
• V valine
• K lysine
• M methionine
• D aspartic acid
• A alanine
• F phenylalanine.
• Numerals show percentage cleavage of the peptide bond a each point indicated by an arrow.
• FIG. 5 is a print of a radiograph of a TLC plate showing cysteine protease activity in fractions purified using DEAE-Trisacryl M ion exchan chromatography from brain homogenates from AD patients. Fractions eluted with a linear NaCl gradient, indicated by the arrow at the lower margin of the Fig., were incubated with radioiodinated PI and separated TLC. The sequences of uncleaved PI and of the cleaved products are sho to the right.
• Fig. 6 is a print showing the influence of dithiothreitol ("DTT”) on AD cysteine protease activity.
• DTT dithiothreitol
• DTT DTT solvent alone (no DTT); B, 5.0 mM DTT; C, 2.5 mM DTT; D, 1.0 mM DTT; E, peptide alone (no DTT, no solvent).
• Fig. 7 is a print showing inhibition by various agents on cysteine protease activity in fractions from brain homogenates from AD patients. Isolation and Purification of Cysteine AD proteolytic factor An AD proteolytic factor according to the invention can be identified in and isolated from a tissue homogenate using, for example, conventional liquid chromatography.
• the protocol includes steps of homogenizing the tissue; making a crude separation using affinity liquid chromatography; further separating using a first DEAE-ion exchange column, followed by a gel filtration column, followed by a second DEAE-ion exchange column; and dialyzing and finally purifying using affinity liquid chromatography.
• the description also includes protocols for characterizing the purified AD proteolytic factor (molecular weight; substrate specificity) and for screening for useful inhibitors of the activity.
• This cysteine AD protease cleaves the PI peptide after Met. It has a molecular weight about 43-68 kDa, and is a metalloprotein, being Ca 2+ - or
• Brain tissue from AD patients is homogenized in ice-cold 5 x (volume/weight) Tris-Cl buffer containing 1% Triton X-100 and 1 mM dithiothreitol ("DTT") in a Wearing blender. After homogenization, the solution is stirred for 30 minutes on ice, and then centrifuged at 100,000 x for 60 minutes.
• the supematants representing the soluble enzyme are subjected to ammonium sulfate fractionation: 0-25%, 25-50%, 50-75%, >75%, by slowly adding ammonium sulfate salt to the supematants while stirring on ice.
• the solution is then stirred for 20 minutes and centrifuged in a Sorval RC-58 refrigerated centrifuge at 10,000 x g for 30 minutes. After the third centrifugation, the three precipitates from the ammonium sulfate fractionation steps are redissolved in Tris-Cl and 1 mM DTT, pH 7.4 buffer, and all fractions are dialyzed extensively against the same buffer before further steps.
• an 126 I-labeled peptide having the sequence HSEVKMDAEF (peptide Tl") was synthesized corresponding to the ⁇ -PP sequence flanking that site.
• the peptide starts five amino acids upstream from the N-terminus (the aspartic acid, "D", is at the N-terminus of the ⁇ -protein) and extends across the putative cleavage site into the ⁇ -protein itself; histidine, "H”, was added for purpose of radioiodination (that is, histidine replaces the isoleucine that appears at that site in the native ⁇ -protein).
• Labeled peptide was incubated with brain fractions of varying purity and the resulting fragments were separated by t.hi-n layer chromatography ("TLC"); N-terminal fragments were detected by autoradiography.
• TLC t.hi-n layer chromatography
• the site of cleavage for an unknown cleavage product is then determined either by direct sequence analysis of the cleavage product, or by comparing the unknown cleavage product with cleavage products resulting from a known enzyme such as cathepsin G.
• Proteolytic activities from AD brain can also be examined by Western blots using full length ⁇ -PP derived from rat brain as a substrate.
• Affigel Blue Affinity Chromatography.
• Affigel Blue is preferred for the first chromatographic step, as it purifies the protease pool from serum albumin and a large number of other protein species.
• Affigel Blue Bio-Ra (1.5 cm x 33 cm) is equihbrated to 50 mM Tris-Cl + 1 mM DTT, pH 7.4, an eluted with a linear gradient of 0 to 0.5 M NaCl. The flow rate is 20 ml/hour, and 3 ml fractions are collected.
• a protease activity-positive pool from the first DEAE-Trisacryl M gel filtration step is concentrated to 2.5 m by ultrafiltration through an Amicon filter (PM-10, 10 kDa cutoff) under nitrogen.
• the concentrated pool is then loaded on a Sephacryl S-200 gel filtration column (2.5 cm x 66 cm), equihbrated, and eluted with 50 mM Tris-Cl + 1 mM DTT, Ph 7.4. The flow rate is adjusted to 25 ml/hour, and 2.2 ml fractions are collected.
• Second DEAE-ion Exchange Chromatography is adjusted to 25 ml/hour, and 2.2 ml fractions are collected.
• the pool from the Sephacryl S-200 column containing the protease activity is chromatographed on a second DEAE-Trisacryl M ion exchange column (1.5 cm x 4.5 cm) with a gradient of 0 to 0.5 M NaCl in 50 mM Tris-Cl + 1 mM DTT, pH 7.4. The flow rate is adjusted to 20 ml/h, and 2.2 ml fractions are collected.
• the molecular weight of the protease can be estimated by gel filtration using a Sephacryl S-200 (2.5 cm x 66 cm) column equihbrated with 50 mM Tris-Cl + 1 mM DTT, pH 7.4. The column is ' ited with the same buffer at 25 l hour, collecting 2.2 ml fractions. The fractions are assayed for proteolytic activity as described above. Protein standards for calibration of the column include ⁇ -amylase (200,000 kDa), alcohol dehydrogenase (150,000 kDa), albumin (66,000 kDa), carbonic anhydrase (29,000 kDa), and cytochrome C (12,400 kDa).
• the apparent molecular weight of the protease can also be determined by SDS-PAGE.
• Molecular weight standards include: Myosin (H-chain) (228,000 kDa), phosphorylase B (109,600 kDa), bovine serum albumin (70,000 kDa), ovalbumin (44,100 kDa) and carbonic anhydrase (27,900 kDa).
• Myosin H-chain
• phosphorylase B 109,600 kDa
• bovine serum albumin 70,000 kDa
• ovalbumin 44,100 kDa
• carbonic anhydrase 27,900 kDa
• proteolytically active sample is incubated with the appropriate amount of a putative inhibitor reagent for
• protease pool To examine the substrate specificity of the purified protease pool, aliquots of the purified active fraction were electrophoresed on SDS- substrate gels, containing 1 mg/ml casein or gelatin. The protease pool was mixed on a 1:1 ratio with 2 x Laemmli sample buffer without mercaptoethanol and loaded on a 12% SDS polyacrylamide gel containing
• Electrophoresis was carried out at 4 °C at 20 mA. After electrophoresis, the SDS was removed by shaking the gel in 2.5% Triton X-100 for 30 min. at 25 °C. The gel was the incubated in 50 mM Tris-Cl + 1 mM CaCL- for 2 days at 37 °C while shaking. The gel was stained in 0.5% Coomas ⁇ ie Blue and destained.
• the protease was labelled with C NEM and the sample was analyzed using SDS-PAGE and autoradiography, as follows. Because
• NEM W C NEM is provided in n-pentane
• the NEM solution was added to an equal volume of double distilled water and the n-pentane was evaporated with a gentle stream of nitrogen gas before use.
• the protease pool solution was incubated with 1 C NEM (6.7 mM final concentration NEM) at 4 °C for 2 hours. After incubation, the solution was mixed with an equal volume of 2x sample buffer and then electrophoresed on a 12% SDS acrylamide gel, generally as described in U.K. Laemmli (1970), Nature, Vol. 227, pp. 680-85.
• the gel was washed in 40% (v/v) methanol, 10% (v/v) acetic acid for 30 min, and then was washed in Enlightning solution (New England Nuclear) for 30 min, and then was dried under vacuum and heat. The gel was then exposed to film for 2 weeks using an intensifying screen.
• the cysteine AD protease becomes radiolabelled by virtue of its being bound to the labelled NEM inhibitor.
• the activity of the protease was tested against full length ⁇ -PP purified from rats by incubating them in 50 mM Tris-Cl, pH 7.4, 1.2 mM DTT, 1.7 mM MgCl ⁇ . The reactions were incubated overnight at 37 °C and then separated on 7.5% SDS-PAGE gels. The separated polypeptides were transferred to PDVF membranes (Millipore) generally as described in Towbin et al. (1979), Proc. Natl. Acad. Sci. USA, Vol. 76, p. 4350.
• the blots were immunoetained with rabbit anti- ⁇ -PP antibody targeted to the area flanking the N-terminus of the ⁇ -peptide, and ⁇ -PP fragments were detected using goat anti-rabbit alkaline phosphatase and the appropriate color substrate.
• Fig. 5 shows cysteine protease activity in fractions purified from brain homogenates from AD patients using DEAE-Trisacryl M ion exchange chromatography as described above.
• Fig. 6 shows the effect of DTT on AD cysteine protease activity.
• Fig. 7 illustrates effects of inhibition by various agents on cysteine protease activity in fractions prepared as described above from brain homogenates from AD patients. The AD cysteine proteolytic factor preparation and the inhibitor (at the indicated concentrations) were incubated at 0 °C for 30 min and then assayed for remaining activity. L identifications in Fig.
• lane A H-.0
• lane B Na iodoaceta 5 mM
• lane C E-64 (Sigma), 0.02 mM
• lane D E-64, 0.01 mM
• lane E p- hydroxymercuribenzoate, 5 mM
• lane F N-ethylmaleimide, 5 mM
• lane G phenanthroline, 4 mM
• lane H o-phenanthroline, 1.8 mM
• lane I ethanol lane J, ethanol/H j O
• lane K PMSF, 5 mM
• lane L bestatin, 0.02 mM
• lan M EGTA, 5 mM
• lane N CaCla
• 2 2 mM
• lane O DTT, 5 mM
• lane P pepti alone.
• AD proteolytic Factor Isolation and Characterization of Serine AD Proteolytic Factor The following is a detailed description of identification and purification of an AD proteolytic factor that includes a Ca 2+ activated seri protease whose PI cleaving activity is inhibited by ACT and PN2.
• Brain fractions were incubated with the iodinated peptide ( 126 I-P1) and treated with disuccinimidyl suberate ('OSS") to crosslink any protein that were in intimate contact with the peptide, i.e., to crosslink any enzy substrate complex (and, in this instance, any protease-substrate complexe). Then, the enzymes were recovered in fractions containing a labelled enzyme-substrate complex, stabilized by the DSS cro ⁇ slinking, and N- terminal fragments were detected by autoradiography on TLC plates, generally as described above in the detailed protocol for the cysteine AD protease.
• 'OSS disuccinimidyl suberate
• AD brain Alzheimer's disease brain
• PBS phosphate buffered saline
• DDT dithiothreitol
• the supernatant was dialyzed against PBS (20 mM phosphate buffer pH 7.0, 20 mM NaCl) and 1 mM DDT, and applied to DE52-cellulose (Whatman) equihbrated with 10 mM Tris-HCl (pH 7.0, 20 mM NaCl) and 1 mM DDT; the column was extensively washed and bound proteins were eluted with 0.5 M NaCl.
• the active fraction was further purified by ammonium sulfate precipitation, followed by dialysis. The purification was monitored by a peptide degradation assay as follows.
• Lane identifications in Fig. 1 are as follows: lane 1, Mr standards; lanes 2, 6, 15, DE52 column fiowthrough fraction; lanes 3, 7, 10, ammoniu sulfate CAS") precipitation, 0-25% saturation; lanes 4, 8, 11, 25-50% AS; lanes 5, 9, 12, 50-75% AS; lane 13, .75% AS; lane 14, untreated 1 6 I-P1.
• the asterisk (*) indicates the minor 30 Kd band.
• Further purification including a 100,000 g spin in PBS followed by solubilization of the pellet in 1% Triton X-100 in PBS and a second spin at 100,000 g, revealed that following these treatments the enzymatic activity is found in both the soluble fraction and the membrane-bound fraction.
• Fig. 2 Re ⁇ ults of purification of a specific serine protease from AD brain by size exclusion chromatography are shown in Fig. 2.
• Fractions demonstrating activity were concentrated and applied to a Sephadex S-300 column (Pharmacia) and eluted with 10 mM Tris-HCl, pH 7.0, 200 mM NaCl, and 1 mM DDT.
• Panels A, B and C in Fig. 2 are as in Fig. 1. Analysis of S-300 column fractions 14 to 32 is shown; fraction numbers are indicated. The first lane in Panel C is untreated 126 I-P1; the second lane is fraction AS 50- 75% (before S-300 separation). Fractions 23-28 were reserved for further analysis.
• FIG. 1 A single protein was radioaffinity labeled (Fig. 1) following initial purification. Subsequent steps of purification resulted in a major protein of approximately 68 Kd (Figs. 1 and 2) and a minor one at 30 Kd (Fig. 1).
• Fig. 3 illustrates effects of inhibitors on the protease activity of the serine AD proteolytic factor isolated as described above.
• EGTA an inhibitor of Ca 2+ dependent proteases
• DFP an inhibitor of serine proteases
• the complex of the AD proteolytic factor protease with PN2 is reversible; compare lane 8 (1 hr incubation) with lane 9 (20 hrs incubation). In contrast, protease nexin 1 and albumin did not influence the activity of the AD proteolytic factor.
• Kunitz-type protease inhibitors are identical to the inhibitor protease nexin 2 ("PN2", described, for example, in W.E. Van Nostrand et al. (1989), Nature, Vol. 341, pp. 546-49; T. Oltersdorf et al. (1989), Nature, Vol. 341, pp. 144-47.
• PN2 inhibitor protease nexin 2
• the calcium-activated AD proteolytic factor cleaves metabolically labelled endogenous ⁇ -PP substrate as well as the PI peptide.
• Labelled 36 S- ⁇ -PP made as described in S. Sisodia et al. (1990), Science, Vol. 248, pp. 492-95 and obtained from S. Sisodia, was incubated with the AD proteolytic factor, and the fragments were separated on gel and the gel was autoradiographed.
• a mixture of proteases appear to be present, and one or more than one of these may play a role in an alternative pathway in the brains of persons having abnormal amyloid deposits.
• the serine protease fraction In addition to being able to degrade the synthetic PI, the serine protease fraction also degraded purified human PN2, and metabolically labeled 38 Met-PN2 secreted into the medium by cells transfected with the human ⁇ -PP770. Other labeled secreted proteins were not affected by the protease.
• ⁇ -PP the ⁇ - protein precursor
• ACT amyloid deposits of the ⁇ -protein type
• ⁇ -PP the ⁇ - protein precursor
• ACT the ⁇ -protein precursor
• the two forms of the ⁇ -PP one having the protease inhibitory domain (751/770 amino acids) and the other lacking it (695 amino acids)
• the different ratios of the two forms may explain the almost unique accumulation of the ⁇ -protein type amyloid in the brain, although ⁇ -protein antibodies can also label skin, intestine and adrenal sections.
• the amounts of the ⁇ -PP are compared between AD and controls they do not seem significantly different, but high levels of abnormal degradative forms of ⁇ -PP are found in AD neurons and neurites and on Western blots using ⁇ -PP antibodies.
• AD Alzheimer's disease
• ⁇ -protein is probably formed as a result of an abnormal proteolytic degradation of a normal protein.
• the finding that the AD cysteine protease cleaves after lysine suggests that it may also be able to cleave at amino acids 15-17 (QKL; Q, gluta ine; L, lysine) of the ⁇ -protein.
• QKL gluta ine
• L lysine
• AD may be treated according to the invention by administering to the patient an inhibitor of the AD proteolytic activity according to the invention.
• an inhibitor can be, for example, a competitive inhibitor, such as a fragment of the ⁇ -PP molecule corresponding to the binding site of the proteolytic enzyme.
• AD protease Likely candidates for effective inhibitors of AD proteolytic activity can be screened by incubating an AD protease according to the invention with a known specific substrate (such as a synthetic oligopeptide having an amino acid sequence corresponding to a sequence spanning the ⁇ -protein N-terminus) in the presence of a candidate agent under conditions in which the AD protease would be expected, in the absence of an inhibitor, to cleave the synthetic oligopeptide near the N-terminus.
• a known specific substrate such as a synthetic oligopeptide having an amino acid sequence corresponding to a sequence spanning the ⁇ -protein N-terminus
• a preferred inhibitor is capable of crossing the blood-brain barrier, so that it can be administered parenterally or orally.
• a preferred inhibitor is a molecule other than a peptide, so that the inhibitor will not be rapidly degraded following administration.
• the preferred inhibitor specifically inhibits the ⁇ -PP cleaving activity of the AD protease, and does not generally inhibit the activity of brain proteases that are essential to normal metabolism.
• Proteolytic fragments resulting from the action on ⁇ -PP of an AD protease according to the invention can be detected using, e.g. , a battery of antibodies directed against the C-terminus and the N-terminus of the ⁇ -PP on Western blots.
• inhibitors may be screened for capacity to arrest formation of the AD protease in vitro.
• Monoclonal antibodies raised against a purified AD protease according to the invention, prepared using standard protocols, can be used to establish (and to quantify) the cellular location of the enzyme in the brain and in other tissues such as skin, kidneys, and hver.
• Such mAb can also be used in the screening of expression libraries, for locating and identifying AD protease-encoding genes, and for cloning such AD protease genes for use in AD protease-producing expression systems.
• AD amyloid deposition
• Tissue or body fluid samples such as, for example, samples of blood, CSF, saliva, urine, can be drawn and assayed for the presence of AD proteases, as an indication of a likelihood of abnormal ⁇ -PP metabolism, producing ⁇ protein and, ultimately, causing amyloid deposition in tissues.
• any crosslinking agent other than DSS can be used in the enzyme purification protocol, provided that the agent is capable of forming crosslinks in an enzyme-substrate complex between portions of the enzyme and the substrate where the enzyme and substrate are in near proximity.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biomedical Technology (AREA)
• Organic Chemistry (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Zoology (AREA)
• Genetics & Genomics (AREA)
• Neurology (AREA)
• Wood Science & Technology (AREA)
• Biochemistry (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Neurosurgery (AREA)
• Molecular Biology (AREA)
• Animal Behavior & Ethology (AREA)
• Gastroenterology & Hepatology (AREA)
• Pharmacology & Pharmacy (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Microbiology (AREA)
• Biotechnology (AREA)
• Psychiatry (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Biophysics (AREA)
• Toxicology (AREA)
• General Engineering & Computer Science (AREA)
• Hospice & Palliative Care (AREA)
• Immunology (AREA)
• Epidemiology (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Peptides Or Proteins (AREA)
• Enzymes And Modification Thereof (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)

Applications Claiming Priority (2)

Publications (2)

Family

ID=24272818

Family Applications (1)

Country Status (5)

Families Citing this family (28)

• 1991
  • 1991-08-19 HU HU9300421A patent/HUT69771A/hu unknown
  • 1991-08-19 AU AU85387/91A patent/AU643835B2/en not_active Ceased
  • 1991-08-19 EP EP9191916872A patent/EP0546084A4/en not_active Withdrawn
  • 1991-08-19 JP JP3515327A patent/JPH06503948A/ja active Pending
  • 1991-08-19 WO PCT/US1991/005932 patent/WO1992003542A1/en not_active Application Discontinuation

Non-Patent Citations (5)

Also Published As

Similar Documents

Legal Events